Techniques for Studying Iron in Health and Disease EMBL COURSE EMBL-ATC May 2-4, 2019 Coordinated by D Swinkels and I Cabantchik

Techniques for Studying Iron in Health and Disease EMBL COURSE EMBL-ATC May 2-4, 2019 coordinated by D Swinkels and I Cabantchik

Introduction to Practicals and experimental Protocols

General introduction 1

Program DAY 1 2 I. Generation and interpretation of assay test results for biomarkers of iron homeostasis and dyshomeostasis. Rian Roelofs, Rachel van Swelm Dorine Swinkels. Radboud University. Nijmegen NL. 3-25 References (pp 25)

Program DAY 2 26 II. Labile 2Fe-2S Clusters. Rachel Nechushtai. Institute of Life Sciences. Hebrew University, Jerusalem, Israel 26-43 References (pp 28-29) II. Biochemical and functional characterization of mitochondrial ISC assembly factors. Oliver Stehling. Institut für Zytobiologie, Philipps- Universität Marburg 44-77 References (pp 76-77)

Program DAY 79 Real-time monitoring of intracellular labile iron with fluorescent metal- sensors. Maya Shvartsman. EMBL HD. Rome. IT 80-87

Labile Iron IN BIOLOGICAL FLUIDS. Breno Pannia Espósito, Institute of Chemistry University of São Paulo, Brazil 88-94

Mitochondrial Iron probing. Charareh Pourzand. Department of Pharmacy & Pharmacology. Centre for Therapeutic Innovation University of Bath, UK 95-103 References (pp 98) Techniques for studying iron in health and disease EMBL

EMBL, Heidelberg, May 2-4, 2019 1st International laboratory course dedicated to analytical methods for assessing iron in living entities in physiological and pathological states Directors: Dorine Swinkels(NL) and Ioav Cabantchik (IL) https://www.embl.de/training/events/2019/BIR19-02/index.html

The workshop is designed to expose the various experimental tools used for: a. tracing iron dynamics in biological systems, from solution to cells to organs, with major emphasis on fluorescence techniques available in most laboratories b. assessing iron status in humans in health and disease and c. exploring methods to study iron sulphur cluster (ISC) proteins (ISP). The 3-day workshop is planned for up to 18 students. Morning sessions comprise lectures and experimental planning and afternoon-evening data analysis and discussions. Bursaries covering the course registration fees are available for selected applicants.

TOPICS DAY I. Thursday May 2 2019. GENERATION AND INTERPRETATION OF ASSAY TEST RESULTS FOR BIOMARKERS OF IRON HOMEOSTASIS AND DYSHOMEOSTASIS Coordinator: Dorine W. Swinkels The aim is to reach a level of understanding for adequately generating and interpreting the results of assays for biomarkers of iron homeostasis and dys-homeostasis in both research and clinical setting. Specifically, the students should acquire a level of understanding to be able to: a. Clinically interpret test results of a broad range of conventional and novel iron biomarkers b. Apply fundamentals of implementing an assay and generating a reliable test result in both the research and clinical setting. In the workshop we will: - Discuss interpretation of test results generated by a fully validated assay for the diagnosis of iron disorders. Students will be challenged to solve case –puzzles in small groups, and present their “diagnosis” for the whole group for discussion. - Discuss design of a validation plan for an assay of an analyte. In the afternoon, students will apply this knowledge by performing a validation of a commercial hepcidin ELISA kit in the lab in small groups. By the end of the day each group will present their analytical findings for the whole group for discussion.

Techniques for studying iron in health and disease EMBL

DAY II. May 3 2019. DYNAMICS OF IRON SULFUR CLUSTER (ISC) PROTEINS (ISP): FROM SOLUTION TO CELLS Coordinator: Coordinators: Stehling, Lill & Nechushtai The aim is to get acquainted with the application of spectroscopic methods for assessing mitochondrial ISP maturation within the cellular environment. I. Biophysical and biochemical analysis of recombinant ISP a. Determination of iron and sulphide content of holo-ISP: ISCA1 and FDX2 as paradigms. b. Assessment of ISC cofactor stability under reducing and oxidizing conditions (UV/Vis spectroscopy). c. Tracing ISC transfer between proteins by native gel chromatography and UV-VIS spectroscopy-CISD2-gene product NAF-1 (wild type and mutants) as paradigm.

II. Assessing the consequences of ISP assembly defects of cells as reflected in: a. morphological and metabolic changes of mitochondria; b. Activity measurements of key mitochondrial ISC enzymes by spectroscopic assays; c. ISC-dependent protein modification (lipoylation of DLAT and DLST). b. ISC transfer between proteins by native gel chromatography and UV-VIS spectroscopy: CISD2-gene product NAF-1 (wild type and mutants) as paradigm. d. the roles of amino acid residues in the ISC domain of NAF-1 on cell properties by fluorescence spectroscopy: a. mitochondrial membrane potential; mitochondrial labile (chelatable) iron; mitochondrial ROS susceptible to iron chelators. Techniques: fluorescence plate reader and microscope imaging, UV-VIS spectroscopy, affinity chromatography and native protein gel chromatography

DAY III. Thursday May 4 2019. FLUORESCENCE MONITORING OF LABILE IRON TRAFFICKING IN CELLS AND IRON SPECIATION IN ANIMAL FLUIDS AND ORGANS. Coordinators: Pourzand, Hider and Cabantchik We will explore how can fluorescence probes be designed and used for sensing iron dynamically in solution (fluorescence plate reader) or in cell compartments. Theoretical aspects of labile iron sensing with the aid of fluorescence probes will be provided and application in solution and in cells demonstrated by: I. Monitoring on line iron ingress (influx) into cytosol and mitochondria compartments with organelle targeted probes using both transferrin-iron and non-transferrin iron as iron substrates and. K562 as model cells. II. Assessing the level of labile iron in biological fluids of clinical relevance: simulants of labile plasma iron as direct target of chelation. Performance of assays and data evaluation. III. The application of a novel mitochondrial targeted iron sensor as a biochemical and pharmacological tool in iron research- applied to cell model. Techniques: fluorescence plate reader, fluorescence microscope imaging and FACS.

Techniques for studying iron in health and disease EMBL

Program DAY 1 (May 2nd)

GENERATION AND INTERPRETATION OF ASSAY TEST RESULTS FOR BIOMARKERS OF IRON HOMEOSTASIS AND DYSHOMEOSTASIS.

Lecturer Topics Allocated time (min)

All Organizers Welcome 9:00-9:15 (15´) Yvonne Yeboah& House notes/ ice breaking activity 9:15- 9:45 (30´) Elisabeth Zielonka Course introduction 9:45- 9:55 (10´) Ioav Cabantchik

9:55-10:15 (20´) COFFEE BRAKE

Generation and interpretation of assay

test results for biomarkers of iron homeostasis and dyshomeostasis 10:15-10:20 (05´) 1) Goal and daily schedule 2) Clinical interpretation of test results 10:15-10:20 (05´) • Presentation 10:35-11:05 (30´) Dorin Swinkels • Case study (in groups) 11:05-11:25 (20´) • Discussion (plenair)

3) Test validation 11:25-11:55 (30´) • Presentation (30´) • Design practical plan (in groups) 11:55-12:25 • Provide groups with final 12:25-12:30 (35´)

practical plan

Lunch 12:30-13:15 (45´)

Rachel van Swelm Practical in Groups 13:30-18:00 (4,5´) Rian Reolofs

Rachel van Swelm Calculation of results and prepare 18:00-19:00 (60´) Rian Reolofs presentation

Dinner 19:00- 20:00 (60´)

Dorin Swinkels Presentation and Discussion 20:00- 21:00 (60´)

Techniques for studying iron in health and disease EMBL

Day 1 Generation and interpretation of assay test results for biomarkers of iron homeostasis and dyshomeostasis. Day coordinator: Dorine W. Swinkels

Audience This course is aimed at PhD students and postdocs in all basic and clinical biomedical specialities including biochemistry, physiology, pharmacology, immunology and molecular biology. There will be a maximum of 18 participants, who will be divided into 6 groups for the practical part. The morning will be devoted to lectures and experimental planning. The experiments will be carried out in the afternoon and analyzed and discussed in the evening.

Aim By the end of this day students will be able to adequately generate and interpret results of an assay for biomarkers of iron homeostasis and dyshomeostasis within both a research and clinical setting. More specifically the students will be able to: c. Clinically interpret test results of a broad range of conventional and more novel iron biomarkers d. Apply fundamentals of implementing an assay and generating a reliable test result in both the research and clinical setting. During the workshop we will: - Discuss interpretation of test results generated by a fully validated assay for the diagnosis of iron disorders. Students will be challenged to solve case –puzzles in small groups, and present their “diagnosis” for the whole group for discussion. - Discuss design of a validation plan for an assay of an analyte. In the afternoon, students will apply this knowledge by performing a validation of a commercial hepcidin ELISA kit in the lab in small groups. By the end of the day each group will present their analytical findings for the whole group for discussion.

Teachers Dorine Swinkels, Radboudumc Center for Iron Disorders, The Netherlands. [email protected] Rachel van Swelm, Radoudumc Center for Iron Disorders, The Netherlands. [email protected] Rian Roelofs, Radboudumc Center for Iron Disorders, The Netherlands. [email protected]

Techniques for studying iron in health and disease EMBL

Recommended Literature: 1. Andreasson et al (2015) A Practical Guide to Immunoassay method validation. Frontiers in Neurology 6:179-186. 2. Armbruster DA, Pry T (2008) Limit of Blank, Limit of Detection and Limit of Quantification. Clin Biochem Rev. 29:S49-52. 3. Biswas S, Saha MK (2015) Uncertainty of Measurement for ELISA in a Serological Testing Laboratory. Immunochem Immunopathol. 1:109-114. 4. Shrivastava A, Gupta VB (2011) Methods for the determination of limit of detection and limit of quantification of the analytical methods. Chron Young Sci 2:21-25. 5. Pfeiffer CM, Looker AC. Laboratory methodologies for indicators of iron status: strengths, limitations, and analytical challenges. Am J Clin Nutr. 2017;106(Suppl 6):1606S-1614S. 6. Cappellini MD, Stanley FL, Swinkels DW. Chapter 38. Hemoglobin, iron and bilirubin. Tietz Textbook of Clinical Chemustry and molecular diagnostics. Nader Rifai, Andrea Rita Horvath and Carl T. Wittwer. 6th edition 2018. Elsevier, St. Louis, Missouri, USA.

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Theoretical part

The morning program will be organized as follows:

1. Clinical interpretation of test results A. Short presentation on clinical interpretation of data generated by validated assay (15 min) B. Case study (30 min) Each student group will make the mini-exam on interpretation of assay results (Table 2). For each case students will assess whether the patient: a. is iron deficient, or ii) has an iron distribution disorder or iii) is iron overloaded b. has one of the following disorders: IRIDA, hereditary hemochromatosis, metabolic syndrome, blood loss, beta-thalassemia intermedia or inflammation C. Discussion of results in a plenary session (20 min) 2. Test validation A. Short presentation on how to validate an assay (30 min) B. In groups design a practical plan for the validation (30 min) C. Prepare for the practical work in the afternoon (5 min)

To optimally prepare, we strongly recommend participants prepare the following literature and Table 1 below.

- Pfeiffer CM, Looker AC. Laboratory methodologies for indicators of iron status: strengths, limitations, and analytical challenges. Am J Clin Nutr. 2017;106(Suppl 6):1606S-1614S. - Cappellini MD, Stanley FL, Swinkels DW. Chapter 38. Hemoglobin, iron and bilirubin. Tietz Textbook of Clinical Chemistry and molecular diagnostics. Nader Rifai, Andrea Rita Horvath and Carl T. Wittwer. 6th edition 2018. Elsevier, St. Louis, Missouri, USA. OR - Ellervik C, Cappellini MD, Stanley FL, Swinkels DW. Chapter 28. Hemoglobin, iron and bilirubin. Tietz Fundamentals of Clinical Chemistry and molecular diagnostics. Nader Rifai, Andrea Rita Horvath and Carl T. Wittwer. 8th edition 2019. Elsevier, St. Louis, Missouri, USA.

Techniques for studying iron in health and disease EMBL

Table 1. Laboratory tests for iron status in adults and their (relative) values in various disorders of iron metabolism. Iron Iron Functional Iron IRIDA Anemia of Iron deficiency Iron overload Reference status deficiency Iron deficiency chronic anemia and in hereditary Interval deficiency anemia disease anemia of chronic hemochromatosis Test disease Proposed

hepcidin Low low very low High for high Normal-high Low for ferritin7 Varies* TSAT6 sTfR (mg/L) high high high High Low-normal Variable Low-normal Varies* sTfR /log ferritin NA NA >22* NA < 12* >22* NA Varies* ZnPP mmol/mol High high high high high high low-normal < 40-80* heme Reticulocyte < 283* < 294* low low Low-normal low High-normal 30.2-35.95* hemoglobin content (pg) bone marrow iron 8 negative Variable Negative positive positive Positive Positive Positive

From Cappellini MD, Stanley FL, Swinkels DW. Chapter 38. Hemoglobin, iron and bilirubin. Tietz Textbook of Clinical Chemustry and molecular diagnostics. Nader Rifai, Andrea Rita Horvath and Carl T. Wittwer. 6th edition 2018. Elsevier, St. Louis, Missouri, USA. *, varies with the methodology used; 1, based on (1); 2, value from (3, 4); 3, For CHr based on (6); 4, for CHr, from (2); 5, for CHr from (5); 6, absolute levels vary, but in the absence of inflammation levels are high for circulating iron levels; 7, absolute levels vary, but levels are low for serum ferritin levels; 8, by Perls staining of bone marrow for iron in reticulocytes according to (7); 9, mostly low normal in untreated patients; 10, in more severe cases < 5 %; 11, from (1); 12, in populations exposed to infections and in patients with renal failure, inflammatory bowel disease, chronic heart failure or other (low grade) inflammatory diseases, threshold values indicating iron deficiency are generally considered to be higher than in those without these diseases (see below). In these situations levels > 100 µg/L generally exclude absolute iron deficiency and for levels between 30 and 100 µg/L other parameters are needed to diagnose ID. 13, some guidelines propose a threshold of 50% for men; NA, not available; IRIDA, iron refractory iron deficiency anemia; TIBC, Total Iron Binding Capacity. Literature of Table 1:

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1. Camaschella C. Iron-deficiency anemia. N Engl J Med 2015;372:1832-1843. 2. Thomas DW, Hinchliffe RF, Briggs C, Macdougall IC, Littlewood T, Cavill I, British Committee for Standards in H. Guideline for the laboratory diagnosis of functional iron deficiency. Br J Haematol 2013;161:639-648. 3. Weiss G, Goodnough LT. Anemia of chronic disease. N Engl J Med 2005;352:1011-1023. 4. Punnonen K, Irjala K, Rajamaki A. Serum transferrin receptor and its ratio to serum ferritin in the diagnosis of iron deficiency. Blood 1997;89:1052- 1057. 5. Piva E, Brugnara C, Spolaore F, Plebani M. Clinical utility of reticulocyte parameters. Clin Lab Med 2015;35:133-163. 6. Mast AE, Blinder MA, Lu Q, Flax S, Dietzen DJ. Clinical utility of the reticulocyte hemoglobin content in the diagnosis of iron deficiency. Blood 2002;99:1489-1491. 7. Gale E, Torrance J, Bothwell T. The quantitative estimation of total iron stores in human bone marrow. J Clin Invest 1963;42:1076-1082.

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Table 2: Mini-exam Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Reference values F, 30yr M, 60yr F, 65yr M, 45yr F, 4yr M, 18yr (red = not standardized)

Hb 9.1 10.5 14.2 13.5 7.9 8.5 Men >13 g/dl, Women>12g/dl

CRP < 5 45 <5 8 < 5 < 5 < 5 mg/L Ferr. 15 103 1700 950 25 2700 Men: 30-300 µg/L; Women: 20-200 µg/L TSAT 10 10 94 21 3 101 15-45 % Hepc. <0.5 13.2 4.2 9.4 3.1 4.3 < 0.5 -14.7 nmol/L sTfR 2.60 0.90 0.90 0.90 2.80 6.90 0.76-1.76 mg/L

ZnPP 89 81 39 75 103 95 < 40-80 mmol/mol heme MCH 1.65 2.00 2.15 2.00 1.20 np 1.67-2.11 fmol RetHb 1.55 2.12 2.25 2.12 1.29 np 1.87-2.23 fmol

Techniques for studying iron in health and disease EMBL

Practical part: analytical and biological test validation

The 18 participants will be divided into 6 groups who will each have the availability of one Hepcidin 25 ELISA kit to practice one particular aspect of analytical assay validation as discussed in the morning lecture. Due to restrictions, we will only be able to cover a selection of all validation and quality aspects. In addition, patient samples from different patient groups, with different amounts of hepcidin will be distributed for biological validation.

The selection of validation aspects that will be performed is described on the following pages. Each group discusses what experiment(s) could be done taking in account the available materials. All samples have to be measured in duplicate.

Subsequently, for practical reasons you will be handed a detailed pre-determinated plan to perform the desired experiments.

Population catagories for n= Label ID biological validation Iron deficiency anemia 4 1, 2, 3, 4 healthy 4 21, 22, 23, 24 Kidney failure 4 84, 87, 90, 91 Diurnal rhythm 1 4 2_8:00 / 2_11:00 / 2_13:00 / 2_15:30

Diurnal rhythm 2 4 39_8:00 / 39_11:00 / 39_13:00 / 39_15:30

Basic 1-2 3-4 5-6 7-8 9-10 11-12 plate layout A standard 0 3 90 39_13:00

B standard 1 4 91 39_ 15:30

C standard 2 21 2_ 8:00

D standard 3 22 2_ 11:00

E standard 4 23 2_ 13:00

F standard 5 24 2_ 15:30

G 1 84 39_ 8:00 Control Low (kit) H 2 87 39_11:00 Control High (kit)

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After finishing the practical work the calculations can be done using Excel and/or Graphpad. With your group you process the results of the analytical and biological validation in a short report which will be presented to all participants (Powerpoint).

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Detailed workflow DRG Hepcidin 25 (bioactive) HS ELISA

ADVISED Instructors: bring all reagents to room temperature and let the samples thaw TIME SCHEDULE If desired, read (parts of) the 'intructions for use', recommended are '2. PRINCIPLE OF THE TEST' and '3. WARNINGS AND PRECAUTIONS' on page 2 and 3. 13:30- REAGENT AND SAMPLE PREPARATION 14:30 Standards (6 vials) and controls 'low' and 'high':

Reconstitute the lyophilized content of each vial with 0.5 ml demineralized water and let stand for 10 minutes in minimum. Mix several times for use by pipetting up and down Preparation of samples

Always mix thawed samples thoroughly on a vortex and spin down Depending on your specific research question, see further instructions Hepcidin calibrator set (calibrator low and calibrator middle; only for sub validation 'trueness'):

Reconstitute the lyophilized content with 0.30 ml water and let stand for 15 minutes carefully mix for 20 minutes on a tube roller bank (see leaflet in appendix 1) Wash solution

Dilute 30 ml of concentrated Wash Solution with 1170 ml demineralized water to a final volume of 1200 ml 14:30 TEST PROCEDURE

Dispense 20 ul of each standard, control and/or sample into appropriate wells Dispense 50 ul Enzyme conjugate into each well, using multichannel pipet with matching reagent reservoir

Thoroughly mix for 10 seconds on a microplate shaker

15:00- INCUBATE FOR 60 MINUTES AT ROOM TEMPERATURE 16:00 Wash the plate, washing procedure:

Briskly shake out the contents of the wells (in a sink)

Add 300 ul of diluted washing solution to the wells

Repeat the two former steps 3 times

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Strike the wells thoroughly on absorbent paper to remove residual droplets

Dispense 100 ul of Enzyme Complex into the cells (using multichannel pipette)

16:20- INCUBATE FOR 30 MINUTES AT ROOM TEMPERATURE 16:50

Wash the plate according to washing procedure

Add 100 ul of Substrate solution to each well (multichannel pipette)

17:10- INCUBATE FOR 20 MINUTES AT ROOM TEMPERATURE 17:30

Stop the enzymatic reaction by adding 100 ul of stop solution (multichannel pipette)

Determine the absorbance (OD) of each well at 450 +/- 10 nm with a microtiter plate reader

It is recommended that the wells be read within 10 minutes after adding Stop solution

17:45 practical part is done till 18:45 CALCULATION OF THE RESULTS

Transport the OD data to excel and make calibrationcurve (in Graphpad) to interpolate the unknowns

Depending on your research question, make your own calculations

Make a presentation of your part of the validation and the patient outcomes 19:00- DINNER 20:00 20:00- PRESENTATION AND DISCUSSION 21:00

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MATERIALS NEEDED BUT NOT PROVIDED IN KIT labcoats, gloves, waste containers spin down centrifuges demineralized water pipette set (range 20-500 ul) multichannel pipettes (50- 100 - 300 ul) disposable reagent reservoir fitting multichannel pipettes timer vortex tube roller bank glassware for dosing 1170 ml and adding concentrated wash solution parafilm for mixing wash solution or magnet stirrer absorbent paper (for instance stacked up Kleenex) Plastic adhesive sealers for 96 wells plate (18 pieces in total) microplate shaker(s) microtiter plate reader 450 +/- 10 nm

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1. Validation _ Precision

Definition: 'The closeness of agreement between independent test results obtained under stipulated conditions'. There are three different types of precision namely repeatability ('r'), intermediate precision ('Rw') and reproducibility ('R). Repeatability (also called 'within run' or 'within day precision') is the variability observed when as many factors as possible (e.g. laboratory, technician, days, instrument, reagent lot) are held constant and the time between the measurements is kept to a minimum. In case of reproducibility all factors are varied and measurements are carried out over several days (for this reason not included in this workshop). For intermediate precision (also called 'between run' or 'between day precision') all factors except laboratory are allowed to vary.

Task: Asses repeatability an intermediate precision.

Available samples: HiQC2016 and LoQC2016. These are pooled and aliquoted samples containing high resp. low concentrations of hepcidin and are used for intern quality system. Note: for determinating 'intermediate precision', make use of data obtained from an extra sample sets HiQC2016 and LoQC2016 from other groups.

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2. Validation _ Trueness

Definition: 'The closeness of agreement between the average value obtained from a large series of test results and an accepted reference value'. Ideally, the reference value is derived directly from a CRM (Certified reference material) or from materials that can be traced to the CRM. A CRM enables to minimize the bias by calibrating your method to the CRM.

The quantity in which the trueness is measured is called bias (b), which is the systematic difference between the test result and the accepted reference value.

Task: Asses the bias.

Available materials: CRM (Hepcidin Calibrator Set (Calibrator Low, Calibrator Middle; see appendix for leaflet)) The bias of the CRM (Bcrm) will be calculated using the following formula: bcrm = Xmean - Xref

Xmean is the measured mean value Xref is the given reference value (see leaflet).

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3. Validation _ Limits of quantification

Definition: 'Highest and lowest concentrations of analyte that have been demonstrated to be measurable with acceptable levels of precision and accuracy'. The working range for a method is defined by the lower and upper limits of quantification (LLOQ and ULOQ). The LLOQ can be either determined based on the signal from the instrument (the analytical detection limits LOB (Limit Of Blanc) and LOD (Limit Of Detection) or the calculated concentrations from samples (LOQ (Limit Of Quantification)).

The LOQ can be defined as the endpoints of an interval in which the duplo CV% is under a specific level. This method of detection determination had a few difficulties. First, many (very low limit) samples have to be measured (plot duplo CV% against concentration) in order to achieve the right limit. Second, it is questionable with CV% value must be taken to determine the lower limit. For this reason it is in general accepted to calculate the LOQ from de LOD.

Task: asses the relevant Limits of quantification: LOB, LOQ, LOD.

Available materials: zero standard (0-point of calibration curve), zero serum* * zero serum is derived from a patient suffering from juvenile hemochromatosis. Normally, patients with juvenile hemochromatosis are characterized by low or limited amount of hepcidin. However, when such patients are in status of iron depletion, hepcidin levels should reach almost zero in order to compensate this.

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4. Validation _ Dilution linearity

Definition: 'Dilution linearity is performed to demonstrate that a sample with a spiked concentration above the ULOQ can be diluted to a concentration within the working range an still give a reliable result'. In other words, it determines to which extent the dose-response of the analyte is linear in a particular diluent within the range of the standard curve. Often, as is also the case here, a commercial kit lacks a (very very) high calibrator which is suitable for spiking experiments.

Nevertheless we can determine the linearity within the working range: 2 serum samples (1 with a high hepcidin concentration and 1 with a low hepcidin concentration) are mixed in different proportions. Based on the measured values of the undiluted samples, the values of the mixtures are calculated. The test should be linear for the evaluated range and the measured values should be equal to the calculated values.

Task: asses the linearity within the working range.

Available samples: sample ‘Linearity High’ (hepcdin in upper range of standard curve) and Sample ‘Linearity Low’ (hepcidin in lower range of standard curve)

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5. Validation _ Parallelism

Definition: 'relative accuracy from recovery test on the biological matrix of diluted matrix against the calibrators in a substitute matrix'. Conceptually parallelism and dilution linearity are similar. The major difference is that in het dilution linearity experiments the samples are spiked with the analyte in such a high concentration that after dilution the effect of sample matrix is likely to be neglibible.

For parallelism, on the other hand, no spiking is allowed but only samples with high endogenous concentrations of the analyte must be used. However, the concentrations must be lower than the ULOQ. The goal of investigating the parallelism is to ascertain that the binding characteristic of the endogenous analyte to the antibodies is the same as for the calibrator and that there is no influence of the sample matrix.

Task: asses parallelism of the assay.

Available samples: Two patient samples with high endogenous hepcidin: ‘parallelism 1’, ‘parallelism 2’

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6. Validation _ Recovery / Selectivity

Definition recovery: 'The recovery of an analyte in an assay is the detector response obtained from an amount of the analyte added to and extracted from the biological matrix, compared to the detector response obtained for the true concentration of the analyte in the solvent'. A spike recovery test is conducted to investigate if the concentration-respons relationship is similar in the calibration curve and the samples. A bad outcome of the test suggest that there are differences between the sample matrix and calibratior diluent that affects the response in signal. As for spike recovery tests we need the calibrator in a very high concentration and this in not included in the kit, it is not possible to do this.

Definition selectivity: 'The ability of the bioanalytical method to measure and differentiate the analyte in the presence of components that may be expected to be present'. On the different validation parameters the selectivity is in principle the only one for which a certain amount of knowledge about the analyte and related substances is demanded. For example, do metabolites of the analyte or post translational modifications of a protein analyte interfere with the assay? Does hemolysis or the presence of lipids in the sample disturb the assay?

In case of ELISA hepcidin it is valuable to know for which part the isoforms Hepcidin -20, -22, -24 and -25 are responsable for the test results.

Task: Asses the recovery of the synthetic hepcidin isoforms -20, -22, -24 and -25.

Available materials: Synthetic hepcidin -20, -22, -24, -25 stocks of 5600 ng/ml in 20% acetonictrile. Acetonitrile helps to prevent sticking of hepcidin to the epp (plastics in general).

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Graphad Prism (version 5.03)_to fit a dose-response curve and interpolate concentrations for the unknown samples.

Background Equation: log(inhibitor) vs. response -- Variable slope Many log(inhibitor) vs. response curves follow the familiar symmetrical sigmoidal shape. The goal is to determine the EC50 of the inhibitor - the concentration that provokes a response half way between the maximal (Top) response and the maximally inhibited (Bottom) response. Many inhibitory dose-response curves have a standard slope of -1.0. This model does not assume a standard slope but rather fits the Hill Slope from the data, and so is called a Variable slope model. It is also called a four-parameter dose-response curve.

Model: Y=Bottom + (Top-Bottom)/(1+10^((LogIC50-X)*HillSlope))

EC50 is the concentration of agonist that gives a response half way between Bottom and Top. This is not the same as the response at Y=50. Depending on which units Y is expressed in, and the values of Bottom and Top, the EC50 may give a response nowhere near "50". Prism reports both the EC50 and its log. HillSlope describes the steepness of the family of curves. A HillSlope of -1.0 is standard, and you should consider constraining the Hill Slope to a constant value of -1.0. A Hill slope more negative than -1 (say -2) is steeper. Top and Bottom are plateaus in the units of the Y axis

Short procedure After opening Graphpad, Choose ‘XY datatable’ (=default) Sample data: Use sample data / Interpolate unknowns from standard curve (RIA) Choose a graph: Points only Subcolumns for replicates or error values: change nothing. Enter ‘CREATE’

In first column fill in the names of the standards and sample ID’s, in the ‘X’ column the corresponding concentrations for the standards and in column A de duplo’s of the measured OD’s.

All the dose-response equations built-in to Prism expect the X values to be the logarithm of concentrations; use Prism to transform the X values (See below).

To interpolate from the standard curve: Go to the transformed results page. 1.Click ‘Analyse’, choose ‘Nonlinear regression’ from the list of XY analyses, choose ‘Dose- response – inhibition’ – panel of equations and choose the equation log(inhibitor) vs response – Variable slope (four parameters). 2. Check the option on the bottom of the first tab of the nonlinear regression dialog: ‘Interpolate unknowns from standard curve’. To find the concentrations corresponding to the unknown values, go to the results subpage ‘Interpolated X mean values’.

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You can transform the interpolated X values from the logarithms to their concentrations again with X=10^X Transforming of values: Select the desired colomn (X) 1. From the ‘Analysis’data table, click ‘Analyse’ 2. On the ‘Transform’ dialog, check the option to transform X, and choose X=log(X). At the bottom of the dialog, check the option ‘Create a new graph of the results’. Note1: because log 0 is not possible, choose another small number instead of zero. Note2: the column title will not automatically change with this transformation.

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Appendix 1

Product certificate secondary reference material Hepcidin

Product name Hepcidin Calibrator Set

Batch number 2016-146X

Calibrator low: 2016.1461 Calibrator middle: 2016.1462

Volume in vial 0.30 mL

Date of issue 2016-06

Expiry date 2021-06 when stored at 2-

8 °C Assigned values

Assigned value (SD) Mean value (SD) Level by of 9 validated methods hepcidinanalysis.com worldwide [nmol/L] / 1 (low) 0.851 (0.049) 2.38 (1.22)

2 (middle) 3.758 (0.090) 7.03 (3.15)

Homogeneity Homogeneity has been determined according to ISO 13528 and meets its criteria.

Hepcidin Calibrator Set Intended purpose The secondary reference material enables laboratories to calibrate their hepcidin assays.

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Product Description The calibrators are prepared from pooled lyophilized native human serum with the addition of stabilizer CLP. There are 2 levels; low and middle. Volume The content of one vial is 0.30 mL lyophilized serum of human origin.

Manufactured by MCA laboratory for Hepcidinanalysis.com, Geert Grooteplein 10 (830), 6525 GA Nijmegen, The Netherlands (www.hepcidinanalysis.com)

Value assignment In absence of a primary reference method, a consensus approach is used for the value assignment of the reference material. The values of these calibrator samples have been measured in triplicate by a selection of laboratories participating in the hepcidin harmonization study 2016.

Storage and stability Specimens should be stored at 2-8 °C. The stability of the samples once reconstituted is equal to patient material. No responsibility is taken for samples that have been frozen after reconstitution.

Instructions for use: Prior to use the samples need to be reconstituted. a. Remove the cap and stopper. b. Add 0.30 mL aqua dest. c. Replace the stopper. d. Leave the vial upright at room temperature for 15 minutes. e. Carefully mix for 20 minutes. f. Process product as patient material.

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Precautions and warnings 1. For in vitro diagnostic use only. 2. Individual donors have been tested and found negative for HIV, hepatitis B and hepatitis C. 3. Specimens should be handled with care, as appropriate for biological materials. Outdated and left-over material should be discarded as potentially infectious material, according to the procedures in your institute. 4. The specimens have been prepared using the protocol of the MCA Laboratory, which has the ISO 13485:2003 certificate for the development, manufacturing, distribution and sales of calibrators, controls, specimens and reference materials used for In Vitro Diagnostic Devices and External Quality Assessment schemes in medical laboratories.

Remarks The stability of the reference material has been assessed according to accelerated stability testing. For questions, comments or suggestions, please do not hesitate to contact the team of Hepcidinanalysis.com: ● E-mail: [email protected] ● Phone: +31 (0) 24 361 4567

References • van der Vorm L, Hendriks J, Laarakkers C, Klaver S, Armitage A, Bamberg A et al. Toward Worldwide Hepcidin Assay Harmonization: Identification of a Commutable Secondary Reference Material. Clinical Chemistry. 2016 Jul;62(7):993-1001. • Laarakkers C et al. Improved Mass Spectrometry Assay For Plasma Hepcidin: Detection and Characterization of a Novel Hepcidin Isoform. PLoS ONE. 2013;8(10): p. e75518. • Diepeveen LE, Weykamp C, Swinkels D et al. Hepcidin Harmonisation Study 2016: Development of a commutable secondary reference material. Report in preparation.

Techniques for studying iron in health and disease EMBL

Program DAY 2 (May 3rd)

DYNAMICS OF IRON-SULFUR PROTEINS - FROM SOLUTION TO CELLS

Lecturer Topics Allocated time (min)

Instructors Overview of the day 9:00 – 9:15

INTRODUCTION TO PRACTICALS 9:15 – 10:45

Native-PAGE an analytical tool to 15’ Mz. Ola Karmi follow 2Fe-2S cluster transfer

Mr. Henri-Baptiste Absorption spectroscopy to follow 15’

Part I Marjault cluster transfer

The role of the NEET proteins in Iron 15’ Dr. Yang-Sung Sohn homeostasis

(Bio)chemical examination of ISC 15’

Vinzent Schulz targeting factors

Part II Analysis of Fe/S enzyme activities in 30’ Oliver Stehling tissue culture material

Break 10:45 – 11:00

PRACTICAL Part I 11:00 – 12:30

Lunch break 12:30 – 13:30

PRACTICAL Part II 13:30 – 14:30

Techniques for studying iron in health and disease EMBL

Lecturer Topics Allocated time (min)

Eukaryotic iron-sulfur protein biogenesis • General overview • Mitochondrial Fe/S protein Lecture I assembly (ISC system) • Cytosolic and nuclear Fe/S protein 14:30 – 15:30 Roland Lill assembly(CIA system) • The connection of Fe/S cluster assembly and cellular iron homeostasis • Medical aspects of Fe/S protein assembly

Break 15:30 – 15:45

PRACTICAL Part III 15:45 – 17:15

Labile [2Fe-2S] clusters • 4CYS Structure of [2Fe-2S] coordination • The 3CYS:1HIS coordination in the NEET proteins Lecture II • The labile NEET’s cluster effects on 17:15 – 18:00 the NEET proteins structures Rachel Nechushtai • How we follow cluster transfer • Effects on mitochondrial-cytosol crosstalk • How can we disrupt the NEET proteins’ cluster transfer

Dinner 18:00 – 19:00

Closing session • Lecturers and instructors Final analyses 19:00 – 20:00 • Data presentation and discussion • Questions • Exchange of ideas

Techniques for studying iron in health and disease EMBL

REFERENCES (DAY 2)

1. Tamir S, et al. (2015) Structure-function analysis of NEET proteins uncovers their role as key regulators of iron and ROS homeostasis in health and disease. Biochim Biophys Acta 1853(6):1294-1315. 2. Bandyopadhyay S, Chandramouli K, & Johnson MK (2008) Iron-sulfur cluster biosynthesis. Biochem Soc Trans 36(Pt 6):1112-1119. 3. Lill R & Muhlenhoff U (2006) Iron-sulfur protein biogenesis in eukaryotes: components and mechanisms. Annu Rev Cell Dev Biol 22:457-486. 4. Ayala-Castro C, Saini A, & Outten FW (2008) Fe-S cluster assembly pathways in bacteria. Microbiol Mol Biol Rev 72(1):110-125, table of contents. 5. Stehling O, Wilbrecht C, & Lill R (2014) Mitochondrial iron-sulfur protein biogenesis and human disease. Biochimie 100:61-77. 6. Ye H & Rouault TA (2010) Human iron-sulfur cluster assembly, cellular iron homeostasis, and disease. Biochemistry 49(24):4945-4956. 7. Rouault TA & Tong WH (2008) Iron-sulfur cluster biogenesis and human disease. Trends Genet 24(8):398-407. 8. Mochel F, et al. (2008) Splice mutation in the iron-sulfur cluster scaffold protein ISCU causes myopathy with exercise intolerance. Am J Hum Genet 82(3):652-660. 9. Amr S, et al. (2007) A homozygous mutation in a novel zinc-finger protein, ERIS, is responsible for Wolfram syndrome 2. Am J Hum Genet 81(4):673-683. 10. Wiley SE, et al. (2007) The outer mitochondrial membrane protein mitoNEET contains a novel redox-active 2Fe-2S cluster. J Biol Chem 282(33):23745-23749. 11. Wiley SE, Murphy AN, Ross SA, van der Geer P, & Dixon JE (2007) MitoNEET is an iron- containing outer mitochondrial membrane protein that regulates oxidative capacity. Proc Natl Acad Sci U S A 104(13):5318-5323. 12. Wiley SE, et al. (2013) Cellular and bioenergetic dysfunction induced by loss of the Wolfram Syndrome protein, Miner1, can be reversed by treatment with N-acetylcysteine. Mitochondrion 13(6):940-940. 13. Lipper CH, et al. (2018) Structure of the human monomeric NEET protein MiNT and its role in regulating iron and reactive oxygen species in cancer cells. Proc Natl Acad Sci U S A 115(2):272-277. 14. Tirrell TF, et al. (2009) Resonance Raman studies of the (His)(Cys)3 2Fe-2S cluster of MitoNEET: comparison to the (Cys)4 mutant and implications of the effects of pH on the labile metal center. Biochemistry 48(22):4747-4752. 15. Wiley SE, et al. (2007) The outer mitochondrial membrane protein mitoNEET contains a novel redox-active 2Fe-2S cluster. J. Biol. Chem. 282(33):23745-23749. 16. Tamir S, et al. (2015) Structure-function analysis of NEET proteins uncovers their role as key regulators of iron and ROS homeostasis in health and disease. Biochim. Biophys. Acta 1853(6):1294-1315. 17. Zuris JA, et al. (2011) Facile transfer of [2Fe-2S] clusters from the diabetes drug target mitoNEET to an apo-acceptor protein. Proc Natl Acad Sci U S A 108(32):13047-13052. 18. Conlan AR, et al. (2009) Crystal structure of Miner1: The redox-active 2Fe-2S protein causative in Wolfram Syndrome 2. J Mol Biol 392(1):143-153. 19. Conlan AR, et al. (2009) The novel 2Fe-2S outer mitochondrial protein mitoNEET displays conformational flexibility in its N-terminal cytoplasmic tethering domain. Acta Crystallogr Sect F Struct Biol Cryst Commun 65(Pt 7):654-659. 20. Tamir S, et al. (2014) A point mutation in the [2Fe-2S] cluster binding region of the NAF-1 protein (H114C) dramatically hinders the cluster donor properties. Acta Crystallogr D Biol Crystallogr 70(Pt 6):1572-1578.

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21. Karmi O, et al. (2018) The unique fold and lability of the [2Fe-2S] clusters of NEET proteins mediate their key functions in health and disease. JBIC Journal of Biological Inorganic Chemistry. 22. Sohn YS, et al. (2013) NAF-1 and mitoNEET are central to human breast cancer proliferation by maintaining mitochondrial homeostasis and promoting tumor growth. Proc Natl Acad Sci U S A 110(36):14676-14681. 23. Holt SH, et al. (2016) Activation of apoptosis in NAF-1-deficient human epithelial breast cancer cells. J Cell Sci 129(1):155-165. 24. Darash-Yahana M, et al. (2016) Breast cancer tumorigenicity is dependent on high expression levels of NAF-1 and the lability of its Fe-S clusters. Proc Natl Acad Sci U S A.

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Part I: Analysis of iron sulfur cluster (ISC) transfer from NAF-1, a NEET protein, to apo- ferredoxin using native gels electrophoresis & UV-VIS spectrophotometry as analytical methods. Demonstration of ISC transfer ability in the WT protein vs. the HC mutated variants (H114C of NAF-1).

Introduction:

Iron-sulfur (Fe-S) proteins are important proteins, present in wide range of biological processes some of which are highly conserved through evolution. The most well-known function of such proteins is to serve as electron transfer proteins, however; there are evidence that Fe-S proteins have additional functions such as sensing of iron or oxygen, substrate binding/catalysis and gene expression regulation (1). In accordance with the hypothesis that [Fe-S] clusters are one of the most ancient types of prosthetic group, the biosynthetic machineries for [Fe-S] cluster biogenesis are widely conserved in all three kingdoms of life, which are localized to both the cytosol, mitochondria (2-4) and chloroplasts (1). The eukaryotic simpler clusters are [2Fe-2S], which are converted to [4Fe-4S]. In the recent years increased numbers of [Fe-S] proteins were identified through different scientific aspects including genetic mutations associated with dysfunction of [Fe-S] cluster biogenesis pathway (5-8), or genes encoding [Fe-S] proteins (9). The three most common structures of the coordination of the [2Fe-2S] clusters to proteins are presented in (Figure 1) below.

Figure 1. Comparison of the [2Fe–2S] cluster coordination binding domain of ferredoxin (PDB ID: 1RFK), Reiske (PDB ID: 2NUM) and human NEET proteins (mNT PDB ID: 2QH7 and NAF-1 PDB ID: 3FNV). (A–B) Structural comparison of the [2Fe–2S] coordination. (C) The absorption peaks of the [2Fe–2S] clusters in the different proteins. UV–vis spectra were distinct (ferredoxin, black; mitoNEET, blue; Rieske, green).

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As shown, the coordination domain of the [2Fe-2S] cluster in Ferredoxin (Fdx) contains 4-cysteine amino acids (Figure 1,A,B-a&d), or Rieske 2-cysteine and 2-histidine group (Figure 1,A,B– b&e). The unique coordination, kind of in between these two proteins, are the recently discovered NEET proteins where their clusters’ coordinating structure is composed of 3-cystine and 1-histidine (3Cys:1His) which is on the surface of the NEET proteins (Figure 1,A,B – c,f) (1). The NEET proteins members are conserved in all forms of living organisms; including archaea, bacteria (Bacterial MiNT), through plants (contains only one copy, e.g. in Arabidopsis At-NEET) through humans (where three members of NEET proteins exist) (Figure 2, A.). All of the NEET proteins share a 39 amino acid sequence; called CDGSH domain, in this domain there is a 16 amino acid stretch that was shown to contain the four ligands of (3Cy:1His) surrounding the [2Fe-2S] cluster (Figure 2, B.). The NEET proteins’ [2Fe-2S] clusters confer their specific absorbance peak at 458nm (Figure 3, A.) that is different than the peak of Fdx and Rieske proteins (Figure 1, C.) (10, 11).

Figure 2. NEET proteins’ structures determined by X-ray crystallography. A. The superposition of the four proteins shows the high structural similarity shared between the NEET proteins. B. The [2Fe-2S] cluster-binding domain of the NEET proteins. The moleculars details of the superposition of the [2Fe- 2S] 3Cys:1His pocket of each protein is highlighted within the box.

In humans, there are three NEET (C-terminal sequence Asn-Glu-Glu-Thr) protein members, encoded by the cisd1, cisd 2 and cisd 3 genes. These proteins are located in the mitochondrial outer membrane (OMM) - mitoNEET (mNT), The Nutrient-deprivation autophagy factor-1 (NAF-1) is located also in the OMM and the ER - mitochondrial associated membrane (MAM) that connects the ER to the OMM (11, 12). The major parts of mNT and NAF-1 proteins are in the cytosol, while a single transmembrane helix anchors them to the membranes where they are situated. The third NEET protein that is mitochondrial inner NEET protein (MiNT) is localized within the mitochondria (Figure 4) (11, 13).

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Figure 3. UV–vis spectra of the NEET proteins. A. Note the similarities of the peak 458nm positions in the visible region reflecting the presence of the same type of [2Fe–2S] clusters and the similarity of their coordination domains. B. Destabilization of NAF-1 [2Fe–2S] cluster: Similar to mNT, the stability of the NAF-1 [2Fe–2S] cluster is dependent on pH.

The finding that NEET proteins have as a fourth coordinating cluster ligand, the His residue, which conveys a pH-dependent-lability to their clusters, is a unique feature of their [2Fe-2S] clusters. Indeed, the latter does not exist in other [2Fe-2S] proteins, e.g. Fdx [2Fe-2S] (4Cys coordination) which has a much higher level of cluster stability under similar buffer conditions (14-16). Moreover, when the coordinating His was replaced with a Cys, (H87C and H114C in mNT and NAF-1, respectively), the [2Fe- 2S] clusters of the NEET proteins were stabilized (between 10-25X), similar to that of the Ferredoxin cluster (Figure 3, B.).

Figure 4. Localization of the NEET proteins in human cells. mNT and NAF-1 shown to be located onto the OMM, ER and MAM. The major part of these proteins is in the cytosol, while a single transmembrane helix anchors each of their monomers to the membranes. MiNT, the monomeric human NEET protein, is localized within the mitochondria.

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In this workshop, you will be thought how to monitor the cluster transfer between an holo-NEET protein (holo-NAF-1; Donor for 2Fe-2S cluster) and Apo-acceptor protein (Apo-Fdx; the acceptor of the 2Fe-2S cluster) (Figure 5). The wt (native form) of NAF-1 transfers its [2Fe-2S] clusters to apo-Fdx proving the labile nature of its cluster. We will also perform a cluster transfer assay with NAF-1-H114C mutant, where the His coordinator of the NAF-1 [2Fe-2S] cluster is substituted with Cys, forming a 4Cys coordination. The HIS to CYS change conveys a stability to the [2Fe-2S] clusters of NAF-1 and prevents its release (Figure 3, B.). We are using Fdx (Apo-form) as an acceptor for the [2Fe-2S] from the holo- WT- NAF-1, since it is a gold standard apo-acceptor for measuring the transfer kinetics. Moreover, since holo-Fdx absorption peak is at 423nm, it is possible to distinguish it, by UV-Vis spectrometry, from that of NAF-1 whose absorption peak is at 458nm (Figure 1, C.) (17).

Figure 5. A schematic representation of cluster transfer from NAF-1 to apo-Fd.

You will be introduced to these two different methodologies – Native gels and absorbance spectroscopy. For testing the ability of accepting or donating the [2Fe-2S] clusters; by using a UV-Vis absorption spectrum to track the movement of the absorption peaks of the [2Fe-2S] clusters, i.e. from 458nm to 423nm. In addition, by using a Native-PAGE gel, an analytical chromatographic separation method will allow tracking the exchange of the protein color from colorless (Apo- form) to a reddish color (Holo-form).

Brief outline of the experiments to be performed:

• Station No. 1: Native-PAGE for following [2Fe-2S] cluster transfer in vitro: Instructor: Mz. Ola Karmi.

WT of the NAF-1 protein and it’s mutant H114C will be incubated at 37C°, with pre-reduced Apo-Fdx for different time length periods. Following the cluster transfer assay, the solution will be electrophoresed on a Native-PAGE (5% acrylamide) gels along with controls. The NAF-1-H114C mutant will be also be tested in the cluster transfer assay (Figure 6).

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Figure 6. Native-PAGE analytical method to follow cluster transfer from holo-NAF-1 protein (donor) to apo-Fd (acceptor) to obtain holo-Fd.

• Station No. 2: UV-Vis Absorption Spectroscopy to determine the [2Fe-2S] cluster transfer process: Instructor: Henri-Baptiste Marjault.

After reduction, the apo-Fdx will be incubated with WT of NAF-1 protein and it’s mutant H114C, and the UV-Vis absorption spectrum from 380nm to 550nm over time will be monitored in situ. Special attention will be given to the absorption peak at 458nm (Holo-NAF-1 - specific) and 423 nm (holo-Fd -specific). At the end of the experiment, the student will compare the absorption spectra in a similar way described in (Figure 7).

Figure 7. Optical spectroscopy analytical method to follow cluster transfer from NEET protein (donor) to apo-Fd (acceptor) to obtain holo-Fd.

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Objectives:

1- Understanding the definition of lability of a [2Fe-2S] clusters. 2- Learning the main principle of different methodologies (Native-PAGE and UV-Vis) through applying them on the cluster transfer process. 3- Appreciating the cluster coordination amino acid role in the lability and activity of the protein. Protocol:

a- Parts prepared in advance:

Bacterial expression of the human soluble form of NAF-1 and M. laminosus’ Fdx:

The human soluble form of NAF-1 and it’s mutant H114C and the Fdx from M. laminosus, were expressed in E. coli as explained in (18-20). The vectors used for the expressions (Figure 8).

Figure 8. Map of the bacterial expression vectors; A. NAF-1 (pET-28a(+)) expression vector, B. Ferredoxin (pET-20b(+)) expression vector. The black arrow represents the open reading frame.

• Holo-NAF-1/ H114C protein purification:

1- Transformed E. coli cells (50g) were homogenized in 500ml of 20mM Tris-HCl pH 8.0, 500mM NaCl and 10mM MgCl2. 3-5 mgs of DNAse and 3-5 mgs of lysozymes were added together with 2.5ml of Proteases inhibitor solution containing 200mM aminocaproic acid, 200mM benzamidine, and 200mM PMSF. 2- The cells were then broken by Microfluidizer at 21000psi. 3- The broken cells were centrifuged in SLA-3000 rotor at 11,000 rpm for 10 minutes to remove all unbroken cells. 4- The obtained supernatant was re-centrifuged as above. 5- The supernatant was then mixed with 30ml (Ni-NTA- Resin), (NiNTA- Resin must be washed with buffer- 20mM Tris-HCl pH 8.0, 500mM NaCl and 5mM Imidazole). 6- Supernatant and washed Ni-NTA- Resin were shacked together at 4°C for 1 hour. 7- The mixture was loaded into a column (2.5X6cm) and all the liquid allowed to flow through.

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8- The column was washed with 50ml buffer containing 20mM Tris-HCl pH 8.0, 500mM NaCl and 5mM Imidazole. Then, the column was washed with 50ml buffer containing 20mM Tris-HCl pH 8.0, 500mM NaCl and 30mM Imidazole. All the liquid flew through. 9- Then to the column’s resin 50ml of Thrombin buffer containing 20mM Tris-HCl pH 8.0, 100mM NaCl and 2.5mM CaCl2 were added along with 3ml Thrombin (0.5K Unit of thrombin). The column was then well sealed and was shacked overnight at 4°C, followed by 3 hours shaking at room temperature. 10- After all the thrombin buffer flew through, the red eluting fractions are collected. 11- To fully elute the NAF-1 from the column it was further washed with buffer containing 20mM Tris-HCl pH 8.0, 500mM NaCl and 30mM Imidazole and the red eluted fractions were collected until the flow through became colorless. 12- The collected red solution was concentrated with Amicon (cut-off filter 10K). 13- The concentrated protein solution was diluted to a final concentration of 40mM NaCl by a 20mM Tris-HCl pH 8.0 solution. 14- The protein was re-concentrated with Amicon to reach a final volume of 25ml. 15- A size-exclusion column (1cmX1m) pre-washed washed with 20mM Tris-HCl pH 8.0 and 100mM NaCl in a GE AKTA-Pure system is prepared. Two (2) ml of the protein sample is loaded on the column and the column was chromatographed with the same buffer. 16- The red peak fractions with an absorption at 458nm were collected in 4ml tubes. 17- Each fraction is analyzed on SDS-PAGE (15% acrylamide) and the fractions with >98% purity are concentrated as PURE- NAF-1 fractions. 18- The concentration of the pure protein is determined by its absorption in 458nm and its extinction coefficient of 5000.

• Holo-Fdx protein purification:

1- E. coli cells (50g) were homogenized in 500ml of 20mM Tris-HCl pH 8.0, 500mM NaCl and 10mM MgCl2. 3-5 mgs of DNAse and 3-5mgs of lysozymes were added together with 2.5ml of Proteases inhibitor solution containing 200mM aminocaproic acid, 200mM benzamidine, and 200mM PMSF. 2- The cells were then broken by Microfluidizer at 21000psi. 3- The broken cells were centrifuged in SLA-3000 rotor at 11,000rpm for 10 minutes to remove all unbroken cells. 4- The obtained supernatant was re-centrifuged as above. 5- The supernatant was then mixed with 30ml (Ni-NTA- Resin) pre-washed with buffer containing 20mM Tris-HCl pH 8.0, 500mM NaCl and 5mM Imidazole. 6- Supernatant and washed Ni-NTA- Resin were shacked together at 4°C for 1 hour. 7- The mixture was loaded into a column (2.5X10cm) and all the liquid flew through. 8- Then, the column was washed with 50ml buffer containing 20mM Tris-HCl pH 8.0, 50mM NaCl and 15mM Imidazole. 9- Fdx was then eluted with 50ml buffer containing 20mM Tris-HCl pH 8.0, 300mM NaCl and 150mM Imidazole. 10- The collected red protein solution was concentrated with Amicon (cut off filter 10K). 11- The concentrated material is diluted to a final concentration of 40mM NaCl by a 20mM Tris- HCl pH 8.0 solution 12- The fraction is re-concentrated with Amicon to reach a final volume of 25ml. 13- A size-exclusion column (1cmX1m) pre-washed washed with 20mM Tris-HCl pH 8.0 and 100mM NaCl in a GE AKTA-Pure system is prepared. Two (2) ml of the protein sample is loaded on the column and the column was chromatographed with the same buffer. 14- The red peak fractions with an absorption at 423nm were collected in 4ml tubes.

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15- Each fraction was analyzed on SDS-PAGE (15% acrylamide) and the fractions with >98% purity were concentrate as PURE- Fdx fractions. 16- The concentration of the pure Fdx protein is determined by its absorption in 423nm and its extinction coefficient of 9500.

• Apo-Fdx preparation:

1- The concentrated holo-Fdx was boiled with 0.5M of DTT and 5mM of EDTA at 95°C for 2 minutes. 2- The DTT was then removed from the Apo-Fdx by dialysis against a buffer containing; 20mM Tris-HCl pH 8.0 and 100mM NaCl. b- In the day of the experiment (3.5.2019):

 Station No. 1: Native-PAGE for following [2Fe-2S] cluster transfer in vitro:  Instructor: Mz. Ola Karmi.

1) Pre-reduction of apo-Fdx: Incubate 200µL of apo-Fdx (300µM) with 2% of β-mercaptoethanol and 5mM of EDTA for 30 minutes. Following the incubation, fractionate into Eppendorf tubes 25µLof the reduced apo-Fdx. 2) Add to the reduced apo-Fdx tube, 2µL of holo-NAF-1 or NAF-1-H114C mutant (concentrated to 2 mM each) to obtain a final concentration of 150µM of the NEET proteins. 3) Start with 60 minutes incubation time point and add the same volume to each tube gradually until time 0’. 4) At the end of 10’ incubation time point, prepare the Native-gel running buffer. 5) Running buffer: To a 250 Erlenmeyer flasks add 25mL of Deriphat reservoir (10X stock), 500µL of SDS (10% stock), 0.5g of Deriphat powder, and 225mL of DDW. Mix it well and add to the electrophoresis apparatus after mounting the gel. 6) Controls preparations: Add 2µL of holo-NAF-1/ holo-Fdx to 25µL of the buffer containing 20mM Tris-HCl pH 8.0 and 100mM NaCl, to have the same concentration as the other samples have. 7) For all tubes, add 5% Glycerol (take 5μl from the 50% Glycerol stock). 8) Load the native-PAGE gel, using the order of the table below. Then run the gel at 100mV for 20min.

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Reduced Apo-Fdx+ Holo-NAF-1 1 2 3 4 5 6 7 8 9 10 Time Time Time Time Time Time Time Holo- Holo- Time 0’ 60’ 0’ 5’ 10’ 15’ 30’ 60’ Fdx NAF-1 H114C H114C

Native-PAGE Gels Preparation: This amount allows preparing 10 gels: 1- DDW 77ml. 2- Acrylamide (37.5:1) 18.5ml. 3- Deriphat Gel Buffer 37ml; composed of (Tris 0.75g, Glycine 3.5g, Deriphat 1g, for a final volume with DDW 250ml). 4- 10X Deriphat Reservoir 13ml; composed of (Tris 15g, Glycine 72g, for a final volume with DDW 1000ml). 5- Ammonium persulfate (APS) (10%) 2ml. 6- TEMED (1,2-Bis(dimethylamino)ethane) 200µL.

Native-PAGE Running Buffer: 1- Deriphat (powder) (0.5g). 2- Deriphat reservoir (10X) 25ml. 3- Sodium dodecyl sulfate SDS (10%) 0.5ml. 4- DDW 225ml.

 Station No. 2: UV-Vis Absorption Spectroscopy to determine the [2Fe-2S] cluster transfer process:  Instructor: Mr. Henri-Baptiste Marjault.

1- Pre-reduction of Fdx: Incubate 450µL of apo-Fdx (300µM) with 2% of β-mercaptoethanol and 5mM of EDTA for 30 minutes. 2- During the pre-reduction of the apo-Fdx, prepare holo NAF-1, holo-Fdx and holo-H114C, as follow: To three (3) Eppendorf tubes add 128µL buffer solution containing 20mM of Tris-HCl and 100mM NaCl and 22µL of holo-NAF-1 or holo-Fdx or holo-H114C, respectively, to a final concentration of 300 µM of each holo-protein. 3- Then load the whole volume of each Eppendorf of holo-Fdx (#3), holo-NAF-1 (#4) and holo- H114C (#5) of the 96 wells plate as described in the table below. 4- Then load 150µL of the reduced-apo-Fdx #6 of the 96 wells plate as describe in the figure bellow. 5- Cluster transfer Assay: From the reduced apo-Fdx load 128µL into the 96 wells plate #1 and #2 as describe in the figure below. Then add 22µL of holo-NAF-1 (300µM) or H114C (300µM) and Mix well using a pipette. 6- Read the spectral absorbance from 380nm until 550nm after incubating at 37°C, with continuous shacking for 0 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes and 60 minutes. 7- Extract the data from the computer then plot the UV-Vis spectra for analysis.

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Please note that every group will load the samples in a different row.

Techniques for studying iron in health and disease EMBL

Part I : The involvement of the NEET proteins in Iron homeostasis demonstrated by mitochondrial iron accumulation in WT and cells with suppressed expression of NEET proteins. A Confocal microscopy DEMO by Dr. Yang-Sung Sohn.

Introduction

NEET proteins, such as mitoNEET (mNT; CISD1), Nutrient-deprivation autophagy factor-1 (NAF-1; CISD2), and mitochondrial inner NEET protein (MiNT; CISD3) are a class of iron-sulfur proteins involved in several human pathologies, including diabetes, cystic fibrosis, Wolfram syndrome 2, neurodegeneration, and muscle atrophy. mNT and NAF-1 are localized to the outer mitochondrial membrane. NAF-1 is also localized to the endoplasmic reticulum. Deficiency in mNT and in NAF-1 causes the accumulation of iron and ROS in mitochondria of mammalian and plant cells (1, 21). mNT and NAF-1 proteins’ suppression (using shRNA) allowed to obtain stable cell lines where reduced levels of these proteins [mNT (-)] and [NAF-1 (-)] lines. The effects of the latter on iron homeostasis of the mitochondria in human breast cancer cells will be monitored with fluorescent metal sensors which undergo fluorescence quenching upon iron binding (22-24).

In the current workshop, the students together with DR. Sohn, will measure mitochondrial iron level in MDA-MB-231, breast epithelial cancer cells with suppressed expression of NEET proteins. They will use the fluorescent metal-sensors rhodamine-B-phenanthroline (RPA; exc.560nm, em.610nm) for detecting iron level in mitochondria. Since RPA has strong iron affinity until we cannot remove bound iron from it even with a high-affinity cell permeant iron chelator, like SIH, we will measure Iron content indirectly using cell permeant iron chelator, Deferiprone (DFP) applying a pre-incubation method. Fluorescence changes will be measured by confocal fluorescence microscopy (22, 23). Please note that upon binding of iron to the phenanthroline of the RPA the rodamin fluorescence is quenched (see below Figure 9).

Figure 9. rhodamine-B-phenanthroline (RPA); A. RPA gives fluorescence emission at 610nm, B. RPA quenched upon binding of iron (Fe+2) to the phenanthroline part.

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Brief outline of the experiment:

Cells will be planted in Petri dish adapted for microscopic images in advance. In the following day the cells will be stained with metal-sensors and the fluorescence difference among different cell lines which are WT, mNT(-), NAF-1(-) will be measured using confocal microscopy. They also will pre incubate cells with DFP for 30 minutes and then stained with RPA for measuring the fluorescence difference. After the experiment, the students will analyze the data with Image-J program as depicted in (Figure 10).

Figure 10. Representative images and quantitative analysis using Image-J. Images (Left) and a quantitative graph (Right) showing over accumulation of iron in mitochondria of mNT() or NAF-1() ii mitochondrial iron accumulation (Right).

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Preparation of cell lines:

All shRNA transfections were performed with 1μg DNA using Lipofectamine2000 (Invitrogen) or Genejuice (EMD Millipore) as a transfection reagent according to the manufacturer’s instructions. Cells were treated with 1μg/mL of puromycin for 48hrs. The efficiency of the transfection was monitored by the expression of GFP at 24hrs after transfection. Stable knockdown lines for mNT [mNT(-)] and NAF-1 [NAF-1(-)] were obtained by FACS after sorting based on GFP fluorescence. The stable lines isolated were characterized for the level of the NEET proteins in them by protein blots.

a- One day before experiment (02.05.19):

1. Prepare RPMI medium supplemented with 10% FCS, L- glutamine, and antibiotics. 2. Cells are detached from the flask (T75) with trypsin. 3. Count the cells. 4. Plant cells at the appropriate cell number (~0.2x106cells/ml) on the special petri-dish for microscopy measurement. 5. Prepare required solutions and reagent stocks (list below).

b- The day of the experiment (03.05.19):

1. Prepare RPA (0.5µM) in DMEM-Hepes. The DMEM-Hepes should be at 37°C. 2. Aspirate cell medium from the petri-dish and add RPA solution for 15 minutes at 37°C. 3. Wash cells with DMEM-Hepes two times and replenish cells with the flesh DMEM-Hepes medium. 4. Take images randomly at least 5 different areas in each cell lines with confocal microscopy. 5. DFP (50-100µM) will be added to all cell lines for 30 minutes of pre-incubation and 1- 4 processes will be repeated for the measurements.

• Data analysis: Extract mean FI of RPA in ROI of the samples using Image-J program.

Required reagent stocks and solutions:

• DMEM-Hepes (1 vial of DMEM no phenol red powder, 4.77g Hepes, DDW to 1L, adjust pH to 7.2-7.4 with NaOH. Filter and keep at 4°C. • DMSO. • RPA 10mM in DMSO. • DFP 50mM in H2O. • Trypsin-EDTA. • Ethanol. • DDW.

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Required equipment:

• Confocal microscope. • Water bath 37°C. • Gilsons and sterile pipet tips. • Timers. • Calculators. • Paper towels. • Containers for biological waste disposal (liquid, solid). • Nitrile gloves. • Plastic beakers and cylinders. • Scales. • pH meter. • Falcon tubes (15ml and 50ml). • Eppendorf tubes. • Vortex. • Aluminium foil.

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DAY 2-part II (May 3rd) Oliver Stehling Biochemical and functional characterization of mitochondrial ISC targeting factors

Introduction Iron-sulfur (Fe/S) clusters are inorganic protein cofactors of rhombic or cubane shape based on an alternating arrangement of iron and sulfur atoms (1). The clusters exhibit versatile properties which allow their host proteins to participate in crucial cellular processes including energy conversion, cofactor biosynthesis, genome homeostasis, protein biosynthesis, viral defense, and iron regulation (2) (Fig. 1). Assembly of Fe/S clusters is assisted by three compartmentalized systems (3, 4). The iron-sulfur cluster assembly (ISC) system resides within the mitochondrial matrix and assists the assembly of all cellular Fe/S proteins, the export system involves the mitochondrial ABC transporter ABCB7 and connects the ISC system with the extra-mitochondrial cytosolic iron-sulfur protein assembly (CIA) sys

Fig. 1: Simplified overview of the localization, function and biogenesis of cellular iron-sulfur proteins. Known iron-sulfur (Fe/S) proteins are localized in mitochondria, cytosol and nucleus. They are involved in numerous cellular functions and pathways. Some (shown in green) execute essential functions by participating in, e.g., protein translation or DNA replication and repair. Classical Fe/S proteins are the mitochondrial aconitase and respiratory complexes I, II, and III, whereas viperin involved in antiviral response has been discovered more recently. The synthesis and insertion of Fe/S clusters in living cells needs complex machinery which is highly conserved in eukaryotes from yeast to man. The mitochondria-localized iron-sulfur cluster assembly (ISC) machinery comprises 18 ISC proteins, and is involved in the biogenesis of all cellular Fe/S proteins. The core ISC proteins assist the cytosolic iron-sulfur protein assembly (CIA) machinery (11 known proteins) by synthesizing an unknown sulfur-containing molecule (X-S) which is exported to the cytosol by the ABC transporter ABCB7. Mitochondrial Fe/S

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protein biogenesis is tightly linked to cellular iron regulation by its role in the maturation of iron regulatory protein 1 (IRP1) in humans. tem which aids the maturation of cytosolic and nuclear Fe/S proteins (Fig. 1). As a basic principle, Fe/S clusters are not directly formed on the respective apo-proteins but assembled de novo on scaffold proteins from where they are subsequently transferred by trafficking and targeting factors (5) (Fig.2). The key event for the assembly of all cellular Fe/S cluster is the de novo formation of a [2Fe-2S] cluster within the mitochondrial matrix on the ISCU scaffold, involving mitochondrial iron supply by mitoferrin 1 / 2 (SLC25A37 / 28) and possibly frataxin (FXN), sulfur release from cysteine by the cysteine desulfurase complex NFS1-ISD11-ACP, and electron supply for reduction of sulfur to sulfide by the electron transfer chain NADH-FDXR- FDX2. Once formed the [2Fe-2S] cluster is released from the scaffold by direct interaction of ISCU with a HSC20-HSPA9 chaperone system and transferred to GLRX5. The dimeric holo- GLRX5 is required for three divergent branches of the assembly pathway, i. e. direct [2Fe-2S] protein assembly, the ISCA1-ISCA2-IBA57-dependent conversion of [2Fe-2S] to [4Fe-4S] clusters, and the export system. The [4Fe-4S] clusters are finally inserted into target apoproteins by specific ISC targeting factors including NFU1 and IND1.

The biosynthesis of cytosolic and nuclear Fe/S proteins is strictly dependent on the early reactions of the mitochondrial ISC assembly pathway and on the mitochondrial inner membrane ABCB7 transporter of the export machinery (4). The compound exported by mitochondria is ill-defined yet but in all likelihood contains a NFS1-derived sulfur moiety that is used by the CIA system for the assembly of a [4Fe-4S] cluster on the cytosolic scaffolding complex NBP35-CFD1 where it is only transiently bound. Reductive power for this process is provided by the electron transfer chain NADPH-NDOR1-CIAPIN1, but its biochemical role is so far ill defined. The transient [4Fe-4S] cluster is released from the scaffold involving the iron- only hydrogenase-like protein CIAO3 and inserted into apo-proteins by specific targeting factors. The majority of cytosolic and nuclear [4Fe-4S] proteins require the CIA targeting complex (CTC) composed of CIAO1-CIAO2B-MMS19 for their maturation. Cluster insertion into iron regulatory protein 1 (IRP1) is mediated by a dedicated branch of the CIA system, namely the CIA targeting factor CIAO2A (6, 7). Interestingly, CIAO2A is also required for the stability of IRP2 although the latter is not known to contain an Fe/S cofactor (6). Thereby, cellular Fe/S protein biogenesis is intimately connected to cellular iron homeostasis.

Genetic defects in components of the mitochondrial ISC system give rise to so-called “Fe/S” diseases with neurological, metabolic and hematological phenotypes (8). Particularly, defects in the mitochondrial ISC assembly system are causative for a broad spectrum of mitochondria- related disorders. Defects in the core ISC machinery required for mitochondrial [2Fe-2S] protein assembly affect both mitochondrial and cytosolic-nuclear Fe/S proteins and frequently result in a combined mitochondrial neuro-muscular phenotype. The prototypical Fe/S disorder is Friedreich’s ataxia which is mainly caused by an insufficient expression of the regulatory core ISC component FXN. Despite the phenotypical heterogeneity of core ISC assembly defects their common denominator is mitochondrial iron accumulation by an ill- defined mechanism involving IRP1/2 and mitoferrin. In contrast, defects restricted to components involved in mitochondrial [4Fe-4S] cluster assembly are not associated with mitochondrial iron accumulation and do not affect extra- mitochondrial Fe/S proteins.

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Rather, they result in severe mitochondrial phenotypes with a wide phenotypic spectrum, and hence are summarized as multiple mitochondrial dysfunction syndromes (MMDS). Affected individuals present with hypotonia, respiratory insufficiency, encephalopathy, brain malformations, and neurological regression. Biochemical hallmarks are impaired

Fig. 2: Detailed working model for the maturation of cellular iron-sulfur proteins in human cells. The mitochondrial ISC pathway can be dissected into three main steps. First, a [2Fe-2S] cluster is de novo assembled on the scaffold protein ISCU. Synthesis of the cluster requires the cysteine desulfurase complex NFS1-ISD11-ACP1 releasing sulfur from cysteine and transferring it to ISCU. Frataxin is proposed to support sulfur transfer and/or supply iron, while the sulfur reduction to sulfide requires the electron transfer chain NADPH-ferredoxin reductase- ferredoxin (FDXR, FDX2). Reduced iron (Fe2+) is imported into mitochondria by the carrier proteins mitoferrin1/2 in a proton-motive force (pmf) dependent fashion. In the second step, the ISCU-bound [2Fe-2S] cluster is released from ISCU by direct binding of dedicated Hsp40- and Hsp70 chaperones (HSC20, HSPA9).

The [2Fe-2S] cluster is transferred to glutaredoxin GLRX5, from where it is directly inserted into [2Fe-2S] apoproteins completing their maturation. In the final step, the synthesis of [4Fe-4S] clusters is catalyzed by fusing the GLRX5-bound [2Fe-2S] clusters, a reaction involving the specialized ISC proteins ISCA1-ISCA2-IBA57.

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In contrast to aconitase, complex Fe/S proteins such as respiratory complexes I and II and the radical SAM protein lipoyl synthase (LIAS) require further, late-acting ISC factors for Fe/S cluster transfer (IND1, NFU1).

The maturation of cytosolic and nuclear Fe/S proteins depends on the core ISC system provi- ding sulfur from mitochondrial NFS1 and generating the unknown sulfur-containing compound X-S. The mitochondrial ABC transporter ABCB7 exports X-S to the cytosol in a glutathione (GSH)-dependent step, where it is used by the CIA machinery for the synthesis of a [4Fe-4S] cluster on CFD1-NBP35 P-loop ATPases serving as a scaffold complex. The latter reaction needs electron input from the flavin-oxidoreductase NDOR1 and the Fe/S protein CIAPIN1. The complex of GLRX3-BOLA2 may facilitate Fe/S cluster insertion into CIAPIN1. The CFD1-NBP35-bound [4Fe-4S] cluster is then released and trafficked to the majority of cytosolic and nuclear target Fe/S apoproteins by the help of CIAO3, and the CIA targeting complex (CIAO1, CIAO2B, and MMS19) which facilitates cluster insertion by direct apoprotein interaction. Interestingly, the translation-associated ATPase ABCE1 additionally needs the ORAOV1-YAE1D1 adapter complex to recruit the apo-ABCE1 to the CIA targeting complex for [4Fe-4S] cluster transfer. Maturation of the iron regulatory protein 1 (IRP1) is unique in that it depends on the early part of the CIA machinery, yet - instead of the CIA targeting complex – it uses CIA2A (a close homolog of CIA2B) for Fe/S cluster insertion, thereby linking cellular Fe/S protein biogenesis and cellular iron regulation. activities of respiratory complexes I, II, and IV as well as of the radical SAM enzyme lipoyl synthase (LIAS), resulting in insufficient lipoyl cofactor formation on the E2 subunits ofpyruvate (PDH), α-ketoglutarate (α-KGDH), α-oxoadipate (α-OADH), and branched-chain ketoacid (BCKDH) dehydrogenases as well as on the H protein of the glycine cleavage system (GCS-H).

The observation that Fe/S protein maturation defects interfere with essential cellular pathways emphasizes the key function of mitochondria as primary sites of the de novo assembly of cellular Fe/S clusters, and provides an explanation why these organelles are essential for eukaryotic life.

Objectives The 2nd part of this workshop is intended for 9 participants and introduces on the one hand biochemical approaches to assess the functionality of mitochondrial ISC targeting factors within the cellular environment and on the other hand techniques to study the biochemical properties of these factors. The participants will pass up to 4 different stations where they i) examine (bio)chemical properties of ISC targeting factors, ii) spectrophotometrically determine the activities of key mitochondrial Fe/S enzymes, iii) analyze the activity of the mitochondrial Fe/S enzyme lipoyl synthase by immunoblotting, and iv) inspect the phenotype of viable ISC targeting factor-deficient cells.

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Practical

DAY 2 (Part II) - STATION 1. (Bio)chemical examination of ISC targeting factors. Instructor: Vinzent Schulz

Introduction The biochemical characterization particularly of Fe/S cluster-coordinating assembly factors is a critical prerequisite for the understanding of the molecular mechanisms underlying the biogenesis of Fe/S proteins in vivo. Recombinant expression and purification of the core ISC assembly factor FDX2 and the ISC targeting factor ISCA2 shed light on indicative properties of the two proteins (9, 10).

In-vitro arrangement: Protein expression and purification The human ISC assembly factors FDX2 and ISCA2 were cloned to carry a N-terminal His-tag and overproduced in E. coli BL21(DE3) cells in 4 L of terrific broth medium (2x 2 L in 5 L flasks) in presence of selection antibiotics. Cultures were inoculated from bacterial overnight suspensions and shaken at 37°C at 160 rpm for about 7 h. Overproduction was induced by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. Subsequently, temperature was decreased to 28°C and cultures shaken overnight (ca. 17 h). Cells were spun down for 10 min at 4°C, and pellets were resuspended in protein buffer (50 mM Tris/HCl, 300 mM NaCl, 5% w/v, pH 7.4). Subsequent to the addition of lysozyme, DNAse I and proteinase inhibitor (cOmplete™ Protease Inhibitor Cocktail) the cells were kept on ice and lysed by sonication. The lysate was cleared by centrifugation at 40,000 x g at 4°C and the cell extract was loaded onto a Histidine-binding column (NiNTA-Agarose) pre-equili- brated with protein buffer. Subsequently, the column was washed with washing buffer (protein buffer + 20 mM imidazole) in order to remove weakly binding contaminations, and eluted in one step with elution buffer (protein buffer + 500 mM imidazole). The eluate was concentrated to a volume of 2 mL using a 10 kDa cutoff filter and further purified by size exclusion chromatography using a HiLoad® 16/600 Superdex® 200 pg column. Desired fractions were combined, concentrated to ca. 1 mL and checked for purity by SDS-PAGE. Protein concentration was determined using the Bradford assay.

Characterization of purified human FDX2 and ISCA2 by UV/VIS-spectroscopy Fe/S clusters display broad, characteristic UV/VIS absorption spectra that may also provide a first hint concerning the type of Fe/S cluster (Fig. 3) (11). As a rule of thumb, spectra of [2Fe-2S] proteins are more complex with several characteristic absorption maxima between 410 and 430 nm, around 470 nm, and occasionally, a relatively broad peak between 550 and 600 nm. Spectra of [4Fe–4S] proteins are less featured and display a characteristic peak around 400–420 nm.

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The intensity ratio of the Fe/S cluster-specific peak at about 420 nm to that of the protein at 280 nm is frequently used as a practical measure to roughly estimate the amount of holoprotein in a preparation. Fig. 3: UV/VIS spectra of representative [2Fe–2S] and [4Fe–4S] proteins. UV/VIS spectra of two [2Fe–2S] proteins, S. cerevisiae Yap5 and human GLRX5, and of two [4Fe-4S] proteins, human NFU1 and the N-terminal domain (1– 97) of S. cerevisiae Rli1. The positions of the major peaks of the UV/vis spectrum of Yap5 are indicated (11).

Equipment • UV/VIS Spectrophotometer (e. g. Amersham Ultrospec 3100 or QIAxpert) • UV cuvettes (or QIAxpert trays) • Ice bucket

Reagents • Dithionite

Spectroscopic Analysis • Place 200 µL-duplicates of 50 µM FDX2 and ISCA2 on ice. • Add 1 mM dithionite to one of each duplicate and incubate on ice for 30 min. • Record spectra.

Iron content determination of purified Fe/S proteins Determining the amount of iron bound to a given Fe/S protein can already provide information about the type of cluster coordinated by the polypeptide chain. In order to get access to the metal, it is first liberated from the protein by treatment with hydrochloric acid. Excess acid is neutralized with ammonium acetate, followed by reduction of Fe3+ to Fe2+ with ascorbic acid. Sodium dodecylsulfate is applied to solubilize precipitated protein, and addition of the iron chelator ferene results in the formation of a blue complex. The increase in color represents a measure of the amount of ferrous iron in solution and can be determined spectrophotometrically (12).

Equipment • UV/VIS Spectrophotometer (e. g. Amersham Ultrospec 3100 or QIAxpert) • UV cuvettes (or QIAxpert trays)

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Reagents • Hydrochloric acid, 1% w/v • Ammonium acetate solution, 7.5% w/v • Sodium dodecylsulfate (SDS), 2.5% w/v • Ascorbic acid, 4% w/v (freshly prepared) • Ferene (3-(2-pyridyl)-5,6-bis(5-sulfo-2-furyl)-1,2,4-triazine, disodium salt 3 H2O), 1.5 % w/v • 0.2 mM (NH4)2Fe(SO4)2.6H2O (Mohr's Salt, 78 mg/L, molecular mass 392.14 g/mol, freshly prepared) • Bradford reagent • Bovine serum albumin (BSA)

Ferene-based iron determination • Prepare iron standard samples from Mohr's Salt stock solution (1 to 100 microliter, i.e. 0.02-20 nmol Fe). • Treat samples with acid: Triplicate samples of an Fe/S protein solution (different volumes of 0-100 microliter) and iron standard samples including two blanks are diluted to 100 microliter with demineralized water. Subsequently add 100 microliter 1% hydrochloric acid and mix gently. • Incubate samples at 80°C for 10 min. • Chelate iron by sequential addition and vortexing: - 500 microliter 7.5% ammonium acetate - 100 microliter 4% ascorbic acid - 100 microliter 2.5% sodium dodecylsulfate - 100 microliter iron chelator • Centrifuge samples for 5 min at 13000 x g to remove precipitates. • Measure absorbance of the solutions at 593 nm against water. • Similarly determine protein concentration of the Fe/S protein solution by the Bradford- method; include BSA standard and blank samples into the analysis.

Calculate the iron concentration of the Fe/S protein samples from the calibration curve. Relate the amount of iron to the corresponding protein amount.

In order to obtain accurate results use Mohr’s salt with proper crystalline appearance (no caking) and prepare iron standard solution freshly. The shape of the calibration curve is usually extremely linear (up to an absorbance of 2-2.5). The color change of the blank samples depends on the quality of the applied solutions.

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0.20 Fig. 4: Example for a ferene Absorbance at 593 nm = 0.0352 nmol Fe + 0.0175 2 calibration curve. The relation R = 0.9997 between the iron amounts in 0.15 reference solutions and the formation of the colored iron- 0.10 ferene complexes is usually straight.

0.05 Absorbance 593 nm at

0.00 0 1 2 3 4 nmol Fe

Determination of acid-labile sulfide in purified Fe/S proteins In addition to iron content, analysis of acid-labile sulfide complements the information about the cluster type coordinated by an Fe/S protein. Denaturation of the Fe/S protein in an alka- line medium containing zinc hydroxide releases sulfide which is simultaneously co-precipitated with Zn(OH)2 as ZnS. After acidification, H2S condenses with two molecules of N,N'- dimethyl-p-phenylenediamine to form the dye methylene blue. The increase in color represents a measure for the amount of released sulfide and can be determined spectrophotometrically (13).

Since sulfide can be oxidized by oxygen it may be (at least partially) lost by evaporation after acidification. Hence, anaerobiosis is the condition of choice for the assay, although the procedure outlined below represents a sufficient compromise for working in presence of atmospheric oxygen. The presence of protein can affect the yield of methylene blue formation. This effect may be tested by the addition of a known amount of sulfide standard to samples of the desired Fe/S protein.

Equipment • UV/VIS Spectrophotometer (e. g. Amersham Ultrospec 3100 or QIAxpert) • UV cuvettes (or QIAxpert trays) • Reagents • Zinc acetate, 1% w/v (freshly prepared from 10 % w/v) • Sodium hydroxide, 7% w/v • N,N'-dimethyl-p-phenylene-diamine (DMPD), 0.1% w/v in 5 M HCl (TOXIC) • 10 mM FeCl3 in 1 M HCl. • Na2S x 9H2O (mol. mass 240.18 g/mol) or Li2S (mol. mass 45.95 g/mol) • 10 mM NaOH • Bradford reagent • Bovine serum albumin (BSA)

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Sulfide standard preparation

Prepare a sulfide standard (≈ 2 mM) gravimetrically from Na2Sx9H2O by blotting a crystal (≈ 0.5 g) on filter paper. Weigh rapidly and add to a 1 liter volumetric flask containing 10 mM argon-purged NaOH. Close flask immediately and stir. The solution is stable for at least 10 h. Alternatively, lithium sulfide can be used (mol. mass 45.95), which is easier to weigh and not as problematic regarding hygroscopic properties.

Methylene blue-based sulfide determination • Prepare sulfide standard samples from the sulfide stock solution (5, 10, 15, 20, and 25 µL i.e. 10-50 nmol S2-). • Prepare two samples of the Fe/S protein preparation to be analyzed and add 10 and 20 µL sulfide stock solution • Dilute standard samples including blanks, the two sulfide-supplemented Fe/S protein samples as well as three untreated Fe/S protein aliquots to 200 µL with water. • Add 600 µL of 1% zinc acetate. • Add 50 µL of 7% NaOH, close reaction tubes, put upside down once to mix. • Incubate tubes for 15 min at room temperature. • Centrifuge for a few seconds at low speed.

Continue assay by quickly treating tubes one by one in order to minimize loss of sulfide: • Add 150 µL DMPD solution by dipping the end of the yellow tip at the bottom of the tube; gently release the DMPD and mix by slowly by rotating the pipette (keep the piston depressed). The zinc hydroxide and sulfide precipitates will dissolve. • Carefully remove the pipette-tip from the tube; add 150 µL FeCl3 solution quickly. • Close the vessel immediately and vortex vigorously for 30 sec; continue with the next tube. • Once all tubes have been treated with DMPD/FeCl3 continue incubation for 20 min with intermittent vigorous shaking. • centrifuge for 5 min at 13,000 x g to remove precipitates and measure absorbance at 670 nm against water. • Determine protein concentration of the Fe/S protein solution by the Bradford-method; include BSA standard and blank samples into the analysis.

Calculate the sulfide concentration of the Fe/S protein samples from the calibration curve. Relate the amount of sulfide to the corresponding protein amount.

Prefer lithium sulfide for the preparation of the sulfide standard solution. Take care for proper grainy appearance without caking. If sodium sulfide is used as a standard, open a new vial with discrete crystals; avoid wet (hygroscopic) material. Stock and diluted sulfide standard solutions have to be prepared freshly. Note that reducing agents (DTT, cysteine, ascorbate, dithionite) and organic compounds reacting with Fe3+ and/or DMPD may lead to erroneous results. Also test whether the buffer of the Fe/S protein solution impacts on the optical density of the methylene blue sample. The effective extinction coefficient is highly sensitive to experimental changes and thus may vary between individual test series.

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The shape of the calibration curve is usually not linear at higher sulfide concentrations. Note -1 -1 -1 - that the final assay volume is 1.15 ml, yielding λ670 nm = 1.15 x 23.2 mM cm = 26.7 mM cm 1

Fig. 5: Example for a methy- Absorbance at 670 nm= 0.0232* nmol S2- - 0.0034 2 lene blue calibration curve. R = 0.9976 0.8 The relation between the sulfide amounts in reference 0.6 solutions and the formation of methylene blue is usually not straight. 0.4

Absorbance 670 nm at 0.2

0.0 0 10 20 30 40 nmol S2-

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DAY 2 (Part II) - STATIONS 2 to 4. Analysis of Fe/S enzyme activities in tissue culture material Oliver Stehling

Tissue culture arrangements Experimental depletion of ISC assembly factors by RNAi RNA interference (RNAi) is a powerful tool to specifically deplete proteins by degrading their mRNAs with the help of small interfering RNAs (siRNAs) (14). In contrast to a complete gene knock-out achieved by CRISPR-Cas9 the RNAi-mediated knock-down phenotype is less severe and reversible, rendering the analysis of ISC assembly factor deficiencies possible even if an entire gene knock-out would be lethal. Since siRNAs for a given target mRNA are usually not equally efficient we recommend to test at least three individual siRNA duplexes for their capability to eventually decrease the cellular levels of a given protein (6, 7). Those siRNAs with the highest activity may then be applied as a pool in order to further improve the degree of depletion. Besides oligomeric siRNA duplexes it is also possible to use plasmid-encoded short- hairpin (sh) RNAs (15), although this approach is sometimes less efficient. Introduction of siRNAs or shRNAs into tissue culture cells is performed via transfection. In our hands, electroporation is superior to chemical transfection, and application of shRNAs by the latter approach hardly yields proper depletion. HeLa cells tolerate electroporation well and are a suitable model system for the analysis of knock-down phenotypes (15). For example, using RNAi for the experimental depletion of FDX2 and ISCA2 provided insights into their cellular function as core ISC assembly and ISC targeting factors, respectively (16, 17). The cellular phenotypes are different from those elicited by individual mitochondrial Fe/S protein deficiencies like LIAS defects. Samples of ISC-deficient cells can be prepared in advance and stored at -80°C until use.

Material • Common tissue culture equipment for HeLa cells including 75-cm2 culture flasks • Electroporation system (e. g. Gene Pulser Xcell Eukaryotic System, Bio-Rad) • Electroporation cuvettes with 4-mm gap • Pasteur pipettes (glassware)

Reagents • Common tissue culture media and reagents (DMEM supplemented with 7.5 % FCS, 1 mM glutamine, Penicillin/Streptomycin) • Sterile filtered electroporation buffer (EB) (21 mM HEPES, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 6 mM dextrose, pH 7.2)

siRNA • Control siRNA - Silencer™ Select Negative Control No. 1 siRNA (Ambion 439084-X) - ON-TARGETplus Non-targeting siRNA #1 (Dharmacon D-001810-01-X) • FDX2-directed siRNA (Ambion Silencer Select s41377, s225482, s225483) • ISCA2-directed siRNA (Ambion Silencer Select s42599, s42600, s42601)

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• LIAS-directed siRNA (ON-TARGETplus Set of 4 LQ-010023-01-X) => Prepare 100 µM stock solutions in RNAse free water and store at -20°C.

Transfection of HeLa cells by electroporation For 75 cm2-flask cultures electroporation requires 6.5 × 106 suspended HeLa cells in a volume of 250 µL EB. Total siRNA concentration in the cell suspension amounts to 2.2 to 2.8 µM. The transfection sample may include 4 µg of the plasmid pVA-I in order to improve cell recovery from the electroporation procedure.

• Culture HeLa cells in a 75 cm2-flask at a volume of 20 mL until confluency and transfer the tissue culture supernatant (conditioned TCS) into a sterile tube. Remove debris e. g. by centrifugation. • Harvest cells by trypsination and wash once in EB. • Count cells and adjust their concentration to about 26 x106 / mL in EB. • Transfer 250 µL of the suspension (6.5 × 106 cells) into a sterile 1.5 mL cup, add siRNA to the desired concentration, and mix thoroughly. • Transfer the entire sample into a 4-mm gap electroporation cuvette (make sure that no individual drops remain at the cuvette walls); immediately continue to avoid settling of the cells (electroporation requires a homogenous cell suspension) • Place cuvette into an electroporator device and start the electroporation process (525 µF, 265 V, unrestricted resistance, single pulse); expect a pulse time of 25 to 28 milliseconds. • Immediately add about 750 µL culture medium into the cuvette • Transfer the entire cell suspension into 75 cm2-flasks containing 20 mL of fresh culture medium supplemented with 20 % conditioned TCS (conditioned TCS supports HeLa cell recovery from electroporation) • If desired, rinse the cuvette with medium in order to recover the entire cell amount.

Efficient protein depletion requires at least one repetition of the electroporation procedure at a three day interval. Final harvest is performed also three days after electroporation. Note that in case of siRNAs no other interval is possible.

Preparation of cell samples for immunoblotting Immunoblotting is a versatile method to examine the depletion efficiency of proteins targeted by the RNAi approach. At the same time immunoblotting may allow for quantitation of cellular steady-state levels of cofactors such as lipoate that are dependent on ISC assembly processes. Protein-bound lipoyl moiety can be visualized by a specific antibody, and represents an indirect determination of the LIAS enzyme activity. Finally, the stability of Fe/S proteins is frequently dependent on a bound cluster; hence the level of a Fe/S protein can serve as an indirect measure of their maturation status (18).

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Reagents • 2-times concentrated Laemmli buffer: 120 mM Tris/HCl, 4 % SDS, 20 % glycerol, 0.1 ‰ bromophenol blue, 10 % 2-mercaptoethanol (2-ME; add always freshly) • Biochemical grade water

Sample preparation • Determine total protein content of 2 to 4 × 106 HeLa cells, wash twice with PBS, and keep the dry cell pellet on ice. • Add distilled water to a final concentration of 2 µg protein per µL and suspend thoroughly. • Add 2-times concentrated Laemmli buffer containing 10 % of 2-mercaptoethanol (2- ME) to the suspended samples. The final protein concentration will be 1 µg / µL in 1- times Laemmli buffer containing 5 % 2-ME. Mix well, the samples may be of high viscosity. • Heat to 95°C for at least 5 min, the viscosity will decrease. • Spin down at 15,000 × g for 5 min and apply samples to SDS-PAGE.

Digitonin-based crude cell fractionation for enzymatic analyses Enzymatic analysis of ISC assembly defects requires the compartment-specific determination of Fe/S protein activities. Tissue culture material allows the separation of cytosolic and mitochondrial Fe/S proteins by digitonin-mediated plasma membrane permeabilization (18, 19). At low concentrations, the steroid glycoside acts as a cholesterol-chelating agent and thus interferes with plasma membrane integrity without impacting on other intracellular membranes. Following tabletop centrifugation, a supernatant fraction containing soluble cytosolic proteins can be separated from a “crude mitochondrial fraction” which additionally contains essentially all other membranous compartments including the plasma membrane.

Reagents • Digitonin (water soluble, e. g. MPBio, Cat. No. 159480), 2 to 5 % in DMSO (store at -20°C) • MitoBuffer (25 mM Tris/HCl, 250 mM sucrose, 1.5 mM MgCl2, pH 7.4; store at -20°C) • Phenylmethylsulfonylfluorid (PMSF), 250 mM in isopropanol (saturated, store at -20°C) • DigiBuffer (0.008 % digitonin in MitoBuffer, 1 mM Phenylmethylsulfonylfluorid - PMSF), prepare always freshly from stocks • Phosphate-buffered saline (PBS) without calcium and magnesium

Fractionation: • Wash cells twice with PBS and resuspend pellet in DigiBuffer at a concentration of 10 × 106 cells/mL; the final protein concentration must not exceed 2 mg/mL. • Incubate for about 15 min on ice. • If desired, remove an aliquot of the lysate for later analysis (usually one 3rd). • Centrifuge for 10 min @ 15000 × g @ 4°C.

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• Collect the supernatant as the cytosolic fraction. • Resuspend the pellet (membrane fraction) in 1 mL of MitoBuffer (washing step). • Centrifuge pellet for 10 min @ 15000 × g @ 4°C. • Discard the supernatant and resuspend pellet thoroughly in its original volume (i.e. the volume of the cytosolic fraction). • Determine protein content of the obtained fractions (lysate,) cytosol, pellet (membranes). • Analyze enzyme activities immediately or • snap-freeze fractions and store @ -80°C.

Note: Activities of cofactor-containing enzymes can be determined only in fresh or 1× thawed samples; Fe/S proteins do not tolerate two rounds of thawing.

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DAY 2 (Part II) - STATION 2. Spectrophotometric analysis of Fe/S enzyme activities Oliver Stehling

Introduction The mitochondrial ISC system assists the maturation of both mitochondrial and extra-mitochondrial Fe/S cluster containing enzymes, including succinate dehydrogenase (SDH), the catalytic part of respiratory chain complex II, as well as both mitochondrial and cytosolic aconitase (18). SDH oxidizes succinate to fumarate and feeds the released electrons into the respiratory chain via ubiquinone. SDH consists of four subunits of which SDHA and SDHB constitute the catalytic core and SDHC and SDHD are the membrane anchor. Of the four subunits only SDHB harbors Fe/S clusters in form of one [2Fe-2S], one [4Fe-4S], and one [3Fe- 4S] cluster which all participate in electron transfer. Accordingly, proper maturation and activity of SDH is dependent on both the [2Fe-2S] cluster-generating core ISC assembly system and the [4Fe-4S] cluster-assembling ISCA-IBA57 targeting factors. Mitochondrial aconitase (mtAco) participates in the tricarboxylic acid (TCA) cycle where it converts citric acid into isocitric acid via the reaction intermediate cis-aconitate. Coordination of the enzyme’s [4Fe- 4S] cofactor is achieved by three cysteine residues of the polypeptide chain and the substrate (or product) acts as a fourth cluster ligand, rendering aconitase activity strictly dependent on the ISC system. Cytosolic aconitase (cytAco) activity is exerted by holo-IRP1 which in its apoform can bind to iron-responsive elements (IREs), i.e. stem-loop structures of mRNAs encoding proteins involved in iron trafficking (e.g., transferrin receptor TFR1) or storage (e.g., ferritin), thereby regulating mRNA stability or translation efficiency. The equilibrium between apo- and holo-IRP1 is dependent on the efficiency of Fe/S cluster assembly and thus on the activity of both the mitochondrial ISC system and the CIA machinery. The consequences of Fe/S cluster assembly defects on Fe/S enzyme activities can be tested in mitochondrial or cytosolic HeLa cell fractions using multi-well plate-based spectrophotometric assays (15).

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In order to discriminate whether impaired Fe/S enzyme activity is caused by Fe/S cluster assembly deficiency or due to general enzymatic defects, the examination of meaningful reference enzymes is necessary (15). Respiratory chain complex IV (cytochrome-c oxidase, COX) does not contain Fe/S cluster cofactors but two heme and two copper centers in its functional core. Due to the presence of these metal cofactors, the enzyme is often used as internal reference for the analysis of mitochondrial Fe/S proteins. However, COX activity is known to be indirectly affected by ISC deficiency, and hence must not be used as a reference. Particularly in MMDS a yet unknown mechanism causes a substantial drop in COX activity (8), making complex IV an useful indicator of ISC deficiency rather than a reference enzyme. Instead, citrate synthase (CS) is a useful alternative to monitor mitochondrial function, as no changes are observed by any tissue culture manipulation. CS participates in the TCA cycle and is thus functionally linked to both the respiratory chain and mitochondrial aconitase. The enzyme functions as a soluble homodimer within the mitochondrial matrix and does not depend on a cofactor to catalyze the condensation of acetyl-coenzyme A (acetyl-CoA) with oxaloacetate to form citrate. The cytosolic enzyme lactate dehydrogenase (LDH) may reliably by used as a reference enzyme for the cytosol. LDH is a soluble tetramer without any cofactor and catalyzes the conversion of pyruvate to lactate to regenerate NAD+ from NADH. Despite its functional relationship to mitochondrial respiration, no effects of an ISC deficiency on LDH activity have been observed yet. According to Lambert-Beer’s law the enzymatic activity of a given enzyme can be determined by measuring the time-dependent change in optical density of a reaction-related dye:

E = ε × c × d with: E extinction, <=> ∆E/∆t = ε × ∆c/∆t × d ε extinction coefficient, <=> ∆c/∆t = (∆E/∆t) / (ε × d) c dye concentration, d length of light path, ∆x/∆t change per time interval

The length of the light path d in a multiplate well can be calculated from the well area AW and total well volume VW (d = VW / AW). The change in dye concentration per time interval is a measure of enzymatic activity, expressed as (µmol / min) / mL or units / mL (U / mL):

[∆c/∆t] = mM / min = U / mL

Taking into account the total solution volume in a given well and the corresponding total protein amount therein both the total enzyme activity per well and the specific enzyme activity per protein amount can be calculated. The specific activity is independent of protein amount and sample volume and given as common denominator of enzymatic activity in form of U / mg.

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General equipment • Microplate reader with kinetic mode (preferentially monochromator-based) settings: - 96-well, flat bottom, transparent - Temperature at 32.5°C - Orbital shaking for 5 sec at 40 rpm • Normal and UV-transmissible 96-well plates, well area 0.36 cm2 • Pipette dispenser • Ice bucket • Cooled support plate

Determination of isolated SDH activity The spectrophotometric determination of isolated SDH activity is based on the oxidation of succinate to fumarate with concomitant reduction of the blue dye 2,6-dichloro-N-(4-hy- droxyphenyl)-1,4-benzoquinoneimine (DCPIP), rendering it colorless, via decylubiquinone as the electron carrier (20). Since DBQ may transfer electrons derived from other sources than SDH activity, non-specific DCPIP reduction can be determined by specific inhibition of SDH using malonate. SDH activity is determined by calculating the difference between total and non-specific DCPIP bleaching.

Succinate Decylubiquinone(ox) DCPIP (colorless) Malonate | SDH

Fumarate Decylubiquinone(red) DCPIP (blue)

Fig. 6: Reduction of DCPIP by SDH. Electrons liberated by oxidation of succinate to fumarate are transferred to DCPIP. The change in color can be spectrophotometrically monitored.

Reagents • Mitobuffer (ice-cold) • SDH basal buffer pH 7.4 (room temperature): 50 mM Tris/SO4, 0.1 mM EDTA, 70 µM DCPIP, 0.1 % Triton X-100 • 5 mM Decylubiquinone (DBQ) in ethanol • 20 % Succinate solution • 20 % Malonate solution Assay • Adjust crude HeLa cell mitochondrial fractions to a concentration of about 0.5 µg/µL with ice-cold MitoBuffer. • Load equal amounts corresponding to 3 to 9 µg protein in each of two wells of a cooled 96-well plate as below (Fig. 7). One well serves the determination of total DCPIP reduction (using SDH reaction buffer), the second aids the measurement of SDH-independent DCPIP reduction (by SDH reference buffer).

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1 2 3 4 5 6 7 8 9 10 11 12 A Sample#1 (3, 6, 9 µg) Sample#1 (3, 6, 9 µg) Spl#1 (0.3, 0.6, 0.9 µg) Spl#1 (0.3, 0.6, 0.9 µg) SDH reaction SDH reference CS reaction CS reference B Sample#2 (3, 6, 9 µg) Sample#2 (3, 6, 9 µg) Spl#2 (0.3, 0.6, 0.9 µg) Spl#2 (0.3, 0.6, 0.9 µg) SDH reaction SDH reference CS reaction CS reference C Sample#3 (3, 6, 9 µg) Sample#3 (3, 6, 9 µg) Spl#3 (0.3, 0.6, 0.9 µg) Spl#3 (0.3, 0.6, 0.9 µg) SDH reaction SDH reference CS reaction CS reference D Sample#4 (3, 6, 9 µg) Sample#4 (3, 6, 9 µg) Spl#4 (0.3, 0.6, 0.9 µg) Spl#4 (0.3, 0.6, 0.9 µg) SDH reaction SDH reference CS reaction CS reference E Sample#5 (3, 6, 9 µg) Sample#5 (3, 6, 9 µg) Spl#5 (0.3, 0.6, 0.9 µg) Spl#5 (0.3, 0.6, 0.9 µg) SDH reaction SDH reference CS reaction CS reference F Sample#1 (2, 3, 4 µg) Sample#3 (2, 3, 4 µg) Sample#6 (2, 3, 4 µg) COX reaction COX reaction COX reaction G Sample#2 (2, 3, 4 µg) Sample#4 (2, 3, 4 µg) COX Blank COX reaction COX reaction H

Fig. 7: 96-well plate loading scheme for the measurement of SDH, COX, and CS activities. Samples for SDH and CS activity measurements are loaded in duplicates, samples for COX activity determination require a blank control only.

• At room temperature prepare reaction and reference buffer from the SDH basal buffer according to the following chart:

Reagent SDH reaction buffer SDH reference buffer DBQ 10 µl / mL 10 µl / mL Succinate 10 µl / mL 10 µl / mL Malonate --- 10 µl / mL

Kinetics for 30min, λ = 600 nm, ε = 21 mM-1 x cm-1

• Remove the well-plate from the cooled support. • Dispense 225 µl of SDH reaction or reference buffer into the corresponding sample wells. • Place well-plate into the reader device and start measurement

After equilibration the extinction will change over time in an almost linear fashion. The SDH- dependent change in the dye concentration can be derived from the difference of total minus non-specific change in optical density.

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Determination of COX activity COX activity can easily be determined in crude mitochondrial fractions by examining the enzyme-dependent oxidation of cytochrome-c (cyt-c), resulting in a color change from reddish to brownish (21, 22). The assay is highly specific but requires the assessment of cyt-c oxidation by atmospheric oxygen in the absence of the enzyme. COX activity is calculated by from the difference between total and non-specific cyt-c oxidation.

+ Cyt-cred O2 + 4 H COX

Cytcox 2 H2O

Fig. 8: COX-mediated oxidation of cyt-c. COX uses cyt-c to provide electrons for the formation of water from molecular oxygen and protons. The oxidation state of cyt-c can be spectrophotometrically determined.

Reagents • Mitobuffer (ice-cold) • COX basal buffer pH 6.6 (room temperature): 50 mM 2-(N-morpholino)ethanesulfonic acid (MES), 50 mM NaCl, 1 % BSA, 0.5 mM (~ 0.025 %) dodecylmaltoside (DDM) • Cytochrome-c (reduced) 400 µM [~ 60 mg/ml]; preparation see below

Assay • Adjust crude HeLa cell mitochondrial fractions to a concentration of about 0.5 µg/µL with ice-cold MitoBuffer. • Load 2 to 4 µg protein into wells of a cooled 96-well plate as in Fig. 7. • At room temperature prepare COX reaction buffer by adding 15 µL reduced cyt-c per mL COX basal buffer. • Remove the well-plate from the cooled support. • Dispense 225 µl of COX reaction buffer into sample wells as well as into three blank wells (serving the determination of non-specific cyt-c oxidation by atmospheric oxygen). • Place well-plate into a reader device and start a kinetics measurement for 30 min at λ = 550 nm

After equilibration the extinction will change over time in an almost linear fashion. The COX- dependent change in cyt-c oxidation can be derived from the difference of total minus non- specific change in optical density (ε = 19.1 mM-1 x cm-1).

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Arrangement: Reduction of cyt-c Cytochrome-c is commercially available, but only in its oxidized state. Before it can be used as COX substrate, the protein has to be reduced by dithionite (15). If long-term storage is desired, purification of reduced cyt-c has to be in an anaerobic environment to prevent re- oxidation by atmospheric oxygen.

Equipment • PD10 desalting column • Anaerobic chamber (optional)

Reagents • Cytochrome-c (lyophilized) • 10 mM phosphate buffer (pH 7.2) • Sodium dithionite (Na2[S2O4])

Preparation of reduced cyt-c • Dissolve oxidized cyt-c in 2.25 ml phosphate buffer at a concentration of about 100 mg/mL. • Add dithionite (1 M in 10 mM phosphate buffer) to a final concentration of 25 mM. The color of the solution will change from dark brownish to intensive red. • Incubate for ~ 30 min at RT • Adjust volume to 2.5 mL with phosphate buffer.

If desired, continue desalting in an anaerobic chamber:

• Equilibrate a PD10 column with de-oxygenated phosphate buffer. • Load the cyt-c solution onto the column and elute with 3.5 mL phosphate buffer. • Collect the cyt-c band and store snap-frozen 100 µL-aliquots anaerobically at -80°C.

Note: Aliquots prepared or opened aerobically are not suitable for long-term storage.

Determination of CS activity CS activity can easily be measured in crude mitochondrial HeLa fractions. The method builds on the hydrolytic cleavage of 5,5 O-dithiobis[2-nitrobenzoic acid] (DTNB, Ellman’s reagent) to form 5-thio-2-nitrobenzoate (TNB) by free sulfhydryl groups of CoA that is liberated on the condensation of acetyl-CoA with oxaloacetate (23).

2 Oxaloacetate 2 Acetyl-CoA 2 TNB-CoA (yellow) CS 2 Citrate 2 Coenzyme-A DTNB (colorless)

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Fig. 9: Cleavage of DTNB subsequent to CS-dependent CoA release. Coenzyme A derived from acetyl CoA contains a free sulfhydryl group that reacts with DTNB to form TNB-CoA. The change in color can be spectrophotometrically monitored.

Reagents • Mitobuffer (ice-cold) • 500 mM DTNB in DMSO (stock solution, store at -20°C) • CS basal buffer pH 8.0 (room temperature): 50 mM TRIS/HCl, 100 mM NaCl, 0.5 mM DTNB, 0.1% Triton X-100 • Acetyl-CoA 10 mg/mL • Oxaloacetate 10 mg/mL Assay • Adjust crude HeLa cell mitochondrial fractions to a concentration of about 50 ng/µL (i.e. 0.05 µg/µL) with ice-cold MitoBuffer. • Load 0.3 to 0.9 µg protein into wells of a cooled 96-well plate as in Fig. 7. • At room temperature prepare reaction and reference buffer from the CS basal buffer according to the following chart:

Reagent CS reaction buffer CS reference buffer Acetyl-CoA 10 µl / mL 10 µl / mL Oxaloacetate 20 µl / mL ---

Kinetics for 30min, λ = 412 nm, ε = 13.3 mM-1 x cm-1

• Remove the well-plate from the cooled support. • Dispense 225 µl of CS reaction or reference buffer into the corresponding sample wells. • Place well-plate into the reader device and start measurement

After equilibration the extinction will change over time in an almost linear fashion. DTNB hydrolysis may proceed for more than 30 min, depending on the protein amount applied. Take care to not load too much protein to avoid depletion of the substrates. The CS-dependent change in the dye concentration can be derived from the difference of total minus non- specific change in optical density.

Determination of aconitase activities Both mtAco and cytAco activity can be measured by the same assay which connects aconitase-mediated conversion of cis-aconitate to isocitrate with the subsequent activity of isocitrate dehydrogenase (IDH) (19). IDH uses the aconitase product isocitrate to catalyze the formation of α-ketoglutarate by subsequent reduction of NADP+ to NADPH, a reaction that can be spectrophotometrically followed.

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Aconitase IDH cis-Aconitate Isocitrate NADP+

α-Ketoglutarate NADPH

Fig. 10: Aconitase-dependent formation of isocitrate and subsequent reduction of NADP+. Aconitase activity is coupled to the isocitrate dehydrogenase (IDH) –dependent reduction of NADP+. The formation of NADPH can be spectrophotometrically monitored.

Equipment • UV-transmissible 96-well plate

Reagents • Mitobuffer (ice-cold) • Aco basal buffer pH 8.0 (room temperature): 100 mM Triethanolamine, 1.5 mM MgCl2, 0.1 % Triton X-100 • IDH (NADP+-dependent) 40 mU/µl (store as aliquots in 100 mM Triethanolamine / 10 % glycerol at -80°C) • NADP+ 100 mM • cis-Aconitate 20 mM

Assay • Adjust crude HeLa cell mitochondrial fractions to a concentration of about 0.5 µg/µL with ice-cold MitoBuffer. Do not dilute cytosolic fractions. • For determination of mtAco activity load equal amounts of crude mitochondrial fractions corresponding to 3 to 9 µg protein in each of two wells of a cooled 96-well plate (Fig. 11). • For determination of cytAco activity load 15, 20, and 25 µL of cytosolic fractions in each of two wells of a cooled 96-well plate as indicated (Fig. 11). • One of the two wells serves the determination of total NADP+ reduction (using Aco reaction buffer), the second well aids the measurement of aconitase-independent NADP+ reduction (by Aco reference buffer).

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1 2 3 4 5 6 7 8 9 10 11 12 A Sample#1 (3, 6, 9 µg) Sample#1 (3, 6, 9 µg) Spl#1 (15, 20, 25 µL) Spl#1 (15, 20, 25 µL) mtAco reaction mtAco reference cytAco reaction cytAco reference B Sample#2 (3, 6, 9 µg) Sample#2 (3, 6, 9 µg) Spl#2 (15, 20, 25 µL) Spl#2 (15, 20, 25 µL) mtAco reaction mtAco reference cytAco reaction cytAco reference C Sample#3 (3, 6, 9 µg) Sample#3 (3, 6, 9 µg) Spl#3 (15, 20, 25 µL) Spl#3 (15, 20, 25 µL) mtAco reaction mtAco reference cytAco reaction cytAco reference D Sample#4 (3, 6, 9 µg) Sample#4 (3, 6, 9 µg) Spl#4 (15, 20, 25 µL) Spl#4 (15, 20, 25 µL) mtAco reaction mtAco reference cytAco reaction cytAco reference E Sample#5 (3, 6, 9 µg) Sample#5 (3, 6, 9 µg) Spl#5 (15, 20, 25 µL) Spl#5 (15, 20, 25 µL) mtAco reaction mtAco reference cytAco reaction cytAco reference F Spl#1 (0.1, 0.2, 0.3 µg) Spl#3 (0.1, 0.2, 0.3 µg) Spl#5 (0.1, 0.2, 0.3 µg) LDH reaction LDH reaction LDH reaction G Spl#2 (0.1, 0.2, 0.3 µg) Spl#4 (0.1, 0.2, 0.3 µg) LDH Blank LDH reaction LDH reaction H

Fig. 11: 96-well plate loading scheme for the measurement of aconitase and LDH activities. Samples (Spl) for mtAco and cytAco activity measurements are loaded in duplicates, samples for LDH activity determination require a blank control only.

• At room temperature prepare reaction and reference buffer from the Aco basal buffer according to the following chart:

Reagent Aco reaction buffer Aco reference buffer NADP+ 12.5 µl / ml 12.5 µl / ml cis-Aconitate 15 µl / ml -- IDH 5 µl / ml --

Kinetics for 60 min, λ = 340 nm, ε = 6.22 mM-1 x cm-1

• Remove the well-plate from the cooled support. • Dispense 225 µl of Aco reaction or reference buffer into the corresponding sample wells. • Place well-plate into the reader device and start measurement

After equilibration the extinction will change over time in an almost linear fashion. The aconitase-dependent increase in NADPH can be derived from the difference of total minus non- specific change in optical density.

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Determination of LDH activity LDH activity can easily be determined in cytosolic HeLa fractions by the pyruvate-dependent oxidation of NADH (24). The assay is highly specific but involves the assessment of non- specific NADH oxidation in the absence of the enzyme. LDH activity is calculated by from the difference between total and non-specific NADH oxidation.

Pyruvate NADH LDH Lactate NAD+

Fig. 12: LDH-mediated oxidation of NADH. LDH uses pyruvate for the regeneration of NAD+ from NADH. The concentration of NADH can be spectrophotometrically determined.

Reagents • Mitobuffer (ice-cold) • LDH basal buffer pH 7.4 (room temperature): 50 mM Tris/HCl, 1 mM EDTA, 0.1 % Triton X-100 • 50 mM Na-Pyruvate • 10 mg/mL NADH

Assay • Adjust cytosolic HeLa fractions to a concentration of about 15 ng/µL (i.e. 0.015 µg/µL) with ice-cold MitoBuffer. • Load 0.1 to 0.3 µg protein into wells of a cooled 96-well plate as in Fig. 11. • At room temperature prepare LDH reaction buffer by adding 15 µL of each pyruvate and NADH solution per mL LDH basal buffer. • Remove the well-plate from the cooled support. • Dispense 225 µl of LDH reaction buffer into sample wells as well as into three blank wells (serving the determination of non-specific NADH reduction). • Place well-plate into a reader device and start a kinetics measurement for 30 min at λ = 340 nm

After equilibration the extinction will change over time in an almost linear fashion. The LDH- dependent change in NADH oxidation can be derived from the difference of total minus non- specific change in optical density (ε = 6.22 mM-1 x cm-1).

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DAY 2 (Part II) - STATION 3. Analysis of lipoyl synthase (LIAS) activity and Fe/S protein stability by immunoblotting Oliver Stehling

Introduction The enzymatic function of mitochondrial lipoyl synthase (LIAS) can be easily, yet indirectly estimated by immunoblotting (18). LIAS belongs to the radical S-adenosyl methionine (SAM) enzyme family and catalyzes the conversion of peptide-bound octanoate into the lipoate (LA) cofactor which is an essential part of the mitochondrial enzymes PDH, α−KGDH, α-OADH, BCKDH, and GCS-H. LIAS coordinates two [4Fe-4S] clusters to conduct LA formation: The SAM- binding cluster catalyzes the formation of a 5’-deoxyadenosyl radical, whereas a so-called auxiliary cluster is decomposed in order to provide the sulfur atoms for the modification of the octanoate precursor to LA (25). Due to the critical relevance of each of the two metalloclusters for LIAS function, any disturbance of the ISC system is decreasing the amount of LA attached to the respective substrate proteins. LA-specific antibodies allow the identification of the relative amount of LA attached to the E2 subunits of PDH (i. e., dihydrolipoamide S-acetyltransferase, DLAT) and α-KGDH (i. e., dihydrolipoamide S-succinyltrans- ferase, DLST). Staining of the other LA-dependent enzymes is possible but due to their low abundance less straightforward. Immunodetection of the corresponding E2 subunit polypeptide chains serves to estimate the degree of substrate lipoylation.

Fe/S clusters not only take part in protein function but also confer structural stabilization of the polypeptide backbone by connecting their cognate amino acid ligands. Disturbances in any Fe/S cluster assembly pathway, either experimentally or in Fe/S disease states, may interfere with correct cluster ligation in a given Fe/S protein, and, as a consequence, may impact on the protein’s structural integrity. As a consequence, the improperly matured protein may become instable and subsequently be degraded. Using common immunoblotting techniques thus permit a rough estimation, if and at which step Fe/S protein assembly might be compromised (18). Since the core ISC assembly machinery is required for the assembly of both mitochondrial and extra-mitochondrial Fe/S proteins, impaired function of one (or more) ISC core components will affect virtually all cellular Fe/S proteins, irrespective of cofactor type (i.e. [2Fe-2S] or [4Fe-4S]) and subcellular localization. In contrast, late-acting ISC targeting factors function downstream of the ISC core system and hence do not impact mitochondrial [2Fe-2S] proteins or cytosolic-nuclear Fe/S proteins.

SDS-PAGE and immunoblotting of tissue culture samples Equipment • Electrophoresis chamber (e. g. Bio-Rad Mini Cell Systems for minigels) • Western blotting semidry transfer system (e. g. Bio-Rad Trans-Blot Turbo Transfer System) • Power supply • Polyacrylamide minigel (e. g. Bio-Rad 4–15% Mini-PROTEAN TGX Precast Gel) • Gel-loading pipet tips • Nitrocellulose Transfer Pack (e. g. Bio-Rad 1704158): contains support sheets (i. e. filter paper) and nitrocellulose membrane (pore size 0.2 µm) • Rocking machine

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Reagents • Electrophoresis buffer: 25 mM Tris, 190 mM glycine, 0.1% SDS; do not adjust pH • Blotting buffer: 20 mM Tris, 150 mM Glycine, 0.1% SDS, 20% (v/v) ethanol; do not adjust pH • 1x Laemmli sample buffer (see above) • Molecular weight standard in Laemmli buffer (Marker) • Tissue culture protein samples in Laemmli buffer (1 µg / µL; preparation see above) • Ponceau solution (0.2 % Ponceau S in 3 % trichloroacetic acid, supplemented with 3 % sulfosalicylic acid)

Running an SDS-PAGE • Label sample slots of the polyacrylamide minigel. • Assemble electrophoresis chamber including the minigel according to the manufacturer’s instructions. • Remove comb of the gel, inspect condition of the sample slots, and add electrophoresis buffer. • Use gel-loading tips to apply 15 µg (i. e. 15 µL) of total tissue culture lysate as well as a suitable amount of molecular weight standard into the sample slots according to Fig. 13. Load 1x Laemmli buffer into empty slots. • Perform SDS-PAGE at 15 mA until samples have entered the separating gel and increase current to 30 mA until the running front has reached the bottom of the gel (45 to 60 min).

Slot 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Samples Samples

#1 #2 #3 #4 #5 #1 #2 #3 #4 #5 Marker Laemmli Laemmli Laemmli Laemmli

Fig. 13: Sample loading scheme for SDS-PAGE. Samples are applied in duplicates in order to allow for analysis of multiple antigens. Laemmli buffer applied to empty wells ensures proper migration behavior during gel run.

Immunoblotting on nitrocellulose • Remove minigel from the electrophoresis chamber and disassemble cassette. • Assemble the transfer stack according to the manufacturer’s instructions. • Place transfer stack into the Bio-Rad Trans-Blot Turbo Transfer System and start protein transfer. • After transfer, wash nitrocellulose at least for 10 min with water; shake gently. • Discard water and visualize protein bands with Ponceau S solution; rinse membrane with water to remove excess dye. • If desired label membrane with a pen.

The nitrocellulose membrane can be immediately used or stored upon drying.

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Immunodetection of blotted antigens on nitrocellulose membranes Equipment • Plastic support plate, scalpel, ruler • Incubation containers for immunostaining • Rocking machine • Chemiluminescence detection system (e. g. Bio-Rad ChemiDoc XRS+)

Reagents • Tris-buffered saline with 0.1 % Tween-20 (TBS-T) • Blocking buffer: 1 % BSA, 1x Rotiblock in TBS-T (add 0.1 % Azide for long-term storage at 4°C) • Primary antibody buffer: 0.5 % BSA, 0.3x Rotiblock in TBS-T (add 0.1 % Azide for long-term storage at 4°C) • Secondary antibody buffer: 0.5 % BSA, in TBS-T (add 0.2 % thimerosal for long-term storage at 4°C) • Primary and horseradish peroxidase (HRP) -conjugated secondary antibodies • Solutions for enhanced chemiluminescence (ECL) –detection of HRP

Antigen detection • Wash membrane successively each with water and TBS-T for 10 min in order to remove the Ponceau dye. • Block non-specific protein binding sites on the nitrocellulose membrane by shaking in blocking buffer for at least 1 h. • Wash membrane in TBS-T for 5 min. • In order to simultaneously detect multiple antigens of different molecular weight, use plastic support plate, scalpel, and ruler to cut the nitrocellulose membrane into corresponding strips. • Prepare the desired antibody dilutions in primary antibody buffer and incubate the respective nitrocellulose strips for at least 1 h under gentle shaking. • Wash the strips 3x for 5 min in TBS-T. • Prepare dilutions of the HRP-conjugated secondary antibodies in the corresponding buffer and incubate nitrocellulose strips for up to 1 h under gentle shaking. (Note: exclude azide in this step as it inactivates HRP) • Wash membrane strips 3x for 5 min in TBS-T. • Re-assemble membrane strips and perform HRP-detection by ECL.

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DAY 2 (Part II) - STATION 4 (optional). Phenotypic characterization of cells with defects in mitochondrial ISC assembly Oliver Stehling

Visual inspection of ISC assembly-deficient HeLa tissue culture cells Mitochondria show a unique morphology caused by their rather plain outer and their highly invaginated inner membrane. Presence of these cristae invaginations has been found to be dependent on respiratory chain supercomplex formation. Particularly, mitochondria deficient in respiratory chain complex assembly and mitochondria with compromised Fe/S protein biogenesis activity frequently exhibit profound changes in mitochondrial structure (16, 24, 26). In case of ISC targeting factor deficiency, mitochondria undergo a profound swelling accompanied by loss of their cristae membranes (17). A similar phenotype is elicited by chloramphenicol which blocks mitochondrial protein synthesis, and hence abrogates assembly of respiratory chain complexes (27-29). Mitochondria become visible even by inverted phase contrast microscopy and confer a sponge-like appearance to the cells (17).

Equipment • Inverted microscope (e. g. EVOS Cell Imaging System)

Inspection of cultured cells HeLa cells have been depleted for ISC assembly factors by three rounds of electroporation as described above, and grown for another three days. They may be grown on glass slides, washed with PBS, fixed (4 % paraformaldehyde in PBS) for 20 min, washed with distilled water, and embedded in Mowiol, or directly inspected in viable cultures.

Determination of lactate formation in tissue culture supernatants Impaired mitochondrial Fe/S protein biogenesis not only interferes with respiratory chain function but also slows down the decay of pyruvate by affecting PDH and α-KGDH function via hampered LIAS-mediated lipoylation as well as inactivation of the TCA cycle enzymes aconitase and SDH. In order to satisfy the energetic needs of the cell and to regenerate NAD+ from NADH, energy metabolism is shifted towards aerobic glycolysis, leading to the break- down of glucose to lactic acid and consequently to a profound acidification of the culture medium. The acidification is well detectable by a color change of the pH indicator phenol red in the medium (17). Lactate levels in the respective tissue culture supernatants (TCS) can be determined by a multi-well-based colorimetric approach.

The assay is based on the reduction of NAD+ due to the action of lactate dehydrogenase which reversibly converts lactate to pyruvate. To shift the equilibrium of the reaction towards the conversion of lactate, diphorase is added to re-oxidize NADH under concomitant reduction of DCPIP which becomes colorless (30). Since the oxidation state of DCPIP is determined at 600 nm, the phenol red content of the medium does not interfere with the measurement. In order to monitor direct TCS effects on DCPIP reduction non-specific DCPIP reduction is determined in reference samples. The absolute amount of lactate in each tissue culture sample can be deduced from the absorbance changes occurring in the lactate reference samples.

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Equipment • Microplate reader with kinetic mode (preferentially monochromator-based) settings: - 96-well, flat bottom, transparent - Temperature at 32.5°C - Orbital shaking for 5 sec at 40 rpm • Normal 96-well plate, well area 0.36 cm2 • Pipette dispenser Reagents • Lactate basal buffer pH 7.4 (room temperature): 70 µM DCPIP in Hanks Balanced Salt Solution (HBSS) • 100 mM NAD+ • Diaphorase 40 U/mL • Lactate Dehydrogenase 1 kU/mL • 20 mM lactate standard solution

Assay • If large amounts of lactate are expected dilute TCS with water. • Load equal TCS volumes of 5 to 20 µL in each of two wells of a 96-well plate as below (Fig. 14). One well serves the determination of total DCPIP reduction (using lactate reaction buffer), the second aids the measurement of lactate-independent DCPIP reduction (by lactate reference buffer). • Also load equal volumes of fresh culture medium ranging from 5 to 20 µL in each of two wells of the 96-well plate (serves the determination of lactate already present in the culture medium) • Apply lactate standard solution (125 µM to 4 mM) into respective wells.

1 2 3 4 5 6 7 8 9 10 11 12 A lactate standard solution (reaction buffer) (0.0, 0.125, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0 mM) B Fresh culture medium Fresh culture medium 5, 10, 15, 20 µl (reaction) 5, 10, 15, 20 µl (reference) C TCS Sample#1 TCS Sample#1 5, 10, 15, 20 µl (reaction) 5, 10, 15, 20 µl (reference) D TCS Sample#2 TCS Sample#2 5, 10, 15, 20 µl (reaction) 5, 10, 15, 20 µl (reference) E TCS Sample#3 TCS Sample#3 5, 10, 15, 20 µl (reaction) 5, 10, 15, 20 µl (reference) F TCS Sample#4 TCS Sample#4 5, 10, 15, 20 µl (reaction) 5, 10, 15, 20 µl (reference) G TCS Sample#5 TCS Sample#5 5, 10, 15, 20 µl (reaction) 5, 10, 15, 20 µl (reference) H

Fig. 14: 96-well plate loading scheme for lactate determination. Samples are loaded in duplicates. Determination of the standard values also includes a blank measurement. • At room temperature prepare reaction and reference buffer from the lactate basal buffer according to the following chart:

Techniques for studying iron in health and disease EMBL

Reagent SDH reaction buffer SDH reference buffer NAD+ 7,5 µl / ml 7,5 µl / ml Diaphorase 2,5 µl / ml -- LDH 2,0 µl / ml --

Kinetics for 90 min, λ = 600 nm • Dispense 225 µl of lactate reaction or reference buffer into the corresponding wells. • Place well-plate into the reader device and start measurement.

The extinction will change over time and eventually reach a plateau when the entire lactate is used up. Calculate the lactate-dependent change in DCPIP reduction from the difference of total minus non-specific change in optical density. Concentrations of lactate in media and TCS can be derived from the standard solution. In order to estimate the lactate-producing activity of cells, subtract the media lactate content from the TCS content, and relate the difference to the amount of cells or total protein of the original tissue culture.

Determination of TFR expression by fluorescently labeled transferrin The core of the mitochondrial ISC assembly system plays a critical role in cellular iron homeostasis (31), but only in part due to its role in the maturation of IRP1. Defects in mitochondrial [2Fe-2S] cluster assembly also activate mitochondrial iron uptake by a still ill- defined mechanism (8), eliciting cytosolic iron deficiency and upregulation of IRP2. In turn, both apo-IRP1 and IRP2 bind and stabilize TFR mRNA, resulting in elevated TFR levels in order to overcome the cytosolic iron shortage by increasing the cellular uptake of transferrin-bound iron by receptor mediated endocytosis. Accordingly, hallmarks of diseases related to core ISC assembly defects are mitochondrial iron accumulation as well as elevated cellular and systemic iron uptake. In contrast, deficiencies of ISC targeting factors only mildly (and possibly indirectly) affect cellular and systemic iron homeostasis, most likely since mitochondrial [4Fe- 4S] cluster assembly is not related to cytosolic Fe/S protein formation. Assessing cellular TFR expression can thus help to discriminate between [2Fe-2S] and [4Fe-4S] cluster assembly defects. In viable cells, relative TFR levels can be assessed by a fluorescence-based assay using FITC-labeled holo-transferrin (32).

Equipment • Microplate reader with absorption and fluorescence reading options • Pipette dispenser

Techniques for studying iron in health and disease EMBL

Reagents • HBSS • 10 mM deferoxamine (DFO) • 10 mM ferric iron citrate (FAC) • 10 mM sodium ascorbate (Asc) • FITC-labeled transferrin (Tf-FITC) 10 mg/mL • 0.1 % Triton X-100 in water • BSA standard solution (2 mg / mL) • BCA protein assay reagents (bicinchoninic acid and Cu2+ solutions)

Tissue culture arrangement Seeding of ISC deficient cells into a 48-well plate • Treat cells by a method of choice (e. g. transfection with siRNA as described above) and grow aliquots in a 48-well plate for three days according to Fig. 15.

1 2 3 4 5 6 7 8 A Sample#1: 40 x 106 to 120 x 106 cells B Wells for BSA standard: DFO-Sample#1: FAC-Sample#1: 0 (Blank) to 180 µg 60 to 120 x 106 cells 60 to 120 x 106 cells C Wells for Sample#2: DFO-Sample#2: FAC-Sample#2: BSA 60 to 120 x 106 cells 60 to 120 x 106 cells 60 to 120 x 106 cells D standard Sample#3: DFO-Sample#3: FAC-Sample#4: 60 to 120 x 106 cells 60 to 120 x 106 cells 60 to 120 x 106 cells E Sample#4: DFO-Sample#4: FAC-Sample#4: 60 to 120 x 106 cells 60 to 120 x 106 cells 60 to 120 x 106 cells F Sample#5: DFO-Sample#5: FAC-Sample#5: 60 to 120 x 106 cells 60 to 120 x 106 cells 60 to 120 x 106 cells

Fig. 15: 48-well plate loading scheme for the analysis of Tf-FITC binding. The cell number of sample#1 is titrated from low to high counts and serves to analyze the correlation between cell number (i. e. total protein per well) and Tf-FITC-binding. In order to determine maximal and minimal TFR expression, DFO (blue wells) and Asc-FAC (red wells) are added the day before analysis.

Tf-FITC labeling of viable cells • At room temperature dilute Tf-FITC in 14 mL HBSS to a final concentration of 110 ng/mL; protect from light. • Withdraw TCS from all wells of the plate. • Wash wells with 400 µL HBSS; remove buffer entirely. • Dispense 300 µL Tf-FITC staining solution into the wells and incubate for 30 min under standard culture conditions. • Withdraw Tf-FITC staining solution from all wells of the plate and wash once with HBSS. • Dispense 500 µL HBSS into the wells and measure fluorescence. • Withdraw HBSS from all wells of the plate. • Add 0.1 % Triton X-100 solution and incubate for 15 min to lyse cells.

Techniques for studying iron in health and disease EMBL

• Apply BSA protein standard to respective wells (Fig. 15). • Prepare 25 mL BCA protein staining solution from stocks and dispense 0.5 mL into each well; mix thoroughly. • Incubate at 37°C and determine OD at 562 nm for a total of 3x at a 20 min interval.

Relative Tf binding to cells can be expressed as the ratio of FITC-fluorescence by total protein content of a well. Titration of reference cells allows to take density-dependent effects on TF- FITC binding into account.

Arrangement Preparation of FITC-labeled Tf Fluorochrome-labeled Tf is commercially available but pricey. Covalent coupling of fluorescein isothiocyanate (FITC) is an cost-efficient alternative.

Equipment • Rotating mixer • PD10 desalting column Reagents • Human holo-Tf (lyophilized) • 20 mg/ml FITC in Ethanol • 850 mM (25%) Na-Citrate solution • 100 mM FeCl2 in 5 M HCl • 600 mM NaHCO3 (coupling buffer) • HBSS • BSA solution (20%)

Coupling FITC to Tf and subsequent charging with Fe3+ Perform coupling/charging at room temperature.

• Add 25 µl FeCl2 solution to 275 µl Na-Citrate and incubate 3 – 6 h. • Dissolve 100 mg (Holo-) Tf in 2 mL coupling buffer. • Add 4 mg FITC dissolved in 200 µl EtOH and incubate for 1 h under mild rotation. • Add 300 µl Fe-Citrate and incubate for 1 h under mild rotation. • Equilibrate a PD-10 column with 40 ml of HBSS. • Load coupling mixture onto the PD-10 column and elute holo Tf-FITC with 3,5 mL HBSS. • Add 500 µl of a 20 % BSA solution and dilute to 10 mL with HBSS. • Filter sterile using a 0,22 µm syringe filter and store at 4°C.

Techniques for studying iron in health and disease EMBL

References 1. Balk, J., et al. (2004) The cell's cookbook for iron--sulfur clusters: recipes for fool's gold?, Chembiochem 5, 1044-1049. 2. Lill, R., et al. (2015) Special issue on iron-sulfur proteins: Structure, function, biogenesis and diseases, Biochim Biophys Acta 1853, 1251-1252. 3. Braymer, J. J., et al. (2017) Iron-sulfur cluster biogenesis and trafficking in mitochondria, J Biol Chem 292, 12754-12763. 4. Lill, R., et al. (2015) The role of mitochondria and the CIA machinery in the maturation of cytosolic and nuclear iron-sulfur proteins, Eur J Cell Biol 94, 280-291. 5. Lill, R. (2009) Function and biogenesis of iron-sulphur proteins, Nature 460, 831-838. 6. Stehling, O., et al. (2013) Human CIA2A-FAM96A and CIA2B-FAM96B integrate iron homeostasis and maturation of different subsets of cytosolic-nuclear iron-sulfur proteins, Cell Metab 18, 187-198. 7. Stehling, O., et al. (2012) MMS19 assembles iron-sulfur proteins required for DNA metabolism and genomic integrity, Science 337, 195-199. 8. Stehling, O., et al. (2014) Mitochondrial iron-sulfur protein biogenesis and human disease, Biochimie 100, 61-77. 9. Webert, H., et al. (2014) Functional reconstitution of mitochondrial Fe/S cluster synthesis on Isu1 reveals the involvement of ferredoxin, Nat Commun 5, 5013. 10. Gourdoupis, S., et al. (2018) IBA57 Recruits ISCA2 to Form a [2Fe-2S] Cluster-Mediated Complex, J Am Chem Soc 140, 14401-14412. 11. Freibert, S. A., et al. (2018) Biochemical Reconstitution and Spectroscopic Analysis of Iron-Sulfur Proteins, Methods Enzymol 599, 197-226. 12. Smith, F. E., et al. (1984) Serum iron determination using ferene triazine, Clin Biochem 17, 306-310. 13. Rabinowitz, J. C. (1978) Analysis of acid-labile sulfide and sulfhydryl groups, Methods Enzymol 53, 275-277. 14. Gaynor, J. W., et al. (2010) RNA interference: a chemist's perspective, Chem Soc Rev 39, 4169-4184. 15. Stehling, O., et al. (2009) Controlled expression of iron-sulfur cluster assembly components for respiratory chain complexes in mammalian cells, Methods Enzymol 456, 209-231. 16. Sheftel, A. D., et al. (2010) Humans possess two mitochondrial ferredoxins, Fdx1 and Fdx2, with distinct roles in steroidogenesis, heme, and Fe/S cluster biosynthesis, Proc Natl Acad Sci U S A 107, 11775-11780. 17. Sheftel, A. D., et al. (2012) The human mitochondrial ISCA1, ISCA2, and IBA57 proteins are required for [4Fe-4S] protein maturation, Mol Biol Cell 23, 1157-1166. 18. Stehling, O., et al. (2018) Biochemical Analyses of Human Iron-Sulfur Protein Biogenesis and of Related Diseases, Methods Enzymol 599, 227-263. 19. Drapier, J. C., et al. (1996) Aconitases: a class of metalloproteins highly sensitive to nitric oxide synthesis, Methods Enzymol 269, 26-36. 20. Hatefi, Y., et al. (1978) Preparation and properties of succinate: ubiquinone oxidoreductase (complex II), Methods Enzymol 53, 21-27. 21. Trounce, I. A., et al. (1996) Assessment of mitochondrial oxidative phosphorylation in patient muscle biopsies, lymphoblasts, and transmitochondrial cell lines, Methods Enzymol 264, 484-509.

Techniques for studying iron in health and disease EMBL

22. Birch-Machin, M. A., et al. (2001) Assaying mitochondrial respiratory complex activity in mitochondria isolated from human cells and tissues, Methods Cell Biol 65, 97-117. 23. Srere, P. A., et al. (1963) The citrate condensing enzyme of pigeon breast muscle and moth flight muscle, Acta Chem Scand 17, 129-134. 24. Biederbick, A., et al. (2006) Role of human mitochondrial Nfs1 in cytosolic iron-sulfur protein biogenesis and iron regulation, Mol Cell Biol 26, 5675-5687. 25. McLaughlin, M. I., et al. (2016) Crystallographic snapshots of sulfur insertion by lipoyl synthase, Proc Natl Acad Sci U S A 113, 9446-9450. 26. Sheftel, A. D., et al. (2009) Human ind1, an iron-sulfur cluster assembly factor for respiratory complex I, Mol Cell Biol 29, 6059-6073. 27. Storrie, B., et al. (1973) Expression of the mitochondrial genome in HeLa cells. IX. Effect of inhibition of mitochondrial protein synthesis on mitochondrial formation, J Cell Biol 56, 819-831. 28. King, M. E., et al. (1972) Respiratory enzymes and mitochondrial morphology of HeLa and L cells treated with chloramphenicol and ethidium bromide, J Cell Biol 53, 127- 142. 29. Teranishi, M., et al. (1999) Effects of coenzyme Q10 on changes in the membrane potential and rate of generation of reactive oxygen species in hydrazine- and chloramphenicol-treated rat liver mitochondria, Arch Biochem Biophys 366, 157-167. 30. Savage, N. (1957) Preparation and properties of highly purified diaphorase, Biochem J 67, 146-155. 31. Sheftel, A. D., et al. (2009) The power plant of the cell is also a smithy: the emerging role of mitochondria in cellular iron homeostasis, Ann Med 41, 82-99. 32. Stehling, O., et al. (2008) Human Nbp35 is essential for both cytosolic iron-sulfur protein assembly and iron homeostasis, Mol Cell Biol 28, 5517-5528.

Techniques for studying iron in health and disease EMBL

Program DAY 3 (May 4th)

FLUORESCENCE MONITORING OF LABILE IRON TRAFFICKING IN CELLS AND IRON SPECIATION IN ANIMAL FLUIDS AND ORGANS.

Lecturer Topics Allocated time (min)

Total metal pool measurement • Atomic absorption spectroscopy • ICP-MS • Laser ablation ICP-MS Wing-Hong Tong 9:00-9:35 In situ analysis in Mitochondria: − Electron Microscopy 35 + 5 − SXRF imaging − Confocal Raman Microscopy

Intracellular iron • Fluorescent probes for iron: • Recognition – based sensors 9:35-10:15 Yoav Cabantchik • Reaction – based sensors 35 + 5 • Intracellular organelles • Cytosol and Mitochondria • Lysosomes and Nuclei 10:15-10:45 COFFEE BRAKE

Extracellular iron 10:45-11:15 • Non transferrin bound iron Bob Hider • Direct measurement 25 + 5 • Indirect measurement DISCUSSION 11:15-11:30

INTRODUCTION TO PRACTICALS Allocated time (min)

Maya Shvartsman Intracellular probing 11:30-11:50

Breno Esposito Extracellular probing 11:50-12:10

Charare Pourzand Mitochondrial Iron probing 12:10-12:30

Techniques for studying iron in health and disease EMBL

REFERENCES (DAY 3)

• Koppenol and Hider (2019). Iron and redox cycling. Do's and don'ts. Free Rad. Biol. Med. 133: 3- 10. • Espósito, B.P., Epsztejn, S. Breuer, W. Cabantchik, Z.I. (2002) A review of fluorescence methods for assessing labile iron in cells and biological fluids. Anal. Biochem. 304:1-18. • Cabantchik, Z.I.et al (2014). Labile iron in cells and body fluids. Physiology, Pathology and Pharmacology. Nat. Front. Pharmacol 4:1 http://journal.frontiersin.org/Journal/10.3389/fphar.2014.00045/full • H. Zhu, J. Fan, B. Wang, X. Peng, Fluorescent, MRI, and colorimetric chemical sensors for the first- row d-block metal ions (2015) Chem. Soc. Rev. 44:4337–4366 doi.org/10.1039/C4CS00285G. • Aron, A.T., Reeves, A.G., Chang, C.J. (2018) Activity-based sensing fluorescent probes for iron in biological systems. Curr. Op. Chem. Biol. 43:113-118. doi.org/10.1016/j.cbpa.2017.12.010 • Hirayama T. (2019) Fluorescent probes for the detection of catalytic Fe(II) ion . Free Radical Biology and Medicine 133 38–45. doi.org/10.1016/j.freeradbiomed.2018.07.004 • Hirayama T, Okuda K, Nagasawa H 2013. A highly selective turn-on fluorescent probe for iron(II) to visualize labile iron in living cells. Chem Sci 4:1250-1256. • Niwa M, Hirayama T, Okuda K, Nagasawa H (2014). : A new class of high-contrast Fe(II) selective fluorescent probes based on spirocyclized scaffolds for visualization of intracellular labile iron delivered by transferrin. Org Biomol Chem 12:6590-6597 • Maiti S, Aydin Z, Zhang Y, Guo M (2015): Reaction-based turn-on fluorescent probes with magnetic responses for Fe2+ detection in live cells. Dalton Trans 44:8942-8949. • Itoa F, Nishiyamaa T, Shia L, Moria M, Hirayamad T, Nagasawad H, Yasuie H, Toyokuni S (2016). Contrasting intra- and extracellular distribution of catalytic ferrous iron in ovalbumin-induced peritonitis. Biochem Biophys Res Commun 476:600-606 • Fontaine SD, DiPasquale AG, Renslo AR (2014) Efficient and stereocontrolled synthesis of 1,2,4- trioxolanes useful for ferrous iron-dependent drug delivery. Org Lett 16:5776-5779. • Abrams RP, Carroll WL, Woerpel KA (2016) Five-membered ring peroxide selectively initiates ferroptosis in cancer cells. ACS Chem Biol 11:1305-1312. 57 • Holmes-Hampton GP, Tong W-H, Rouault TA. “Biochemical and biophysical methods for studying mitochondrial iron metabolism.” Methods Enzymol. 2014, 547:275-307.

Techniques for studying iron in health and disease EMBL

DAY 3- STATION 1. REAL-TIME MONITORING OF LABILE IRON INGRESS IN K562 CELLS USING CYTOSOLIC AND MITOCHONDRIAL METAL-SENSORS Maya Shvartsman Ioav Cabantchik

Introduction

Iron is an essential metal for many cellular processes, including catalysis, DNA synthesis, redox reactions and oxygen transport. Mammalian cells import iron in a variety of chemical forms, either as transferrin-iron (TfFe), ionic non-transferrin iron (NTI) or heme iron, using a different import system for each form [1-2]. After import, the iron is redistributed within a cell according to the momentary metabolic needs. Part of the metal goes to a labile iron pool, where it is readily accessible for utilization, and part of it goes for storage in ferritin protein.

The labile iron pool (LIP) is defined as the redox-active, exchangeable and chelator-accessible iron either within a cell or a cellular organelle [2-4]. Mammalian cells have 0.1-1uM iron in their labile pools, depending on cell type, iron import rate and metabolic state [2]. Since both iron deficiency and iron excess in a cell or an organelle could be potentially deleterious, monitoring changes in labile iron pools is an important and interesting aspect of iron biology. Such changes can be measured in real-time with the aid of fluorescent metal-sensors, which undergo stoichiometric fluorescence quenching upon iron binding (Fig.1A) [2]. The changes in metal-sensor fluorescence can be measured kinetically in any cell type by a flow cytometer, a cuvette spectrofluorimeter, a fluorescence plate reader or a microscope, depending if the cells are suspension or adherent [3,4].

In the current workshop, the students will measure changes in mitochondrial and cytosolic labile iron pools of K562 human erythroleukemia cells in response to different iron sources, like TfFe and NTI. For this purpose, they will use the fluorescent metal-sensors rhodamineB-phenantroline (RPA; exc.560nm, em.610nm) for mitochondria and calcein-green (CALG; exc.488nm, em.520nm) for cytosol (Fig.1A). Fluorescence changes will be measured by flow cytometry, since the K562 are suspension cells.

Brief outline of the experiment (Fig.1B): The students will stain the cells with metal-sensors, measure initial baseline fluorescence, supply the cells with iron as TfFe or NTI and measure fluorescence for 30 minutes. Then, they will stop iron ingress Into cells by adding a high-affinity cell- impermeant iron chelator, like DFO or DTPA. In the end, a high-affinity cell permeant iron chelator, like SIH, will be added to remove bound iron from cytosolic calcein and allow better quantification of the cytosolic labile iron pool. After the experiment, the students will analyze the data by extracting the mean fluorescence values of RPA and CALG, and plotting them as a function of time (Fig.2).

Objectives: • Understand the definition of labile iron pool and the principle behind its measurement with fluorescent metal-sensors. • Understand the pitfalls and limitations of the fluorescent metal-sensor assay. • Learn how to run an experiment of iron ingress measurement in real time. • Learn how to analyze the data of such experiment.

Techniques for studying iron in health and disease EMBL

Figure 1: Principle of action of fluorescent metal-sensors. A. A free fluorescent molecule of metal-sensor gets quenched by binding iron; the reaction is reversed by a high-affinity chelator which takes the iron from the metal-sensor. Taken from [2]. B. Intracellular localization of the red mitochondrial metal-sensor RPA and the green cytosolic metal- sensor CALG. C. Fluorescence plot as a function of time for cytosolic CALG and mitochondrial RPA in K562 cells. The small dots represent normalized mean fluorescence values +/- standard deviation (n=5) measured by spectrofluorimetry, the big overlaid dots represent data from flow cytometry experiment. Taken from [3].

Techniques for studying iron in health and disease EMBL

Figure 2:

Principles of data analysis of a real-time iron ingress flow cytometry experiment.

A. Representative flow cytometry plots of K562 cells stained with red mitochondrial RPA (vertical axis) and green cytosolic CALG (horizontal axis). After 30 minutes with TfFe 5uM, the fluorescence of both metal-sensors drops as seen by a shift of the cloud down for RPA and left for CALG. The addition of the high-affinity cell permeant iron chelator SIH fails to recover the fluorescence of mitochondrial RPA, but recovers cytosolic CALG as seen by the shift of cell cloud to the right. Unpublished raw data from Shvartsman, Fibach and Cabantchik. B. From the histograms of CALG fluorescence, the mean fluorescence values are extracted, normalized and plotted as a function of time. From [3].

References: 1. Winter WE, Bazydlo LAL, Harris NS. The molecular biology of human iron metabolism. Lab Med Spring (2014) 45:92-102 2. Cabantchik ZI. Labile iron in cells and body fluids: physiology, pathology and pharmacology. Frontiers Pharmacol (2014) 45: 1-11 3. Shvartsman M, Fibach E and Cabantchik ZI. Transferrin-iron routing to the cytosol and mitochondria as studied by live and real-time fluorescence. Biochem J (2010) 429: 185-193 4. Shvartsman M, Kikkeri R, Shanzer A, Cabantchik ZI. Non-transferrin-bound iron reached mitochondria by a chelator inaccessible mechanism: biological and clinical implications. Am J Physiol Cell Physiol 293: C1383-C1394

Techniques for studying iron in health and disease EMBL

Two days before experiment (02.05.19): • Prepare DMEM-low glucose + 10% FBS + 1% Pen-Strep + 1% L-Gln. • Thaw the K562 cells into a T75 flask.

The day before experiment (03.05.19): • Count the cells. • Split into fresh flasks (T25 or T75), to keep the culture in log growth (~0.5x106 cells/ml). • Prepare required solutions and reagent stocks (list below). • Calibrate the flow cytometer.

The day of the experiment (04.05.19):

6. Collect a T75 flask of K562 cells. Count cells. Centrifuge 5min 1200rpm and discard supernatant. Prepare ~20ml of K562 suspension in DMEM-Hepes to a concentration of 2x10^6 cells/ml. The DMEM-Hepes should be at 37C. 7. Prepare 3 x FACS tubes. Put 2ml of the cell suspension in each tube. These will be the single stain controls for instrument calibration. Put one of the tubes aside in the ice box (unstained control). Set two tubes aside at 37C (single-stain controls).

8. Double-staining of K562 cells (RPA 1uM + CALG-AM 0.125uM): • Take the remaining cell suspension and add DFO to a final concentration of 50uM. • Mix well. • Add RPA to a final concentration of 1uM. Incubate in the 37C shaker for 15 min. • Wash with warm DMEM-Hepes. • Resuspend in warm DMEM-Hepes and add CALG-AM to a final concentration of 0.125uM. Incubate in the 37C shaker for 10 min. • Wash with warm DMEM-Hepes and add probenecid to 0.5mM final concentration. • At the same time, stain the single stain controls (RPA only and CALG only).

IMPORTANT: During ALL staining and kinetics, keep the samples covered from light with aluminium foil, to avoid photobleaching of metal sensors!

Iron ingress kinetic measurements: 9. Disribute your double-stained cell suspension into a 6-well plate at 2ml/well. Keep the plate in the 37C shaker at all times. Put all single stained controls on ice. 10. Label each of the wells of your 6-well plates according to treatment (see plate map below). 11. If needed, add pretreatment to some wells according to the plate map (chloroquine 20uM or hinokitiol 1uM final). Put the plate back in the 37C shaker.

Techniques for studying iron in health and disease EMBL

Plate maps: Plate 1: Endocytosis inhibitors (group of 3 students)

Control no Fe TfFe 5uM FeHQ 5uM

Control no Fe + TfFe 5uM + chloroquine FeHQ 5uM + chloroquine chloroquine 20uM 20uM 20uM

Plate 2: Modulating transmembrane ingress (group of 3 students)

Control no Fe TfFe 5uM FAS 5uM

Control no Fe + hinokitiol TfFe 5uM + hinokitiol 1uM FAS 5uM + hinokitiol 1uM 1uM

12. Prepare sampling FACS tubes with labels of time and treatment: 0’, 10’, 20’, 30’, 40’, 50’, 60’,70’. A total of 48 tubes per 6 well plate. 13. Also prepare FACS tubes with strainer cap, or separate cell strainers and FACS tubes for each timepoint. 14. Fill the tubes 0’-30’ with 200 ul of DMEM-Hepes-probenecid/tube. Fill the tubes 40’-70’ with 200 ul of DMEM-Hepes-probenecid+DTPA 50uM final. Put all tubes in the ice bucket. 15. Take out your sample of 0’: 200 ul from each well of your 6-well plate. Mix well before sampling. Put it into the respective 0’ FACS tube. Get the tubes back on ice, pass sample through strainer, measure on flow cytometer. 16. Add iron to the plates as TfFe 5uM, FeHQ 5uM or FAS 5uM final, according to your plate map. Put the plate back in the 37C shaker and run 10min on your timer. 17. Take out the 10’ sample into the FACS tubes on ice. Measure. 18. Take out the 20’ sample into FACS tubes. Measure. 19. Take out the 30’ sample into FACS tubes. Measure. 20. Add DTPA to all wells of your plate to 50uM final concentration. 21. Take out the 40’ sample into FACS tubes. Measure. 22. Take out the 50’ sample into FACS tubes. Measure. 23. Prepare SIH 2mM stock as follows: 40ul of SIH 50mM + 210ul DMSO + 750ul HBS. Vortex! Add SIH to all wells of your plate at 50uM final. 24. Take out the 60’ sample into FACS tubes. Measure. 25. Take out the 70’ sample into FACS tubes. Measure.

Data analysis. 26. Extract mean FI of RPA and CALG, plot FI(t). Normalize on F(t=0), and plot normalized FI(t).

Techniques for studying iron in health and disease EMBL

References • Shvartsman M, Kikkeri, R, Shanzer A, Cabantchik ZI (2007).Iron accesses mitochondria from a cytosolic pool of non-labile iron. Biological and clinical implications. Am J Physiol Cell Physiol 293: C1383-C1394 • Shvartsman, M., Fibach, E. and Cabantchik, Z.I. (2010). Transferrin-iron routing to the cytosol and mitochondria as studied by live and real-time fluorescence. Biochem. J.429:185–193. • Shvartsman, M. and Cabantchik, Z.I. (2012) Intracellular iron trafficking. Role of cytosolic ligands. Biometals (2012) 25:711–723 DOI 10.1007/s10534-012-9529-7 • Cabantchik, Z.I.et al (2014). Labile iron in cells and body fluids. Physiology, Pathology and Pharmacology. Nature Front. Pharmacol 4:1 http://journal.frontiersin.org/Journal/10.3389/fphar.2014.00045/full

Required reagent stocks and solutions: • HBS (Hepes 2.38gr, NaCl 4.38gr, dH2O to 500ml, adjust pH to 7.2-7.4 with 5M NaOH. Store at RT/4C.) • DMEM-Hepes (1 vial of DMEM no phenol red powder, 4.77 gr Hepes, dH2O to 1L, adjust pH to 7.2-7.4 with NaOH. Filter and keep at 4C.) • DMSO • RPA 10mM in DMSO. • CALG-AM 1 or 10mM in DMSO. • DFO 10mM in HBS • DTPA 50mM in HBS • SIH 50mM in DMSO • FeCl3 10mM in DMSO • 8’-hydroxyquinoline 10mM in DMSO • Hinokitol 1mM in DMSO • Probenecid 5mM or 10mM in HBS • L-ascorbate 10mM in dH2O • FAS 10mM in dH2O (make fresh!) • TfFe in HBS

Required equipment: • Water bath 37C • Table top incubator-shaker 37C • Centrifuge 5810R • Table top eppendorf centrifuge • Sterile pipettes and pipetboys • Gilsons and sterile pipet tips • FACS tubes regular • FACS tubes with strainer cap • 6-well tissue culture plates (not treated) • Timers • Calculators • Paper towels • Containers for biological waste disposal (liquid, solid) • Ice buckets and ice

Techniques for studying iron in health and disease EMBL

• Flow cytometer + analysis program • Nitrile gloves • Plastic beakers and cylinders • Scales • pH meter • Falcon tubes (15ml and 50ml) • Eppendorf tubes • Vortex • Aluminium foil!!!!!

Techniques for studying iron in health and disease EMBL

DAY 3- STATION 2. LABILE IRON IN BIOLOGICAL FLUIDS Breno Pannia Espósito Ioav Cabantchik

Introduction

Non-Transferrin Bound Iron (NTBI) is traditionally quantified by colorimetric methods or HPLC, which are generally low-throughput and time consuming. Fluorescence-emitting systems in general allow for lower detection limits when compared to colorimetric systems. Fluorescence can be registered in high throughput, low cost commercial microplate readers that usually carry the filters for monitoring common fluorophores (rhodamine, fluorescein).

Labile Plasma Iron (LPI) is the redox-active fraction of NTBI, mostly composed of Fe(III) species. This is the NTBI fraction that must be controlled in a chelation regimen. Therefore, a method was devised to quantify LPI by means of a fluorimetric probe, dihydrorhodamine (DHR). In the presence of physiologically relevant ascorbate levels in aqueous buffers, LPI starts a redox cycle generating Reactive Oxygen Species which are detected by the oxidative conversion of non-fluorescent DHR into fluorescent rhodamine (Figure 1 a).

Figure 1. (a) Chemical principle of the fluorimetric detection of LPI by means of the autoxidation of ascorbic acid (Hasc) catalysed by iron. Reactive oxygen species are generated which convert dihydrorhodamine (DHR) into fluorescent rhodamine (Rhod). (b) Treatment with a strong iron chelator (deferiprone; brown square) halts the iron reduction step and hence the whole cascade of reactions, preventing DHR to be oxidized.

Techniques for studying iron in health and disease EMBL

It is ensured that the assay responds only to the free iron present in the plasma sample by running a parallel determination in the presence of a strong iron chelator (deferiprone; Figure 1 b). The ferric deferiprone complex has a unfavorable reduction potential, that cannot be reached by Hasc. Hence, the difference between the rate of DHR oxidation in the absence and presence of deferiprone is a direct measurement of the iron-dependent oxidation. This is an important precaution DHR since is a low specific probe and other oxidants (e.g., atmospheric O2) can also oxidize it, which would lead to false positive readings of LPI.

The rate of DHR oxidation is directly proportional to LPI levels, which can be quantified by running a parallel calibration curve with a suited LPI standard (Fe(nta), ferric nitrilotriacetate; Figure 2).

Techniques for studying iron in health and disease EMBL

Figure 2. Calibration for the LPI experiment. Kinetic curves of DHR oxidation are obtained for known concentrations of Fe(nta), the LPI standard, in the absence (a) or presence (b) of deferiprone, a strong iron chelator that allows for the unequivocal assessment of iron-dependent-only oxidation of DHR. DHR oxidation rates are obtained as slopes (m) for each kinetic curve; in the presence of deferiprone m tends to zero. (c) Oxidation rates in the absence of deferiprone (def) are linearly dependent on iron concentration. (d) The difference between oxidation rates in the absence and presence of def is also linearly dependent on iron concentration, and it is the calibration curve used to quantify LPI in unknown samples according to equation I.

Chelation therapy with high affinity chelators (DFO, deferasirox or deferiprone) traps iron into non- redox active forms, so LPI can be used to assess the efficacy of any chelation regimen and tailor it to specific individual needs. This is the main benefit of LPI quantification.

However, wrong choice of chelators can actually increase the amount of redox-active species by mobilizing otherwise inert plasma forms of NTBI.

In this practical, students will understand the basic principles of the fluorimetric method of detection of LPI. Simulated iron-overloaded samples in artificial human plasma will be provided for quantification of LPI by the students. In addition, the effect of iron species and different chelators on the analytical signal will be demonstrated.

Objectives

1. Understand the principles behind the analysis of LPI 2. Conduct a LPI quantification in artificial samples 3. Understand the limitations of LPI quantification in terms of iron source and presence of chelators

Protocol

a) General remarks

• LPI analysis cannot be performed on serum samples obtained in edta. • HBS/Chelex: Hepes Buffer Saline (Hepes 20 mM; NaCl 150 mM; pH 7.4) washed with Chelex®100 (1 g/100 mL) in order to remove traces of contaminant iron. • PLM: Plasma-like medium (hepes 20 mM, NaCl 150 mM, Human Serum Albumin 40 mg/mL, sodium phosphate dibasic 1.2 mM, sodium citrate 120 uM, sodium bicarbonate 10 mM; pH 7.4). Sodium citrate and sodium bicarbonate should be prepared fresh and added to the buffer only in the day of the experiment. • Serum samples can be frozen (-20oC) and thawed multiple times. • DHR (100 mM in DMSO) and Hasc (40 mM in water) stock solutions should be prepared and frozen (-20oC) in small aliquots. These aliquots should be thawed only once to prepare the DHR/Hasc solution, which should be used to prepare the microplate within 15 minutes.

Techniques for studying iron in health and disease EMBL

b) Quantification of LPI

• Four students will spike samples of PLM with a known volume of iron salt, keeping the values secret (samples s1 to s4). Two of these samples will contain also 20 uM of DFO, deferasirox or deferiprone to simulate real-world chelation therapy. • 10 uL aliquots of Fe(nta) (0 – 20 uM in PLM) will be transferred to a 96-well microplate according to Plate Map (Figure 3) • 10 uL aliquots of the samples will be transferred to a 96-well microplate according to Plate Map (Figure 3) • Transfer 190 uL of DHR/Hasc solution (+ or – deferiprone 100 uM in DMSO) in HBS to the wells for the determination of LPI, according to Plate Map (Figure 3) o • Register fluorescence for 1 h (exc/emis = 485/520 nm) at 37 C

c) Nature and Properties of labile iron and its fluorimetric detection

• Transfer 4 x 1 mL samples of PLM to microconcentration tubes • Spike each tube with 10 uM (final concentration) of Fe3+ (e.g., ferric chloride, Fe(nta), FeCIT and FeNP) • Transfer 10 uL of the contents of each iron-spiked solution to a 96-well microplate according to Plate Map (Figure 3) • Transfer 10 uL of each chelator solution (0 – 60 uM) to the microplate according to Plate Map (Figure 3) • Transfer 180 uL of DHR/Hasc solution to all the wells o • Register fluorescence for 1 h (exc/emis = 485/520 nm) at 37 C

chelators: 0 60 µM

FeNTA: 1 2 3 4 5 6 7 8 9 10 11 12 0 A s1 - def edta FeCl3 10 µM B s2 - def dfo FeCIT 10 µM C s3 - def edta FeNTA 10 µM D s4 - def dfo FeNP 10 µM E s1 + def edta C/C - def C/C F C/C + def s2 + def dfo G s3 + def edta 20 µM H s4 + def dfo

(a) LPI (b) properties of Fe and chel Figure 3. Plate Map for the practical with final concentrations of the studied solutions.

Analysis

Part a) A spreadsheet will be used to build the calibration curve and calculate the values of LPI.

Part b) The kinetic curves will be built in Excel in order to evaluate the effect of different iron sources and chelators on the fluorescence signal over time.

Techniques for studying iron in health and disease EMBL

Evaluation

With these experiments it should be realized that the presence of edta in plasma samples will lead to overestimation of LPI. Also, it is preferred to use rates of oxidation instead of endpoints of oxidation, since fluctuations of fluorescence emission are better corrected over time, thus leading to smaller standard deviations.

In clinical samples, LPI values > 0.5 uM are considered significant. Hemolysis does not affect the test as heme iron is unable to participate in the redox cycle prompted by Hasc. Also, iron bound to transferrin is inert in the LPI test (Figure 4).

Some iron overloaded patients may not display overt LPI, however other potentially readily available forms may be occluded in the NTBI pool. In these cases, these forms are revealed by pre-treatment of a mild mobilizing agent (e.g. NTA at 0.5 mM) before the LPI measurement. This “enhanced” LPI (eLPI), together with overt LPI, provide a better understanding of the chelation requirements of the patient.

The major artifacts in the LPI experiment are (1) blood drawn in edta-containing vials, which may give false positives and (2) turbidity, which may screen off some or most of the emitted fluorescence and give false negatives.

) 80 -1 Control HH 60 F.U. min F.U. (

20 DHR oxidation DHR rate ∆ 0 0 15 80 [HbO ] in serum (mM) 2 Figure 4: (a) Relationship between DHR oxidation and transferrin saturation. A 23 uM human apo- transferrin solution in HBS containing 10 mM sodium bicarbonate was allowed to bind Fe(nta) to give final Fe concentrations of 0 – 82.5 uM, for 20’ at room temperature and then tested for formation of Fe-transferrin by absorption at 465 nm (right scale, filled symbol) and for DHR oxidation rate (left scale, Fluorescence Units/min, F.U. x min-1, open symbol). The concentration of Fe required to reach maximal A465, corresponding to 100% transferrin saturation was 45 uM (theoretical 46 uM, based on two iron- binding sites per transferrin). (b) Effect of red cell lysate on chelator-sensitive DHR oxidation. Sera from a control subject and a hemochromatosis (HH) patient were mixed with red blood cell lysate to yield the indicated hemoglobin concentrations. DHR oxidation rates are given as the difference between rates obtained in the absence and presence of deferiprone.

Techniques for studying iron in health and disease EMBL

References • LPI assay: Esposito, et al (2003). Labile plasma iron in iron overload: redox activity and susceptibility to chelators. Blood 102: 2670-2677. • eLPI assay: Breuer et al (2012). Non-transferrin bound iron in Thalassemia: differential detection of redox active forms in children and older patients. Am. J. Hematol. 87:55–61. • Espósito, B.P., Epsztejn, S. Breuer, W. Cabantchik, Z.I. (2002) A review of fluorescence methods for assessing labile iron in cells and biological fluids. Anal. Biochem. 1;304(1):1-18. • Zanninelli, G., Breuer, W. and Cabantchik, Z.I. (2009). Daily labile plasma iron as an indicator of chelator activity in Thalassemia major patients. Br. J. Hematol. 147, 744–751 • Cabantchik, Z.I. et al (2014). Labile iron in cells and body fluids. Physiology, Pathology and Pharmacology. Nature Front. Pharmacol 4:1 http://journal.frontiersin.org/Journal/10.3389/fphar.2014.00045/full

Abbreviations

DFO Desferrioxamine DHR Dihydrorhodamine DMSO Dimethylsulfoxide edta Ethyilenediaminetetraacetate Fe(nta) Ferric nitrilotriacetate FeCIT Ferric citrate FeNP Magnetite nanoparticles Hasc Ascorbic acid HBS Hepes Buffer Saline HPLC High Performance Liquid Chromatography LPI Labile Plasma Iron nta Nitrilotriacetate NTBI Non Transferrin-Bound Iron PLM Plasma-Like Medium

List of chemicals, consumables and equipment

. Microplate reader (BMG Clarion, interfaced to a PC with Microsoft Excel) . Water bath . Analytical balance . Plastic (preferably) micro spatulas . Vortex . Fridge . Freezer (-20o will do) . pH meter (with electrode and calibration solutioins) . Access to deionized water . Microplates (transparent, flat, 96 wells, not treated for cell adhesion) – 10 . Pipettes (10, 100, 1000 uL, 5000 uL) – at least 1 of each; 2 of each (10, 100) would be better . Multichannel pipettes (10, 300 uL) – 1 of each at least . Multichannel pipette reservoirs – 10 . A few plastic or glass beakers (50 mL) . Tips (10, 100, 1000, 5000 uL) and their respective racks . Microconcentrator tubes (“Eppendorf vials”) 1.5 mL . Microtube and falcon tube racks/holders . Falcon tubes (15 mL; 50 mL) – a dozen each . Parafilm®

Techniques for studying iron in health and disease EMBL

. Aluminum foil . Scissors . Marker pen . Reagents: - Ascorbic acid *not sodium ascorbate* - Sodium phosphate dibasic - Sodium bicarbonate - Human serum albumin - Deferiprone - DFO - DMSO - Sodium citrate - Sodium chloride - DHR hydrochloride - Sodium nitrilotriacetate *not nitrilotriacetic acid* - HBS/CHELEX - Hepes - Ferric chloride - Ferric citrate

Techniques for studying iron in health and disease EMBL

DAY 3. STATION 3. MITOCHONDRIAL LABILE IRON POOL MONITORING USING A HIGHLY SELECTIVE MITOCHONDRIA- TARGETED FLUORESCENT IRON CHELATOR

Dana Beiki Robert C Hider Charareh Pourzand

Introduction

Iron is an important metal involved in many essential cellular processes. This is because of its facile redox chemistry and the high affinity of both redox states (ironII and ironIII) for oxygen. These same properties make iron toxic especially when present in excess in its redox-active chelatable ‘labile’ form. Indeed in contrast to iron bound to proteins, the intracellular labile iron (LI) can be potentially toxic especially under conditions of oxidative stress and certain pathologies. This is because LI in the presence of excess reactive oxygen species (ROS) can catalyse the formation of highly reactive oxygen- derived free radicals such as the hydroxyl radical that can ultimately overwhelm the cellular antioxidant defense mechanisms and lead to cell damage. Originally the intracellular labile iron pool (LIP) was thought to be mainly cytosolic. However recent studies have shown that mitochondrial organelles contain an important pool of LI which renders these organelles particularly susceptible to oxidative stress, notably after exposure to hydrogen peroxide and ultraviolet A radiation. [1, 2] In addition, mitochondrial iron-overload has been recently implicated in the development and progression of neurodegenerative disorders such as Friedreich’s ataxia (FRDA). Cultured skin fibroblasts from FRDA patients show defects in antioxidant mechanisms and are more sensitive than their healthy counterparts to various forms of chemically-induced oxidative stress, including exogenous iron and H2O2 [e.g. 3, 4]. These studies clearly highlight the need for the development of highly specific probes for mitochondrial LIP monitoring. The ideal probe should have high affinity and specificity for iron and be delivered to mitochondria without affecting the cytosolic LI. We have recently developed a series of highly sensitive fluorescent iron sensors based on the class of small “SS peptides”, which selectively target the inner mitochondrial membrane [5, 6]. Such peptides are amphiphilic in nature and are composed of alternating aromatic and basic groups. Although the peptides carry several positive charges, they have been shown to penetrate readily membranes in a non-saturable manner and to rapidly accumulate in the inner mitochondrial membrane. Figure 1 shows the mitochondria-targeted iron sensor BP19 (i.e. compound 13 in ref 5) which will be used in this workshop. BP19 is a chimeric peptide based on a 3-hydroxypyridin-4-one (HPO) iron(III)-selective chelator linked to SS-like peptides and a dansyl (DNS) fluorophore. In these chimeric molecules, complexation with iron results in a turning-off of the fluorophore by energy transfer, as illustrated in Figure 2. The turning-off of the iron sensor can be measured kinetically in any cell type by flow cytometry or spectrofluorimetry, when applicable.

Using the highly sensitive mitochondrial iron sensor BP19, we have recently demonstrated that skin fibroblasts derived from FRDA patients display higher levels of mitochondrial LI (up to 6-fold on average compared to healthy counterparts) and higher sensitivity to ultraviolet A radiation [7]. They also exhibit higher increase in mitochondrial ROS generation after UVA irradiation (up to 2-fold on average), consistent with their differential sensitivity to UVA [7].

Techniques for studying iron in health and disease EMBL

Figure 1. BP19, a fluorescent mitochondria-targeted peptide incorporating an iron chelator

Figure 2. Mitochondria-targeted chimeric peptides incorporating an iron chelator. Our approach involves the linking of short positively charged mitochondria-specific peptides (SS-peptides) to a chelator moiety (a HPO for BP19) and a fluorescent tag (DNS for BP19) for sensing and subcellular localization. In these chimeric molecules, complexation with iron results in a turning-off of the fluorophore by energy transfer.

In this workshop, the mitochondrial LI will be monitored with BP19 in skin FEK4 fibroblasts and HaCaT keratinocytes that have differential sensitivity to UVA and H2O2 treatments [8] as outlined below:

Brief outline of the experiments: Cells will be seeded at a density of 180000 per 6 cm plate in Phenol free EMEM supplemented with 15% FCS, Glutamine and Penicillin/ Streptomycin to reach 80% confluency 48 h after seeding. Cells will then be pre-treated (or not) for 6-7 h with 100 uM of the hexadentate chelator desferrioxamine (DFO), with the purpose to deplete maximally the intracellular LI. Cells will then be washed twice with PBS (or serum-free phenol-free EMEM) and incubated overnight at 37ºC with 50 uM of the fluorescent iron sensor BP19. The next day (day 3 after seeding), cells will be harvested by trypsinization, resuspended in buffer F (containing 10 mM HEPES, pH 7.3 and 150 mM NaCl) and counted (see Figure 3). Equal numbers of cells will then be transferred to a 96 well plate for fluorescence reading (excitation =330-340 nm and emission= 550- 580 nm) with a fluorescent plate reader (e.g. Clariostar BMG Labtech). The difference in fluorescence between the DFO+BP19- and BP19-treated samples will then be taken as a measure of the concentration of the mitochondrial LI, which will be quantified using an ex situ calibration curve (see Figure 4a and 4b).

Techniques for studying iron in health and disease EMBL

Figure 3. Summary protocol for the measurement of mitochondrial LIP using BP19

F2 F2: maximum fluorescence of ∆ F BP19, fully dequenched by DFO F1

[] F1: fluorescence of BP19

Fluorescence

Cells incubated with: BP19 DFO + BP19

BP19 change

fluorescence fluorescence Mito Fluorescence

Fe3+ Fe3+

Figure 4 - (a) The difference in fluorescence (F) between the cells treated with BP19 alone (F1) and those treated with DFO+BP19 (F2) as a measure of the concentration of the mitochondrial LI, when quantified using an ex situ calibration curve (b).

Techniques for studying iron in health and disease EMBL

References: 1. Aroun A, Zhong JL, Tyrrell RM, Pourzand C. Iron, oxidative stress and the example of solar ultraviolet A radiation. Photochem Photobiol Sci. 2012 ;11(1):118-134.

2. Reelfs O, Abbate V, Hider RC, Pourzand C. A Powerful Mitochondria-Targeted Iron Chelator Affords High Photoprotection against Solar Ultraviolet A Radiation. J Invest Dermatol. 2016;136(8):1692-1700.

3. Lim CK, Kalinowski DS, Richardson DR. Protection against hydrogen peroxide- mediated cytotoxicity in Friedreich's ataxia fibroblasts using novel iron chelators of the 2-pyridylcarboxaldehyde isonicotinoyl hydrazone class. Mol Pharmacol. 2008;74(1):225-235.

4. Wong A, Yang J, Cavadini P, Gellera C, Lonnerdal B, Taroni F, Cortopassi G. The Friedreich's ataxia mutation confers cellular sensitivity to oxidant stress which is rescued by chelators of iron and calcium and inhibitors of apoptosis. Hum Mol Genet. 1999; 8(3):425-430.

5. Abbate V, Reelfs O, Hider RC, Pourzand C. Design of novel fluorescent mitochondria- targeted peptides with iron-selective sensing activity. Biochem J. 2015; 469(3):357- 366.

6. Abbate V, Reelfs O, Kong X, Pourzand C, Hider RC. Dual selective iron chelating probes with a potential to monitor mitochondrial labile iron pools. Chem Commun (Camb). 2016; 52(4):784-787.

7. Reelfs O, Abbate V, Cilibrizzi A, Pook MA, Hider RC, Pourzand C. The role of mitochondrial labile iron in Friedreich's ataxia skin fibroblasts sensitivity to ultraviolet A. Metallomics. 2019 Feb 19. doi: 10.1039/c8mt00257f.

8. Zhong JL, Yiakouvaki A, Holley P, Tyrrell RM, Pourzand C. Susceptibility of skin cells to UVA-induced necrotic cell death reflects the intracellular level of labile iron. J Invest Dermatol. 2004;123(4):771-80.

Techniques for studying iron in health and disease EMBL

List of chemicals, consumables and equipment

. Fluorescent microplate reader (BMG Clarion, interfaced to a PC with Microsoft Excel) . Inverted microscope (e.g. model AE2000 from Motic Deutschland GmbH, Wetzlar, Germany) . Neubauer Haemocytometer for cell counting . Water bath . Analytical balance . Plastic micro spatulas . Vortex . Fridge . Freezer (-20o C) . pH meter . Access to deionized water (MilliQ, if possible) . Black-walled 96 well plates (Greiner #655090: black polystyrene wells flat bottom, with micro-clear bottom) – (Quantity: 8 x 96 well plates) . Gilson/Eppendorf Pipettes (10, 200, 1000 uL) – (Quantity: 2 of each) . Gilson/Eppendorf tips (for 10, 200 and 1000 uL pipettes)- (Quantity: At least 3 full racks of 10 and 1000 uL and at least 4 full racks of 200 uL tips) . Eppendorf tubes 1.5 mL – At least 60 . 15 mL falcon sterile centrifuge tubes – (Quantity: 30) . 50 mL falcon sterile centrifuge tubes – (Quantity: 15) . 15 mL Falcon tubes holders . 50 mL falcon tube holders . Centrifuge with holders for 15 and 50 mL falcon tubes. . Multichannel pipettes (300 uL) – (Quantity: 1 x at least) . Multichannel pipette reservoirs – (Quantity: 10) . Parafilm® . Aluminum foil . Scissors . Marker pens (3-4) . 60 mm cell culture plates, Thermo scientific NunclonTM Delata surface cat # 150288 (Quantity: at least 20 plates) . Thermo Scientific™ Nunc™ Serological Pipettes 5, 10 and 25 mL pipettes (Fisher scientific, cat# 4489, 4488 and 4487, respectively (Quantity: At least one bag of each) . Pipetteboy (Quantity: 3) . Timers . Calculators . Tape . T75 mL tissue culture flasks (TPP, Helena Biosciences cat # 90075) (Quantity: 8) . Disposable Gloves Small, medium and large size (e.g. Kimberly-Clark™ KIMTECH Science™ PURPLE NITRILE™ Gloves) . CO2 incubator . 3 x 250 mL beakers . 3 x 500 mL

Techniques for studying iron in health and disease EMBL

. Reagents: - DFO (desferrioxamine mesylate, Sigma-Aldrich, cat # D9533) - DMSO (sterile, tissue culture grade, Sigma-Aldrich, cat # D2650) - HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid) - Phenol red-free EMEM (Life technologies/Thermo fisher scientific, cat # 51200046) (Quantity: 1 x 500 mL) - Phenol red-free DMEM (Life technologies/Thermo fisher scientific, cat # 31053- 044) (Quantity: 1 x 500 mL) - EMEM(Life technologies/Thermo fisher scientific, cat # 21090-022) (Quantity: 1 bottle of 500 mL) - DMEM (Life technologies/Thermo fisher scientific, cat # 41965039) (Quantity: 1 x 500 mL) - 200 mM Glutamine (Life technologies/Thermo fisher scientific, cat # 25030024) (Quantity: 100 mL) - Penicillin/streptomycin (Life technologies/Thermo fisher scientific, cat #15140122) (Quantity: 100 mL) - FCS - Trypsin 2.5 % (Life technologies/Thermo fisher scientific, cat # 15090046) (Quantity: 1 x 100 mL) - PBS (Life technologies/Thermo fisher scientific, cat # 10010023) (Quantity: 3 x 500 mL) - NaCl - Fe solution in HNO3 from Fisher (Fisher Chemical J/8030/05) (Quantity: 1x 100 mL - Nitrilotriacetic acid, trisodium salt monohydrate, from Sigma Aldrich (cat # 72565) (Quantity: 1x 250 g) - MOPS - EDTA - BP19

. Solutions required: o Phenol red-free EMEM completed with 15%FCS, Glutamine and Penicillin/Streptomycin o Phenol red-free DMEM completed with 10%FCS, Glutamine and Penicillin/Streptomycin o EMEM completed with 15% FCS, Glutamine and Penicillin/Streptomycin o DMEM completed with 10% FCS, Glutamine and Penicillin/Strepttomycin o PBS o 0.125-0.25% trypsin o Trypsin/EDTA o Fixing solution (10 mM HEPES pH7.3, 150 mM NaCl): (for 50 mL: 43.5 mL MQwater + 1.5 mL 5M NaCl + 5 mL 100 mM HEPES 7.4) o 50mM DFO stock in MQwater (Eppendorf size volume); o 50mM BP19 (MW 982.1, di-acetate salt) stock in 1:1 DMSO:MQwater (Eppendorf size volume) o Fe solution in HNO3 from Fisher (#J/8030/05; also coded: #10533142).

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Techniques for studying iron in health and disease EMBL

o 17.4 mM NTA in MQwater (nitrilotriacetic acid, trisodium salt monohydrate, from Sigma (#106305). o 100 mM MOPS pH 7.4 (ideally chelexed)

Cells: (1) Human skin fibroblasts (FEK4) ; (2) Human skin Keratinocytes (HaCaT).

The design and evaluation of mitochondrial iron sensors as well as the principle of the assay will be discussed in a lecture in a morning session.

Objectives: • Understand the definition of labile iron pool and the principle behind its measurement with fluorescent metal-sensors. • Understand the pitfalls and limitations of the fluorescent metal-sensor assay. • Learn how to run an experiment using the BP19 for mitochondrial labile iron measurement. • Learn how to analyze the data of such experiment using an ex situ calibration curve.

Procedure: The participants will be split into two groups: • Group 1 will measure the mitochondrial labile iron in skin fibroblasts (FEK4) using an ex situ calibration curve. • Group 2 will measure the mitochondrial labile iron in skin keratinocytes (HaCaT) using an ex situ calibration curve.

Cell seeding (3 days before the start of the workshop): 1) Seed each cell line with the following experimental conditions in mind: (a) Unstained cells; (b) BP19-treated cells; (c) DFO + BP19-treated cells.

2) Grow for 48h, at 37°C, 5% CO2

Treatment day (1 day before the start of the workshop): 1) In the morning (10 am) Remove and keep conditioned medium, 2) Add DFO to cells in conditioned medium (100 µM) 3) Incubate for 6 h, at 37°C, 5% CO2 4) At 4pm, aspirate medium, wash twice with PBS or Phenol red-free serum-free medium 5) Add BP19 (we use 50uM) to cells in conditioned medium 6) Incubate O/N (ca 18-20h), at 37°C, 5% CO2

Analysis (the day of the station 3 workshop). 1) Trypsinize/collect and count cells*. 2) Spin and resuspend cells in 700 uL buffer containing 10 mM HEPES pH7.3, 150 mM NaCl. 3) Transfer 200 uL of cells in triplicates on a blackwalled 96 well plate (Greiner #655090: black polystyrene wells flat bottom, with micro-clear bottom) already containing the reacted calibration curve mixes**

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4) Read fluorescence on a fluorescence plate reader (37°C temperature is preferable if measures are likely to take a long time): excitation wavelength 330nm, emission wavelength 550nm. We use CLARIOstar plate reader from BMG Labtech or equivalent.

* Counting may be done after the fluorescence reading. **Procedure for the preparation of Fe:NTA complex:

i. Fe: solution in HNO3 from Fisher (#J/8030/05). Calculate the titre of Fe from the indication on the label. ii. 17.4 mM NTA in MQwater (nitrilotriacetic acid, trisodium salt monohydrate, from Sigma Aldrich, cat #106305). iii. Mix Fe and NTA in a 1:3 molar ratio, from Fe and NTA solutions prepared in milliQ water, i.e. 40 uL of 17.4 mM Fe solution + 120 uL of 17.4 mM triNa-NTA. [Fe] = 4.34 mM final iv. Let stand 1h/RT for the complex to form, v. Dilute the complex in MOPS solution, so as to reach [Fe] in the range of 15-20 µM. This allows to minimize the volumes used to construct the calibration curve: 1:3 complex: mix 10 uL complex + 395 uL MOPS ( gives a 107.16 uM Fe solution) Then: mix 80 uL of 107.16uM solution + 720 uL MOPS ( gives a 10.716 uM Fe solution). vi. Prepare the wells (in triplicates/quadruplicates) containing incremental amounts of Fe, in a total volume of 50 uL. For this Prepare premixes (as for 5wells) in eppendorfs containing incremental concentrations of Fe.

Load 50 uL each/well, in quadruplicate, as follows, e.g.: Each well [Fe] = 0 will contain: 50 uL MOPS only, Each well [Fe] = 0.2 uM will contain: 1 uL of 20 uM FeNTA + 49 uL MOPS, etc.

Example of 96 well plate layout:

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 buffer only

Cells + BP19 Cells 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 unstained cells unstained cells+BP19+DFO 0.8 0.9 1.0 Legends: 0.8 0.9 1.0 Calibration curve: final [Fe] in uM 0.8 0.9 1.0 Samples to measure 0.8 0.9 1.0

Smaller/bigger increments, others than the ones indicated can be used.

µµ

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uL of uM [Fe] final in uM [Fe] FeNTA MOPS 5well MOPS well, once mixed in (10.716 uM to 50ul premix with 50 uM BP19 premixes in Fe) per well 0 0 50 0.0 250 0.1 0.93 49.07 4.65 245.35 0.2 1.86 48.14 9.3 240.7 0.3 2.79 47.21 13.95 236.05 0.4 3.73 46.27 18.65 231.35 0.5 4.66 45.34 23.3 226.7 0.6 5.59 44.41 27.95 222.05 0.7 6.53 43.47 32.65 217.35 0.8 7.46 42.54 37.3 212.7 0.9 8.39 41.61 41.95 208.05 1.0 9.33 40.67 46.65 203.35 i. Prepare a 20 µM BP19 (MW 982.1) solution from 100mM stock (in DMSO) by dilution in chelexed MOPS. Make up a 100uM solution: 2 uL of 50 mM BP19 + 998 uL MOPS, Make up a 20uM solution: 800uL of 100uM + 3.2mL MOPS ii. Add to the calibration curve wells of the plate: 50 µl of 20 µM BP19/well. This brings each well to a final volume of 100ul and BP19 to a final concentration of 10 µM, mix (e.g. with multichannel pipette), incubate 10-15min (when quenching of the fluorescence is achieved) at 37°C. iii. Continue to point 4) above.

Note: The result ([µM] Fe) obtained for delta Fluorescence can then be normalized per number of cells (e.g. 106 cells) or per µg cell proteins (after measurement of protein content in each well, by Bradford assay for example).

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