Evidence of Post Translational Modification Bias Extracted from the Trna and Corresponding Amino Acid Interplay Across a Set Of

Introduction

Methods

Results

Conclusions Evidence of post translational modiﬁcation bias extracted from the References tRNA and corresponding amino acid interplay across a set of diverse Thanks To organisms

Oliver Bonham-Carter, Ishwor Thapa, Dhundy (Kiran) Bastola

School of Interdisciplinary Informatics University of Nebraska at Omaha, Omaha, NE, 68182 USA

20 September 2014

1/26 A Tailored Customization

Introduction

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Conclusions

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Thanks To

1/26 PTMs in the Central Dogma of Biology

Introduction

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Thanks To

Post Translational Modiﬁcations (PTMs)arestepsin biosynthesis to create functional proteins

2/26 PTMs?

Introduction

Methods

Results

Conclusions

References

Thanks To Protein regulators Quick protein adaptation: Enable protein to overcome stress conditions Restore protein to original form after stress has elapsed Similar kinds of functionality across PTMs: Gene expression: acetylation, glycosylation, etc. Protein regulating: phosphorylation, sumoylation, etc.

3/26 Transfer RNAs (tRNAs): Amino Acid Delivery

Introduction

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Thanks To

Each Amino Acid (AA) is added by a unique tRNA to protein sequence during translation

4/26 A Unique tRNA For Each AA

Introduction

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tRNAs have unique codons to deliver the same AAs If a tRNA is unavailable, the delivery of its AAs in protein sequence is impossible 5/26 Distribution of tRNAs

Introduction

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Adapter molecules between the mRNA sequence and peptide sequence (protein) Di↵erent tRNA compositions across genomes (Codon Usage Database: http://www.kazusa.or.jp/codon/) 6/26 PTMs Interact With Protein at Reaction Sites

Introduction

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Reaction Site: a point of reaction between proteins and PTMs (lysine for acetylation). Lysine is a speciﬁc amino acid that targets PTM interaction 7/26 PTMs Are Not Used Consistently Across Organisms

Introduction

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Results

Conclusions

References

Thanks To

Khoury et al. Proteome-wide post-translational modiﬁcation statistics. http://selene.princeton.edu/PTMCuration/

8/26 A Connection Between tRNAs, AAs and PTMs

Introduction

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Unique tRNAs responsible for delivering speciﬁc AAs AAs form the speciﬁc reaction sites where PTMs interact with protein The availability of tRNAs may impact which PTMs are able to interact with protein.

9/26 Cellular Stress and Mt

Introduction

Methods

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Mt is ﬁrst and then nuclear processes respond to stresses (heat shock, etc.) Energy is required to drive the PTM step to adapt proteins

Zhoa et al. “A mitochondrial speciﬁc stress response in mammalian cells” 10 / 26 Research Interest

Introduction

Methods

Results Conclusions tRNA usage a↵ects AA composition which makes it References possible for PTMs to react with protein Thanks To Stress Mt: Energy makers and ﬁrst to respond to stress Protein adaptation by PTMs

What is the di↵erence between AA, PTMs and tRNAs of ﬁrst and second responders (Mt and non-Mt)? In terms of AA, tRNA, PTMs: proﬁle Mt and non-Mt proteins.

11 / 26 Global Snap-Shot: Diverse Organisms of Mt

Introduction

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Diverse organisms of Mt: Observed trends may be global 12 / 26 Data Collection

Introduction

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Collected over mitochondrial (Mt) and nonMt proteins 13 / 26 Three Frequency Equations for Three Questions

Introduction Composition of PTM content in Mt versus nonMt Methods protein? Results count(PTM(i,j)) freq(PTM )= N Conclusions (i,j) (PTMs) i=1 count(PTM(i,j)) References P Thanks To Composition of AA content in Mt versus nonMt protein?

count(aminoAcid(i,j)) freq(aminoAcid(i,j))= N(actSites) i=1 count(actSite(i,j)) P Composition of PTM reaction sites in Mt versus nonMt protein?

count(aminoAcid(i,j)) freq(ReactionSite(i,j))= N P roteins Seq | i=1 (i,j)| P For (i, j)=(element[i],organism[j]) N = size of set count() function returns number of an element in set of

14 / 26 size N First Equation

Introduction

Methods

Results

Conclusions

References

Thanks To Composition of PTM content in Mt versus nonMt protein?

count(PTM(i,j)) freq(PTM(i,j))= N(PTMs) i=1 count(PTM(i,j)) P

15 / 26 PTMs: Types and Magnitudes Across Organisms Mt: More signiﬁcant PTMs; Glycosylation and Phoshoserine predominance

Introduction

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x-axis: Organisms, y-axis: PTMs, Grid: Freqs > 0.1 value threshold. 16 / 26 Second Equation

Introduction

Methods

Results

Conclusions

References

Thanks To Composition of AA content in Mt versus nonMt protein?

count(aminoAcid(i,j)) freq(aminoAcid(i,j))= N(actSites) i=1 count(actSite(i,j)) P

17 / 26 AA: Types and Magnitudes Across Organisms Mt: Fewer reaction sites, similar freqs: H. Sapiens to R. norvegicus

Introduction

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x-axis: Organisms, y-axis: PTMs, Grid: Freqs > 0.1 value threshold. 18 / 26 Third Equation

Introduction

Methods

Results

Conclusions Composition of PTM reaction sites in Mt versus nonMt References protein? Thanks To count(aminoAcid(i,j)) freq(ReactionSite(i,j))= N P roteins Seq | i=1 (i,j)| P

Equation 3 Provides Edges: Describe relationships between AAs, tRNAs and PTMs

19 / 26 Caenorhabditis elegans: Mt and non-Mt Networks Mt: Less PTMs, AA

Introduction

Methods N6−lipoyllysine Tele−8alpha−FAD S histidine Results N6−biotinyllysine Q H Conclusions N−acetylserine References N6−acetyllysine K N5−methylglutamine Thanks To N6−(pyridoxal Mt phosphate)lysine

Phenylalanine O−(pantetheine amide Phosphoserine 4’−phosphoryl)serine Phosphothreonine N6−methyllysine Phosphotyrosine R N6−methylated S Pyrrolidone lysine T N6−carboxylysine carboxylic Q W N6−biotinyllysine acid Pyruvic acid (Ser) U N6−acetyllysine S−8alpha−FAD P N6−(pyridoxal cysteine Y phosphate)lysine Selenocysteine Tryptophan N N6,N6−dimethyllysine amide Tyrosine A N6,N6,N6−trimethyllysine amide M 1−thioglycine C N5−methylglutamine 2,3−didehydroalanine (Ser) N−acetylthreonine 3−oxoalanine K (Cys) D 5−glutamyl H N−acetylserine glycerylphosphorylethanolamine G E N−acetylmethionine Alanine F amide Asymmetric N−acetylaspartate dimethylarginine Cysteine N−acetylalanine methyl Lipidation ester Hypusine Diphthamide Hydroxyproline Disulfide Glycyl bond adenylate Glutamine Glycosylation Glycine amide amide non-Mt

Nodes: PTMs (left) node size is freq magnitude, tRNAs (right) Edges: active site freq, thickness magnitude of active site interactions

20 / 26 Arabidopsis thaliana: Mt and non-Mt Networks Mt: Less PTMs, AA

Introduction Phosphoserine Phosphohistidine Phosphothreonine T O−(pantetheine S−glutathionyl S 4’−phosphoryl)serine cysteine N Methods N6−lipoyllysine S−nitrosocysteine V

Tele−8alpha−FAD N6−carboxylysine Results histidine M A 2,3−didehydroalanine N6−biotinyllysine (Cys) Conclusions K C N6−(pyridoxal Disulfide phosphate)lysine bond H References G N6,N6,N6−trimethyllysine Glycosylation

N−acetylvaline N−acetylalanine Thanks To N−acetylserine N−acetylglycine N−acetylmethionine Mt

Phosphoserine Phosphohistidine Phosphothreonine Omega−N−methylated arginine Phosphotyrosine O−(pantetheine Pros−8alpha−FAD 4’−phosphoryl)serine histidine R S T N6−methyllysine Pyrrolidone N6−carboxylysine carboxylic Pyruvic V N6−biotinyllysine acid acid (Ser) Q N6−acetyllysine S−(dipyrrolylmethanemethyl)cysteine N6−(pyridoxal S−4a−FMN Y phosphate)lysine cysteine S−8alpha−FAD P N6,N6−dimethyllysine cysteine S−glutathionyl N6,N6,N6−trimethyllysine A cysteine N N2−acetylarginine S−nitrosocysteine C N−methylalanine Sulfotyrosine M N−formylmethionine 1−thioglycine K D N−acetylvaline 2’,4’,5’−topaquinone H G 2,3−didehydroalanine N−acetylthreonine (Ser) N−acetylserine 4−aspartylphosphate

N−acetylmethionine 4−hydroxyproline

N−acetylglycine ADP−ribosylarginine Asymmetric N−acetylalanine dimethylarginine Cysteine N,N−dimethylalanine methyl N,N,N−trimethylalanine Cysteine ester Lipidation sulfenic Cysteine Hypusine acid sulfinic Hydroxyproline (−SOH) acid Glycyl Deamidated (−SO2H) adenylate asparagine Glycosylation Disulfide non-Mt bond

Nodes: PTMs (left) node size is freq magnitude, tRNAs (right) Edges: active site freq, thickness magnitude of active site interactions 21 / 26 Homo sapiens: Mt and non-Mt Networks Mt: Less PTMs, AA

Introduction Phosphoserine Phosphotyrosine Phosphothreonine Pyrrolidone Omega−N−methylarginine carboxylic O−(pantetheine acid Pyruvic S 4’−phosphoryl)serine R Nitrated acid (Ser) T Methods tyrosine S−8alpha−FAD N6−succinyllysine cysteine Q Y S−glutathionyl N6−methyllysine cysteine U S−nitrosocysteine Results N6−malonyllysine Selenocysteine N A Symmetric N6−lipoyllysine dimethylarginine Tele−8alpha−FAD N6−carboxylysine M C Conclusions histidine N6−biotinyllysine ADP−ribosylcysteine K G Asymmetric H N6−acetyllysine dimethylarginine Cysteine N6−(pyridoxal Cysteinemethyl phosphate)lysine References sulfinic ester N6,N6−dimethyllysine acid (−SO2H) Deamidated N6,N6,N6−trimethyllysine asparagine N5−methylglutamine Disulfide Thanks To bond N−acetylthreonine Glycosylation N−acetylserine Lipidation N−acetylmethionine N−acetylglycine Methylhistidine N−acetylcysteine N−acetylalanine Mt O−acetylserine O−(pantetheine Omega−N−methylarginine 4’−phosphoryl)serineO− AMP−tyrosine O−acetylthreonine Omega−N−methylated O−AMP−threonine arginine O−(2−cholinephosphoryl)serine Phenylalanine Nitrated amide tyrosine PhosphohistidinePhosphothreonine N6−succinyllysine Phosphoserine N6−methyllysine Phosphotyrosine N6−methylated PolyADP−ribosyl R S T lysine Proline N6−malonyllysine glutamic acid amide Q V N6−crotonyl−L−lysine Pros−methylhistidine N6−carboxylysine W N6−biotinyllysine Pyrrolidone Pyrrolidone carboxylic P N6−1N6−carboxyethyl−acetyllysine carboxylic Pyruvic acid (Glu) Y lysine acid acid (Ser) N6−(retinylidene)lysine S−(dipyrrolylmethanemethyl)cysteine N6−(pyridoxal S−8alpha−FAD N U phosphate)lysine N6,N6−dimethyllysine cysteine S−cysteinyl N6,N6,N6−trimethyllysine cysteine S−glutathionyl M N5−methylglutamine A cysteine N4,N4−dimethylasparagine S−methylcysteine N−pyruvate S−nitrosocysteine 2−iminyl−valine Selenocysteine C N−methylserine Sulfoserine L N−methylglycine Sulfotyrosine Symmetric N−acetylvaline K D dimethylarginine N−acetylthreonine Tele−methylhistidine I E N−acetylserine Thyroxine H F N−acetylproline Triiodothyronine G Tyrosine N−acetylmethionine amide Valine N−acetylglycine amide N−acetylglutamate (3R)−3−hydroxyasparagine N−acetylcysteine (3R)−3−hydroxyaspartate N−acetylaspartate (3S)−3−hydroxyasparagine N−acetylalanine (3S)−3−hydroxyaspartate N,N−dimethylserine (3S)−3−hydroxyhistidine N,N−dimethylproline 1−thioglycine N,N−dimethylglycine 2’,4’,5’−topaquinone 2,3−didehydroalanine N,N,N−trimethylserine (Ser) N,N,N−trimethylglycine 3−hydroxyasparagine N,N,N−trimethylalanine 3−hydroxyproline Methionine 3−oxoalanine sulfoxide Methionine (Cys) amide 4−carboxyglutamate Methionine Lysine 4−hydroxyproline (R)−sulfoxide amide 5−glutamyl Leucine glycerylphosphorylethanolamine Lipidation 5−hydroxylysine methyl Isoleucine ADP−ribosylarginine Leucine ester ADP−ribosylasparagine amide amide Hydroxyproline ADP−ribosylcysteine Hypusine ADP−ribosylserine Glycosylation Glycyl Glutamine Arginine Allysine Glycine adenylate amide amide Aspartate amide Asparagine1 −(chondroitin Glutamic amide Asymmetric 4−sulfate)− acid Cysteine ester 1−amide Dimethylated persulfide dimethylarginine Disulfide Beta−decarboxylated arginine bond aspartate Deamidated Diphthamide Blocked asparagine amino end Deamidated Citrulline (Ser) glutamine Cysteine Blocked Cysteine methyl amino end sulfenic ester (Thr) acid non-Mt (−SOH)

Nodes: PTMs (left) node size is freq magnitude, tRNAs (right) Edges: active site freq, thickness magnitude of active site interactions 22 / 26 Some of the Conclusions

Introduction

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Results

Conclusions

References Particulars Thanks To Protein PTMs Reaction Sites Networks Type per PTM Mt Few Few Sparse and organized Non-Mt Many Many Dense, disorganized and messy

23 / 26 Some of the Conclusions

Introduction

Methods

Results

Conclusions

References

Thanks To Overall PTMs reacting with AAs of few tRNAs (ex: Tryptophan W) also reacted with other reaction sites Study: Proﬁle of Mt and non-Mt PTMs and reaction sites Future work: To study ﬁrst e↵ects of stress on protein reaction sites by PTMs

24 / 26 References: Cited in this Presentation

Introduction

Methods

Results

Conclusions Zhao, Quan, et al. “A mitochondrial speciﬁc stress References

Thanks To response in mammalian cells.” The EMBO journal 21.17 (2002): 4411-4419. Khoury, George A., Richard C. Baliban, and Christodoulos A. Floudas. “Proteome-wide post-translational modiﬁcation statistics: frequency analysis and curation of the swiss-prot database.” Scientiﬁc reports 1 (2011). Codon Usage Database: http://www.kazusa.or.jp/codon/

25 / 26 Introduction We would like to thank the support students, faculty and sta↵ in the Methods UNO-Bioinformatics Core Facility. This project has been funded by Results the grants from the National Center for Research Resources Conclusions (5P20RR016469), the National Institute for General Medical Science References (NIGMS) (8P20GM103427). Thanks To This study also funded by the NASA Nebraska Space Grant (2014). Thank You! Questions?

[email protected] IS&T Bioinformatics http://bioinformatics.ist.unomaha.edu/

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