MMP-25 Metalloprotease Regulates Innate Immune Response Through NF- Κb Signaling Clara Soria-Valles, Ana Gutiérrez-Fernández, Fernando G

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MMP-25 Metalloprotease Regulates Innate Immune Response through NF- κB Signaling Clara Soria-Valles, Ana Gutiérrez-Fernández, Fernando G. Osorio, Dido Carrero, Adolfo A. Ferrando, Enrique Colado, This information is current as M. Soledad Fernández-García, Elena Bonzon-Kulichenko, of September 27, 2021. Jesús Vázquez, Antonio Fueyo and Carlos López-Otín J Immunol 2016; 197:296-302; Prepublished online 3 June 2016;

doi: 10.4049/jimmunol.1600094 Downloaded from http://www.jimmunol.org/content/197/1/296

Supplementary http://www.jimmunol.org/content/suppl/2016/06/01/jimmunol.160009 Material 4.DCSupplemental http://www.jimmunol.org/ References This article cites 41 articles, 15 of which you can access for free at: http://www.jimmunol.org/content/197/1/296.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

MMP-25 Metalloprotease Regulates Innate Immune Response through NF-kB Signaling

Clara Soria-Valles,* Ana Gutie´rrez-Fernańdez,* Fernando G. Osorio,* Dido Carrero,* Adolfo A. Ferrando,† Enrique Colado,‡ M. Soledad Fernańdez-Garcıá,x Elena Bonzon-Kulichenko,{ Jesu´sVa´zquez,{ Antonio Fueyo,‖ and Carlos Lo´pez-Otıń*

Matrix metalloproteases (MMPs) regulate innate immunity acting over proinflammatory cytokines, chemokines, and other immune- related proteins. MMP-25 (membrane-type 6-MMP) is a membrane-bound enzyme predominantly expressed in leukocytes whose biological function has remained largely unknown. We have generated Mmp25-deficient mice to elucidate the in vivo function of this protease. These mutant mice are viable and fertile and do not show any spontaneous phenotype. However, Mmp25-null mice exhibit a defective innate immune response characterized by low sensitivity to bacterial LPS, hypergammaglobulinemia, and reduced secretion of proinflammatory molecules. Moreover, these immune defects can be tracked to a defective NF-kB activation observed in Mmp25-deficient Downloaded from leukocytes. Globally, our findings provide new mechanistic insights into innate immunity through the activity of MMP-25, suggesting that this proteinase could be a potential therapeutic target for immune-related diseases. The Journal of Immunology, 2016, 197: 296–302.

nflammation is part of the complex biological response of their structure in secreted and membrane-anchored proteases. Among innate immunity, which confers immediate defense against in- the membrane-type (MT)-MMPs, there are three different groups I fection protecting multicellular organisms from pathogens (1). depending on the way these proteases interact with the membrane. http://www.jimmunol.org/ Septic shock, induced by the LPS of Gram-negative bacteria, is a This interaction may be through a transmembrane domain, consequence of a disproportionate stimulation of host immune cells. through a GPI moiety, or through an N-terminal signal anchor (6). Leukocytes have a pivotal role on sensing the pathogens that invade The metalloprotease MT6-MMP or MMP-25 is anchored to the cell the organism and initiate the inflammatory response (1). However, membrane through a GPI moiety and is predominantly expressed in their activation must be precisely controlled to regulate the duration leukocytes, lung, and spleen (7, 8). Previous works have also shown and extension of the inflammation, and therefore protect the host that both classical and alternative activation of macrophages in- from tissue damage. crease MMP-25 steady-state mRNA levels (9). In vitro functional Matrix metalloproteinases (MMPs) are a family of zinc-dependent experiments have revealed that this metalloprotease is able to clear by guest on September 27, 2021 endopeptidases that cleave extracellular matrix components with wide components of the extracellular matrix such as type IV collagen, substrate specificity (2, 3) and are involved in multiple pathological fibronectin, fibrin, and gelatin, and its activity is tightly regulated by processes (4, 5). These enzymes have been classified according to tissue inhibitor of metalloproteinase-1 (10). MMP-25 has also been linked with some pathological processes such as multiple sclerosis or cancer, but its biological role has remained largely unknown (11, *Departamento de Bioquı´mica y Biologıá Molecular, Facultad de Medicina, Instituto 12). In this work, and as part of our long-term studies aimed at Universitario de Oncologıá, Universidad de Oviedo, 33006 Oviedo, Spain; †Institute for generating mouse models of protease deficiency (5, 13–16), we Cancer Genetics, Columbia University, New York, NY 10032; ‡Servivio de Hematologıá, Hospital Universitario Central de Asturias, 33011 Oviedo, Spain; xServicio de Anatomıá describe the generation of Mmp25-deficient mice and evaluate the Patolo´gica, Hospital Universitario Central de Asturias, 33011 Oviedo, Spain; {Laboratorio biological function of MT6-MMP in innate immunity. de Proteo´mica Cardiovascular, Centro Nacional de Investigaciones Cardiovasculares, 28029 Madrid, Spain; and ‖A´ rea de Fisiologıá, Departamento de Biologıá Funcional, Facultad de Medicina, Instituto Universitario de Oncologıá, Universidad de Oviedo, 33006 Oviedo, Materials and Methods Spain Generation of Mmp25 knockout mice ORCIDs: 0000-0001-7869-838X (D.C.); 0000-0001-8675-8207 (E.C.); 0000-0003- 0852-7520 (E.B.-K.); 0000-0003-1461-5092 (J.V.). Mmp25-targeted ESCs clones from Texas A&M Institute for Genomic Medicine were microinjected into C57BL/6 mouse blastocysts to produce Received for publication January 19, 2016. Accepted for publication May 2, 2016. chimeric mice that were then subsequently crossed with C57BL/6 mice to This work was supported by grants from Ministerio de Economıá y Competitividad generate Mmp25-heterozygous mice. Mice genotyping was performed by (Spain) and Red Tema´tica de Investigacioń Cooperativa de Centros de Cancer-Spain. PCR with the following oligonucleotides: 59- CCTGATCAAGTTCTTG- The Instituto Universitario de Oncologıá is supported by Fundacioń Cajastur-Asturias. CTTGC-39;59-ATGGCTCGGAGTCTTTAAAC-39; and 59-CCAATAA- This work was also supported through the generous support of J.I. Cabrera. C.L-O. is an investigator of the Botin Foundation supported by Banco Santander through its Santander ACCCTCTTGCAGTTGC-39. The PCR products consisted in fragments of Universities Global Division. E.B.-K. and J.V. are supported by La Red de Investigacioń 309 bp (knockout-allele) and 531 bp (wild-type allele). Cardiovascular–Las Redes Tema´ticas de Investigacioń Cooperativa en Salud, Fondo de Investigaciones Sanitarias, and Instituto de Salud Carlos III. Mice procedures Address correspondence and reprint requests to Dr. Carlos Lo´pez-Otıń, Departamento Eight- to 10-wk-old male mice received an i.p. injection of LPS (20 mg/kg, de Bioquı´mica y Biologıá Molecular, Facultad de Medicina, Universidad de Oviedo, serotype O55:B5; Sigma-Aldrich) dissolved in saline buffer, and survival 33006 Oviedo, Spain. E-mail address: [email protected]. was evaluated. Isolation of neutrophils from mouse bone marrow was The online version of this article contains supplemental material. performed as described previously (17). Briefly, after flushing bone marrow cells from mice tibias, they were layered on a Percoll gradient Abbreviations used in this article: HA, hemagglutinin; MMP, matrix metalloprotease; 3 MT, membrane-type; SLAM, signaling lymphocyte activation molecule; TRAF6, (80:65:50%), centrifuged for 30 min at 500 g, and recovered from the TNFR-associated factor 6; UEV1A, ubiquitin-conjugating enzyme E2 variant 1A. third layer of the gradient. For the isolation of peritoneal macrophages, mice were injected with 1 ml 10% Brewer thioglycollate medium as de- Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 scribed previously (18). Three days after injection, peritoneal macrophages www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600094 The Journal of Immunology 297 Downloaded from

FIGURE 1. Generation of Mmp25-deficient mice. (A) Schematic representation of Mmp25-targeted allele. Genotyping strategy is indicated as well as the position of the inserted cassette. (B) PCR analysis using Mmp25-specific oligonucleotides, genomic DNA from Mmp25+/+, Mmp25+/2 and Mmp252/2 mice were used as template. (C) Representative picture of 6-mo-old wild-type and Mmp25-deficient mice. Original magnification 30.3. (D) Longevity analysis of +/+ 2/2 E wild-type and Mmp25-deficient mice. Kaplan–Meier survival plot of Mmp25 (n = 10) and Mmp25 (n = 10) mice. ( ) Quantitative RT-PCR of Mmp25 http://www.jimmunol.org/ in bone marrow, lung, and spleen from wild-type (n = 4) and Mmp25-deficient (n = 4) mice. Mean relative mRNA levels are shown. *p , 0.05, **p , 0.01, two-tailed Student t test. were obtained by washing of the peritoneal cavity with RPMI 1640 medium from Addgene. HEK-293T cell line was purchased from the American Type containing 10% FBS and heparin. Determination of nitrite production in Culture Collection and maintained in culture following the provider’s macrophages was performed using Griess Reagent (Molecular Probes) fol- specifications. Transient transfections were performed using Lipofectamine lowing manufacturer instructions. Bone marrow transplantations were per- reagent (Life Technologies), and proteomic analyses were performed 48 h formed as described previously (19). Briefly, recipient mice were treated after transfection. Firefly luciferase under NF-kB promoter was transduced with 25 mg/kg/day busulfan (Sigma-Aldrich) for 4 d, followed by injection in HEK-293T cell line either with pcDNA-MMP25 or pcDNA3. of 200 mg/kg cyclophosphamide (Sigma-Aldrich). Twenty-four hours after by guest on September 27, 2021 last injection, bone marrow was collected from femurs and tibias of donor RNA and protein analysis mice by flushing the dissected bones with HBSS. Cell suspension was fil- Nuclear extracts and EMSA were performed as described previously (22). m 3 6 tered through 100- m filters and counted. A total of 2 10 cells were Briefly, NF-kB consensus oligonucleotides (Promega) were radiolabeled resuspended in HBSS and then injected in recipient animals via the jugular by T4 polynucleotide kinase (New England Biolabs) in the presence of vein. Eight weeks later, the transplanted animals received an i.p. injection of g-[32P]ATP. Labeled oligonucleotides were incubated with 15–20 mgnuclear LPS (20 mg/kg), and survival was evaluated. Hydrodynamic injection of a extracts in 13 binding buffer (43 glycerol, 20 mM Tris-HCl [pH 8], 60 mM plasmid encoding firefly luciferase under NF-kB promoter was carried out as NaCl, 5 mM MgCl2, 1 mM DTT, and 13 protease inhibitor mixture) in the described previously (20). For histological analysis, tissues were fixed in 4% presence of poly(deoxyinosinic-deoxycytidylic) acid. Complexes were run buffered paraformaldehyde solution. Paraffin sections were stained with H&E. in nondenaturing 6% acrylamide gels and exposed to X-ray detector Fuji All of the animal experiments were performed in accordance with the guidelines phosphorimager (Fujifilm Global). Total RNA from cells and tissue samples of the Committee for Animal Experimentation of the Universidad de Oviedo. were extracted using TRIzol reagent (Life Technologies) and processed FACS and blood analysis through alcohol precipitation. RNA pellets were then washed in cold 75% ethanol and resuspended in nuclease-free water (Life Technologies). The Blood was extracted directly from the mandibular sinus after anesthetizing samples were quantified and evaluated for purity (260/280-nm ratio) with a mice with isoflurane. Analysis of primitive cells (Lin2SCA-1+c-KIT+;LSK+), NanoDrop ND-1000 spectrophotometer. cDNA was synthesized with 1–4 mg committed progenitors (Lin2SCA-12c-KIT+;LSK2), and signaling lym- total RNA with the ThermoScript RT-PCR system (Invitrogen). RT-quantitative phocyte activation molecule (SLAM)-enriched long-term hematopoietic PCR was performed in triplicate for each experimental condition using stem cells (21) was performed by FACS using the following mAbs: PE anti- either TaqMan PCR Mastermix or SYBR Green PCR Master mix (Applied mouse CD117 (105807), PerCP/Cy5.5 anti-mouse CD45 (103132), PE/Cy7 Biosystems), according to the manufacturer’s instructions. To normalize anti-mouse CD150 (115914), allophycocyanin anti-mouse CD127 (135012), mRNA levels, GAPDH or ACTB probes were used. Protein lysates for allophycocyanin/Cy7 anti-mouse CD48 (103432), Brilliant Violet 421 anti- Western blot were prepared in radioimmunoprecipitation buffer; equal mouse Ly-6A/E (Sca-1) (108127), anti-mouse CD34 (11-0341-82; eBio- amounts of total proteins were loaded onto SDS-polyacrylamide gels. science), and Lineage mixture (558074; BD Pharmingen). FACS data were After electrophoresis, gels were electrotransferred onto nitrocellulose acquired using a FACScanto II flow cytometer (BD Biosciences) and ana- membranes and incubated overnight with the different primary Abs used. lyzed using Infinicyt software (Cytognos, Santa Marta, Spain). A represen- Finally, blots were incubated for 1 h with secondary Abs conjugated with tative FACS analysis gating scheme is shown in Supplemental Fig. 1. Blood HRP to develop immunoreactive bands. The primary Abs used in the study proteins separation was carried out using manual agarose gels (Hydragel were as follows: GFP (632592; BD Clontech), TLR4 (sc-293072; Santa K20; Sebia) and quantified by densitometry analysis. Serum IL-1a was Cruz Biotechnology), TNFR-associated factor 6 (TRAF6) (sc-7221; Santa determined by ELISA. A pool of four animal sera per condition was hy- Cruz Biotechnology), ubiquitin-conjugating enzyme E2 variant 1A (UEV1A) bridized onto mouse L308 glass slide arrays (RayBiotech). Data were (ab101476; Abcam), HA (3F10; Roche), IkBa (9242; Cell Signaling Tech- clustered using Cluster 3.0 software, and the heat maps were created using nology), and a-tubulin (T6074; Sigma-Aldrich). All the Abs were used at TreeView 1.1.5 software. 1:1000 dilution. Cell culture and transient transfection Immunoprecipitation MMP25 and TRAF6 cDNAs were amplified by PCR and either cloned into MMP-25 immunoprecipitation experiments were performed in the HEK- pcDNA3 or pEGFP. Hemagglutinin (HA)-ubiquitin plasmid was obtained 293T cell line. To this end, cells were transfected with either pEGFP-MMP25 298 MMP-25 REGULATES INNATE IMMUNITY

FIGURE 2. Characterization of Mmp25-deﬁcient mice. (A) Mean relative cell number in bone marrow from wild-type (n = 5) and knockout animals (n =5).(B)LSK+/LSK2 ratio and percentage of SLAM cells in LSK compartment of Mmp25+/+ (n =5)and Mmp252/2 (n =5)mice.(C) Blood proteinogram from Mmp25+/+ (n =4) and Mmp252/2 (n =4)animals.(D) IL-1a levels in peripheral blood from Mmp25+/+ (n =5)andMmp252/2 (n = 6) mice. Values were normal- ized to total viable cellularity. (E) Heat map represents alterations in proinﬂammatory cytokines protein levels between Mmp25+/+ (n =4)and Mmp252/2 (n =4)animals.Dataare Downloaded from displayed as log2-transformed expression signals. Error bars indicate SEM (*p , 0.05, **p , 0.01, two-tailed Student t test). GRA, granulocyte; LYM, lymphocyte; MONO, monocyte. http://www.jimmunol.org/

plasmid or pEGFP empty vector. Cells were lysed with coimmunoprecipi- and then incubated with anti-GFP–conjugated dynabeads for 1 h at 4˚C. tation buffer (150 mM NaCl, 20 mM Tris-HCl [pH 7.4], 1% Nonidet P-40, Beads were washed three times with lysis buffer, and bound proteins were 1 mM MgCl2,10%glycerol,and13 complete protease inhibitors). Protein released from beads by boiling in 23 Laemmli sample buffer. Immuno- extracts were precleared for 2 h at 4˚C with dynabeads (Life Technologies) precipitates and input samples were resolved by SDS-PAGE or subjected to by guest on September 27, 2021

FIGURE 3. Reduced innate immunity in Mmp25-deﬁcient mice. (A) Kaplan–Meier survival plot of Mmp25+/+ (n =8)andMmp252/2 (n =8)malemice after injection with 20 mg/kg LPS. p , 0.01, log-rank/Mantel–Cox test. (B) Representative images from H&E staining of lung sections of LPS- treated and untreated Mmp25+/+ (n = 6) and Mmp252/2 (n = 6) mice. Plot represents inﬂammation and edema histological score in this mice cohort. Scale bar, 50 mm. (C) Peripheral blood levels of glutamate-pyruvate transaminase (GPT), glutamic oxaloacetic transaminase (GOT), creatinine, and blood urea nitrogen (BUN) in LPS-treated and untreated Mmp25+/+ and Mmp252/2 mice (n = 6 mice/ group). Mean values are represented. (D) Kaplan–Meier survival plot of wild-type males transplanted with knockout cells (n = 5) and knockout males transplanted with wild-type bone marrow (n = 5) after injection with 20 mg/kg LPS. p , 0.01, log-rank/ Mantel–Cox test. Error bars indicate SEM (*p , 0.05, **p , 0.01, two- tailed Student t test). The Journal of Immunology 299 Downloaded from

FIGURE 4. Defective NF-kB activation in Mmp25-deficient mice. (A) NF-kB EMSA analysis in bone marrow neutrophils from untreated and LPS- 2 2 2 2 treated Mmp25+/+ and Mmp25 / mice (pool of five animals per group). (B) Representative image of bioluminescence signal in Mmp25+/+ and Mmp25 / http://www.jimmunol.org/ mice 3 d after hydrodynamic transfection. Original magnification 30.3. Plot represents bioluminescence signal quantification (n = 4/genotype). Mean values are represented as relative values of photon flux per second and square centimeter. (C–F) Quantitative RT-PCR of Csf2, Tnfa, Cox2, and Il1a in Mmp25+/+ and Mmp252/2 neutrophils after stimulation with LPS (10 mg/ml, 2 h). Mean relative mRNA levels of three replicates are shown. Error bars indicate SEM (*p , 0.05, **p , 0.01, two-tailed Student t test). protein digestion followed by nanoliquid chromatography coupled to mass The fact that Mmp25 is predominantly expressed in WBCs spectrometry for protein identification and quantification by peptide counting prompted us to analyze the hematological system in Mmp25- (23). For ubiquitination experiments, HEK-293T cells were transfected with deficient mice, but no significant differences between Mmp25+/+ HA-ubiquitin, pcDNA3-MMP25 and pEGFP-TRAF6 plasmids, and total ly- 2/2 by guest on September 27, 2021 sates were precipitated with anti-GFP Ab and immunostained with anti-HA Ab. and Mmp25 animals were observed neither in the distribution of blood cell populations (Fig. 2A) or in the hematopoietic stem Statistical analysis cell compartment (LSK+ cells and SLAMs) (Fig. 2B, Supplemental All the experimental data were collected from experiments performed in Fig. 1). However, additional analysis of blood parameters related technical triplicate; each experiment was repeated three times (unless with immune response revealed that knockout animals spontane- noted), and statistically significant results were obtained in independent ously developed hypergammaglobulinemia (Fig. 2C), showed re- biological replicates. Experimental conditions were blinded randomized, and no statistical method was used to predetermine sample size. Differences duced levels of IL-1a (Fig. 2D), an important mediator of the between groups were assayed using Microsoft Excel and SPSS statistical immunological response mainly producedbyleukocytes,and package. In all the cases, experimental data assumed t test requirements exhibited a severe reduction of several proinflammatory molecules (normal distribution and similar variance); in those cases where the as- (Fig. 2E). These findings addressed us to evaluate the potential role sumption of the t test was not valid, a nonparametric statistical method was of MMP-25 metalloprotease in immune response. used (e.g., Wilcoxon signed-rank test). Results were expressed as means and error bars indicate the SEM, as indicated in the figure legends. Mmp25-deficient mice show an impaired innate immune response Results On the basis of the above results as well as on previous data describing Mmp25-deficient mice are viable but show alterations related the ability of this metalloprotease to cleave key components of innate with innate immunity immunity such as galectin-1 or CD16 (24, 25), we focused on the To investigate the biological role of MT6-MMP, we have generated analysis of innate immunity in Mmp25-null mice. To this end, we Mmp25-deficient mice using an insertion strategy (Fig. 1A, 1B). The performed an endotoxin shock protocol based on the injection of modified allele generates a null allele through splicing to a trapping Mmp25-wild-type and knockout animals with 20 mg/kg LPS, and we element contained in the targeting cassette (Fig. 1A, 1B). Mmp25- evaluated mice survival, with the finding of a remarkable impairment targeted ESCs were microinjected into C57BL/6 mouse blastocysts in the innate immune response in knockout animals. Thus, whereas the to generate chimeric mice that were subsequently crossed with C57BL/6 survival rate of control mice was ∼23%, the survival rate of Mmp25- mice to generate Mmp25-heterozygous mice. Mmp25-deficient an- deficient mice was .63% (Fig. 3A). Accordingly, mutant mice also imals were born at the expected Mendelian ratio and developed showed reduced signs of lung inflammatory damage and intersticial normally (Fig. 1C); both males and females were fertile and did not edema (Fig. 3B) as well as reduced blood levels of liver and kidney show significant differences in survival rates as compared with the damage markers, such as glutamate-pyruvate transaminase, glutamic wild-type littermates (Fig. 1D). As expected, Mmp25-knockout oxaloacetic transaminase, and blood urea nitrogen (Fig. 3C). In animals did not produce the protease in tissues like bone marrow, agreement with the prominent expression of this metalloprotease in lung, or spleen, where wild-type animals showed the appropriate WBCs (26), reciprocal wild-type to mutant and mutant to wild-type Mmp25 expression levels in all these tissues (Fig. 1E). transplantations resembled the effect of primary donors (Fig. 3D). 300 MMP-25 REGULATES INNATE IMMUNITY Downloaded from http://www.jimmunol.org/

FIGURE 5. MMP-25 promotes TRAF6 ubiquitination. (A) Bioluminescence signal quantification of HEK-293T cells transduced with NF-kB luciferase reporter gene and either pcDNA3-MMP25 or pcDNA-empty vector. Mean relative values are represented. (B) Western blot analysis of UEV1A in total lysates and anti-MMP25-GFP immunoprecipitates from HEK-293T cells either transfected with pMMP25-GFP or pEGFP-N1. (C) Western blot analyses of TLR4, TRAF6, UEV1A, IkBa, and a-tubulin in LPS-treated (10 mg/ml, 2 h) and untreated wild-type and Mmp25-deficient cells. (D) Anti-HA Western blot analysis of GFP immunoprecipitates from HEK-293T cells transiently cotransfected with GFP-TRAF6 and HA-ubiquitin plus either MMP25 cDNA or empty vector (pcDNA3). Signal intensity was quantified, and mean values from three independent experiments are represented. Error bars indicate SEM (**p , 0.01, two-tailed Student t test).

NF-kB impaired signaling in Mmp25-deﬁcient mice marrow neutrophils isolated from wild-type and knockout ani- by guest on September 27, 2021 NF-kB signaling is a critical mediator of innate immunity upon a mals. As expected, exposure of wild-type mice to LPS resulted in plethora of stress signals (27). Among them, NF-kB’s role in LPS a remarkable activation of NF-kB in neutrophils (Fig. 4A), response has been reported extensively (18, 28, 29). Therefore, we whereas Mmp25-deﬁcient mice had a severely impaired activation decided to study the activity of this signaling pathway in bone upon LPS stimulation (Fig. 4A). Moreover, we studied in vivo the

FIGURE 6. Impaired macrophages activation in Mmp25-deﬁcient mice. (A) Relative nitrite production of Mmp25+/+ and Mmp252/2macrophages from four animals of each genotype after stimulation with 10 mg/ml LPS during 24 h. Mean relative units of three replicates are shown. (B)NF-kBEMSAanalysis in macrophages from Mmp25+/+ (n =6) and Mmp252/2 (n = 6) mice incubated or not with 10 mg/ml LPS during 1 h. (C) Quantitative RT-PCR of Csf2, Lif, and Cxcl10 in Mmp25+/+ (n =4)and Mmp252/2 (n = 4) macrophages after stimulation with LPS (10 mg/ml, 6 h). Mean relative mRNA levels of three replicates are shown. Error bars indicate SEM (*p , 0.05, **p , 0.01, two- tailed Student t test). The Journal of Immunology 301

NF-kB activity in both mutant and control mice using a reporter- new aspects of the functional relevance of MT6-MMP, demon- based assay and hydrodynamic transfection approaches. Consistent strating a critical role for this metalloproteinase in innate immunity with the above results, we found that Mmp25-deficient animals are through the control of WBCs activation. These results agree with unable to properly activate NF-kB (Fig. 4B). According to NF-kB previous findings describing the upregulation of MMP-25 in clas- defective activation, we observed a reduced expression of several sical and alternative macrophage activation pathways, pointing to key genes associated with inflammatory response, such as Csf2, the essential role of this membrane protease in these processes (9). Tnfa, Cox2,andIl1a,inMmp25-deficient mice (Fig. 4C–F). Furthermore, we have provided in this paper experimental proof about the mechanistic role of MMP-25 in NF-kB activation, MMP-25 promotes TRAF6 ubiquitination to activate NF-kB promoting the ubiquitination of TRAF6 through its direct inter- To get mechanistic insight about MMP-25 regulation of NF-kB action with the E2 ligase UEV1A. Previous works have described signaling pathway activity, we first transduced HEK-293T cells the function of MMPs in different physiological and pathological with a plasmid encoding MMP25 revealing that the expression of situations related with immune system and inflammation, such as MMP-25 in this cell line was associated with an increase in NF-kB responses to infectious and autoimmune diseases, cancer pro- activity, supporting the validity of this experimental model (Fig. 5A). gression, and wound healing (14, 34–37). Although further studies Immunoprecipitation experiments on HEK-293T cells revealed a should address the specific biochemical nature of this cross-talk complex interaction pattern of MMP-25, which included the protein between MMP-25 and NF-kB, this work has described new reg- UEV1A, a critical regulator of TRAF6 ubiquitination that activate ulatory functions for this family of metalloproteinases that could NF-kB signaling pathway (Fig. 5B, Supplemental Table 1) (30–32). be promising for exploring the functions of MMPs on different

We then analyzed some of these key components of LPS-induced mechanisms mediated by NF-kB, including those associated with Downloaded from NF-kB activation pathway, such as TLR4, TRAF6, UEV1A or the modulation of inflammation during aging processes (38–40). IkBa, in wild-type and Mmp25-deficient mice, observing a remark- Moreover, our results describing novel MMP-25 interactions may able increase of TRAF6 expression because of Mmp25 deficiency contribute to identify new specific therapeutic targets in sepsis and (Fig. 5C). These results made us hypothesize about a direct regulation other disorders related to innate immunity impairment that could of MMP-25 over TRAF6 activity and ubiquitination. To test this, we be more effective and avoid the alteration of the beneficial effects

overexpressedaGFP-TRAF6fusionproteininHEK-293Tcells that this MMP may have in different physiological contexts. Fi- http://www.jimmunol.org/ along with HA-tagged ubiquitin, and either with pcDNA-MMP25 or nally, although it is known that leukocytes can modulate the im- pcDNA-empty vector, observing a .2-fold increase in TRAF6 ubiq- munological response by secreting MMPs, the roles of MT-MMPs uitination as a consequence of MMP-25 expression (Fig. 3D). These had not been deeply explored yet (41). In this sense, Mmp25-deﬁcient results ﬁrst describe the precise molecular mechanism through which mice may constitute an extremely valuable experimental model to MMP-25 may regulate NF-kB activation. further study innate immunity regulation through the ability of this MMP-25 regulates macrophage activation through NF-kB metalloprotease to modify WBCs activity. signaling Acknowledgments

Because MMP-25 is highly expressed in macrophages and these by guest on September 27, 2021 We thank F. Rodrı´guez, A. Moyano, R. Feijoo, S. A´ lvarez, D. A. Puente, cells show an important role in the initiation of the innate im- and C. Garabaya for excellent technical assistance and the Servicio de mune response (33), we also analyzed peritoneal macrophages of +/+ 2/2 Histopatologıá (Instituto Universitario de Oncologıá) for histological Mmp25 and Mmp25 mice. Thus, macrophages from wild- preparations. type and Mmp25-deficient mice were isolated, primed with IFN-g, and exposed to LPS (10 mg/ml). Nitrite production was assessed in Disclosures the cultures to monitor macrophage activity (29), observing that The authors have no financial conflicts of interest. the nitrite production in Mmp252/2 macrophages was 2-fold lower than in wild-type macrophages, which suggests intrinsic defects in macrophage activation capacity (Fig. 6A) caused by NF-kBim- References 2 2 pairment (Fig. 6B). We also observed that Mmp25 / macrophages 1. Newson, J., M. Stables, E. Karra, F. Arce-Vargas, S. Quezada, M. Motwani, M. Mack, show a compromised production of inflammatory mediators con- S. Yona, T. Audzevich, and D. W. Gilroy. 2014. Resolution of acute inflammation bridges the gap between innate and adaptive immunity. Blood 124: 1748–1764. trolled by NF-kB after LPS stimulation. Thus, we analyzed some 2. Bonnans, C., J. Chou, and Z. Werb. 2014. Remodelling the extracellular matrix macrophage LPS-characteristic inflammatory mediators such as in development and disease. Nat. Rev. Mol. Cell Biol. 15: 786–801. 3. Puente, X. S., L. M. Sańchez, C. M. Overall, and C. Lo´pez-Otıń. 2003. Human and Csf2, Lif,andCxcl10 (Fig. 6C) and found a reduced expression of mouse proteases: a comparative genomic approach. Nat. Rev. Genet. 4: 544–558. these genes in Mmp25-deficient macrophages as compared with 4. Soria-Valles, C., A. Gutie´rrez-Fernańdez, M. Guiu, B. Mari, A. Fueyo, controls. R. R. Gomis, and C. Lo´pez-Otıń. 2014. The anti-metastatic activity of collagenase-2 in breast cancer cells is mediated by a signaling pathway involving decorin and miR-21. Oncogene 33: 3054–3063. 5. Gutie´rrez-Fernańdez, A., C. Soria-Valles, F. G. Osorio, J. Gutie´rrez-Abril, Discussion C. Garabaya, A. Aguirre, A. Fueyo, M. S. Fernańdez-Garcıá, X. S. Puente, and Globally, our results reveal that the lack of the membrane-bound C. Lo´pez-Otıń. 2015. Loss of MT1-MMP causes cell senescence and nuclear metalloproteinase MT6-MMP impairs NF-kB activation, a criti- defects which can be reversed by retinoic acid. EMBO J. 34: 1875–1888. 6. Fanjul-Fernańdez, M., A. R. Folgueras, S. Cabrera, and C. Lo´pez-Otıń. 2010. cal mediator of immune response (1), connecting defective immune Matrix metalloproteinases: evolution, gene regulation and functional analysis in response with reduced cytokine release in WBCs, and therefore mouse models. Biochim. Biophys. Acta 1803: 3–19. explaining the higher survival rate of Mmp25-deficient mice after 7. Velasco, G., S. Cal, A. Merlos-Sua´rez, A. A. Ferrando, S. Alvarez, A. Nakano, J. Arribas, and C. Lo´pez-Otıń. 2000. Human MT6-matrix metalloproteinase: LPS administration. To raise this conclusion, it has been necessary identification, progelatinase A activation, and expression in brain tumors. Cancer to generate this new strain of mutant mice because the mouse model Res. 60: 877–882. 8. Kojima, S., Y. Itoh, S. Matsumoto, Y. Masuho, and M. Seiki. 2000. Membrane- deficient in MMP-25 (MT6-MMP) was one of the very few in vivo type 6 matrix metalloproteinase (MT6-MMP, MMP-25) is the second glycosyl- murine models of MMP deficiency that was not yet available. phosphatidyl inositol (GPI)-anchored MMP. FEBS Lett. 480: 142–146. Similar to most MMP-deficient mice (13, 14), Mmp25-null mice are 9. Huang, W. C., G. B. Sala-Newby, A. Susana, J. L. Johnson, and A. C. Newby. 2012. Classical macrophage activation up-regulates several matrix metalloproteinases fertile and viable and do not exhibit apparent phenotype abnor- through mitogen activated protein kinases and nuclear factor-kB. PLoS One malities. However, it is remarkable that our studies have unveiled 7: e42507. 302 MMP-25 REGULATES INNATE IMMUNITY

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Peptide counting FastaProtein MMP25 GFP >sp|P49327|FAS_HUMAN Fatty acid synthase OS=Homo sapiens GN=FASN PE=1 SV=3 37 12 >sp|P21333|FLNA_HUMAN Filamin‐A OS=Homo sapiens GN=FLNA PE=1 SV=4 31 8 >sp|Q14204|DYHC1_HUMAN Cytoplasmic dynein 1 heavy chain 1 OS=Homo sapiens GN=DYNC1H1 PE=1 SV=5 28 0 >sp|Q00610|CLH1_HUMAN Clathrin heavy chain 1 OS=Homo sapiens GN=CLTC PE=1 SV=5 25 6 >sp|P53396|ACLY_HUMAN ATP‐citrate synthase OS=Homo sapiens GN=ACLY PE=1 SV=3 24 11 >sp|P78527|PRKDC_HUMAN DNA‐dependent protein kinase catalytic subunit OS=Homo sapiens GN=PRKDC PE=1 SV=3 22 0 >sp|O75643|U520_HUMAN U5 small nuclear ribonucleoprotein 200 kDa helicase OS=Homo sapiens GN=SNRNP200 PE=1 SV=2 22 2 >sp|P42704|LPPRC_HUMAN Leucine‐rich PPR motif‐containing protein, mitochondrial OS=Homo sapiens GN=LRPPRC PE=1 SV=3 22 3 >sp|P07814|SYEP_HUMAN Bifunctional glutamate/proline‐‐tRNA ligase OS=Homo sapiens GN=EPRS PE=1 SV=5 20 2 >sp|P35579|MYH9_HUMAN Myosin‐9 OS=Homo sapiens GN=MYH9 PE=1 SV=4 20 9 >sp|P11586|C1TC_HUMAN C‐1‐tetrahydrofolate synthase, cytoplasmic OS=Homo sapiens GN=MTHFD1 PE=1 SV=3 18 9 >sp|O00410|IPO5_HUMAN Importin‐5 OS=Homo sapiens GN=IPO5 PE=1 SV=4 17 2 >sp|O75369|FLNB_HUMAN Filamin‐B OS=Homo sapiens GN=FLNB PE=1 SV=2 17 2 >sp|Q15029|U5S1_HUMAN 116 kDa U5 small nuclear ribonucleoprotein component OS=Homo sapiens GN=EFTUD2 PE=1 SV=1 17 6 >sp|Q9Y490|TLN1_HUMAN Talin‐1 OS=Homo sapiens GN=TLN1 PE=1 SV=3 16 0 >sp|Q92616|GCN1L_HUMAN Translational activator GCN1 OS=Homo sapiens GN=GCN1L1 PE=1 SV=6 16 2 >sp|P54136|SYRC_HUMAN Arginine‐‐tRNA ligase, cytoplasmic OS=Homo sapiens GN=RARS PE=1 SV=2 15 6 >sp|O15067|PUR4_HUMAN Phosphoribosylformylglycinamidine synthase OS=Homo sapiens GN=PFAS PE=1 SV=4 13 2 >sp|P26640|SYVC_HUMAN Valine‐‐tRNA ligase OS=Homo sapiens GN=VARS PE=1 SV=4 13 2 >sp|O75533|SF3B1_HUMAN Splicing factor 3B subunit 1 OS=Homo sapiens GN=SF3B1 PE=1 SV=3 13 3 >sp|P35580|MYH10_HUMAN Myosin‐10 OS=Homo sapiens GN=MYH10 PE=1 SV=3 13 5 >sp|P49588|SYAC_HUMAN Alanine‐‐tRNA ligase, cytoplasmic OS=Homo sapiens GN=AARS PE=1 SV=2 13 6 >sp|Q9NPA2|MMP25_HUMAN Matrix metalloproteinase‐25 OS=Homo sapiens GN=MMP25 PE=1 SV=1 12 0 >sp|Q8TEX9|IPO4_HUMAN Importin‐4 OS=Homo sapiens GN=IPO4 PE=1 SV=2 12 0 >sp|Q86VP6|CAND1_HUMAN Cullin‐associated NEDD8‐dissociated protein 1 OS=Homo sapiens GN=CAND1 PE=1 SV=2 12 3 >sp|Q08945|SSRP1_HUMAN FACT complex subunit SSRP1 OS=Homo sapiens GN=SSRP1 PE=1 SV=1 12 5 >sp|P41252|SYIC_HUMAN Isoleucine‐‐tRNA ligase, cytoplasmic OS=Homo sapiens GN=IARS PE=1 SV=2 11 2 >sp|P53618|COPB_HUMAN Coatomer subunit beta OS=Homo sapiens GN=COPB1 PE=1 SV=3 11 2 >sp|P53621|COPA_HUMAN Coatomer subunit alpha OS=Homo sapiens GN=COPA PE=1 SV=2 11 2 >sp|P35998|PRS7_HUMAN 26S protease regulatory subunit 7 OS=Homo sapiens GN=PSMC2 PE=1 SV=3 11 3 >sp|Q01518|CAP1_HUMAN Adenylyl cyclase‐associated protein 1 OS=Homo sapiens GN=CAP1 PE=1 SV=5 11 5 >sp|Q13200|PSMD2_HUMAN 26S proteasome non‐ATPase regulatory subunit 2 OS=Homo sapiens GN=PSMD2 PE=1 SV=3 11 5 >sp|P30153|2AAA_HUMAN Serine/threonine‐protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform OS=Homo sapiens GN=PPP2R1A PE=1 SV=4 10 2 >sp|Q01082|SPTB2_HUMAN Spectrin beta chain, non‐erythrocytic 1 OS=Homo sapiens GN=SPTBN1 PE=1 SV=2 10 2 >sp|P27708|PYR1_HUMAN CAD protein OS=Homo sapiens GN=CAD PE=1 SV=3 10 3 >sp|P61221|ABCE1_HUMAN ATP‐binding cassette sub‐family E member 1 OS=Homo sapiens GN=ABCE1 PE=1 SV=1 10 5 >sp|Q14974|IMB1_HUMAN Importin subunit beta‐1 OS=Homo sapiens GN=KPNB1 PE=1 SV=2 10 5 >sp|Q8WUM4|PDC6I_HUMAN Programmed cell death 6‐interacting protein OS=Homo sapiens GN=PDCD6IP PE=1 SV=1 10 5 >sp|O14654|IRS4_HUMAN Insulin receptor substrate 4 OS=Homo sapiens GN=IRS4 PE=1 SV=1 90 >sp|P46940|IQGA1_HUMAN Ras GTPase‐activating‐like protein IQGAP1 OS=Homo sapiens GN=IQGAP1 PE=1 SV=1 9 0 >sp|P55060|XPO2_HUMAN Exportin‐2 OS=Homo sapiens GN=CSE1L PE=1 SV=3 90 >tr|I3L0J9|I3L0J9_HUMAN Pre‐mRNA‐processing‐splicing factor 8 (Fragment) OS=Homo sapiens GN=PRPF8 PE=2 SV=1 9 0 >sp|P19367|HXK1_HUMAN Hexokinase‐1 OS=Homo sapiens GN=HK1 PE=1 SV=3 92 >sp|P78347|GTF2I_HUMAN General transcription factor II‐I OS=Homo sapiens GN=GTF2I PE=1 SV=2 92 >sp|Q14008|CKAP5_HUMAN Cytoskeleton‐associated protein 5 OS=Homo sapiens GN=CKAP5 PE=1 SV=3 9 2 >sp|P05023|AT1A1_HUMAN Sodium/potassium‐transporting ATPase subunit alpha‐1 OS=Homo sapiens GN=ATP1A1 PE=1 SV=1 9 3 >sp|P15924|DESP_HUMAN Desmoplakin OS=Homo sapiens GN=DSP PE=1 SV=3 93 >sp|Q7L2E3|DHX30_HUMAN Putative ATP‐dependent RNA helicase DHX30 OS=Homo sapiens GN=DHX30 PE=1 SV=1 8 0 >sp|Q00341|VIGLN_HUMAN Vigilin OS=Homo sapiens GN=HDLBP PE=1 SV=2 82 >sp|O15355|PPM1G_HUMAN Protein phosphatase 1G OS=Homo sapiens GN=PPM1G PE=1 SV=1 83 >sp|P18621|RL17_HUMAN 60S ribosomal protein L17 OS=Homo sapiens GN=RPL17 PE=1 SV=3 83 >sp|P52209|6PGD_HUMAN 6‐phosphogluconate dehydrogenase, decarboxylating OS=Homo sapiens GN=PGD PE=1 SV=3 8 3 >sp|Q92841|DDX17_HUMAN Probable ATP‐dependent RNA helicase DDX17 OS=Homo sapiens GN=DDX17 PE=1 SV=2 8 3 >sp|O95373|IPO7_HUMAN Importin‐7 OS=Homo sapiens GN=IPO7 PE=1 SV=1 70 >sp|P47897|SYQ_HUMAN Glutamine‐‐tRNA ligase OS=Homo sapiens GN=QARS PE=1 SV=1 70 >sp|P10515|ODP2_HUMAN Dihydrolipoyllysine‐residue acetyltransf component of pyruvate dehydrogenase complex OS=Homo sapiens GN=DLAT PE=1 SV=3 7 2 >sp|P56192|SYMC_HUMAN Methionine‐‐tRNA ligase, cytoplasmic OS=Homo sapiens GN=MARS PE=1 SV=2 7 2 >sp|Q9BXJ9|NAA15_HUMAN N‐alpha‐acetyltransferase 15, NatA auxiliary subunit OS=Homo sapiens GN=NAA15 PE=1 SV=1 7 2 >sp|O00299|CLIC1_HUMAN Chloride intracellular channel protein 1 OS=Homo sapiens GN=CLIC1 PE=1 SV=4 7 3 >sp|O95757|HS74L_HUMAN Heat shock 70 kDa protein 4L OS=Homo sapiens GN=HSPA4L PE=1 SV=3 73 >sp|P33993|MCM7_HUMAN DNA replication licensing factor MCM7 OS=Homo sapiens GN=MCM7 PE=1 SV=4 7 3 >sp|Q14566|MCM6_HUMAN DNA replication licensing factor MCM6 OS=Homo sapiens GN=MCM6 PE=1 SV=1 7 3 >sp|Q9BQG0|MBB1A_HUMAN Myb‐binding protein 1A OS=Homo sapiens GN=MYBBP1A PE=1 SV=2 73 >sp|Q9Y262|EIF3L_HUMAN Eukaryotic translation initiation factor 3 subunit L OS=Homo sapiens GN=EIF3L PE=1 SV=1 7 3 >sp|O00429|DNM1L_HUMAN Dynamin‐1‐like protein OS=Homo sapiens GN=DNM1L PE=1 SV=2 60 >sp|P10155|RO60_HUMAN 60 kDa SS‐A/Ro ribonucleoprotein OS=Homo sapiens GN=TROVE2 PE=1 SV=2 6 0 >sp|P27694|RFA1_HUMAN Replication protein A 70 kDa DNA‐binding subunit OS=Homo sapiens GN=RPA1 PE=1 SV=2 6 0 >sp|P35573|GDE_HUMAN Glycogen debranching enzyme OS=Homo sapiens GN=AGL PE=1 SV=3 60 >sp|Q15021|CND1_HUMAN Condensin complex subunit 1 OS=Homo sapiens GN=NCAPD2 PE=1 SV=3 60 >sp|Q9UQ35|SRRM2_HUMAN Serine/arginine repetitive matrix protein 2 OS=Homo sapiens GN=SRRM2 PE=1 SV=2 6 0 >sp|O00232|PSD12_HUMAN 26S proteasome non‐ATPase regulatory subunit 12 OS=Homo sapiens GN=PSMD12 PE=1 SV=3 6 2 >sp|P11413|G6PD_HUMAN Glucose‐6‐phosphate 1‐dehydrogenase OS=Homo sapiens GN=G6PD PE=1 SV=4 6 2 >sp|P22695|QCR2_HUMAN Cytochrome b‐c1 complex subunit 2, mitochondrial OS=Homo sapiens GN=UQCRC2 PE=1 SV=3 6 2 >sp|Q7Z6Z7|HUWE1_HUMAN E3 ubiquitin‐protein ligase HUWE1 OS=Homo sapiens GN=HUWE1 PE=1 SV=3 6 2 >sp|Q9HAV4|XPO5_HUMAN Exportin‐5 OS=Homo sapiens GN=XPO5 PE=1 SV=1 62 >sp|Q8TEQ6|GEMI5_HUMAN Gem‐associated protein 5 OS=Homo sapiens GN=GEMIN5 PE=1 SV=3 50 >sp|P14923|PLAK_HUMAN Junction plakoglobin OS=Homo sapiens GN=JUP PE=1 SV=3 52 >sp|P30419|NMT1_HUMAN Glycylpeptide N‐tetradecanoyltransferase 1 OS=Homo sapiens GN=NMT1 PE=1 SV=2 5 2 >sp|P42285|SK2L2_HUMAN Superkiller viralicidic activity 2‐like 2 OS=Homo sapiens GN=SKIV2L2 PE=1 SV=3 5 2 >sp|P50213|IDH3A_HUMAN Isocitrate dehydrogenase [NAD] subunit alpha, mitochondrial OS=Homo sapiens GN=IDH3A PE=1 SV=1 5 2 >sp|Q13148|TADBP_HUMAN TAR DNA‐binding protein 43 OS=Homo sapiens GN=TARDBP PE=1 SV=1 52 >sp|Q2VIR3|IF2GL_HUMAN Putative eukaryotic translation initiation factor 2 subunit 3‐like protein OS=Homo sapiens GN=EIF2S3L PE=5 SV=2 5 2 >sp|Q6UB35|C1TM_HUMAN Monofunctional C1‐tetrahydrofolate synthase, mitochondrial OS=Homo sapiens GN=MTHFD1L PE=1 SV=1 5 2 >sp|Q99460|PSMD1_HUMAN 26S proteasome non‐ATPase regulatory subunit 1 OS=Homo sapiens GN=PSMD1 PE=1 SV=2 5 2 >sp|Q99798|ACON_HUMAN Aconitate hydratase, mitochondrial OS=Homo sapiens GN=ACO2 PE=1 SV=2 5 2 >sp|Q9BSJ8|ESYT1_HUMAN Extended synaptotagmin‐1 OS=Homo sapiens GN=ESYT1 PE=1 SV=1 52 >sp|Q9UJS0|CMC2_HUMAN Calcium‐binding mitochondrial carrier protein Aralar2 OS=Homo sapiens GN=SLC25A13 PE=1 SV=2 5 2 >sp|O43172|PRP4_HUMAN U4/U6 small nuclear ribonucleoprotein Prp4 OS=Homo sapiens GN=PRPF4 PE=1 SV=2 4 0 >sp|O60701|UGDH_HUMAN UDP‐glucose 6‐dehydrogenase OS=Homo sapiens GN=UGDH PE=1 SV=1 40 >sp|O60716|CTND1_HUMAN Catenin delta‐1 OS=Homo sapiens GN=CTNND1 PE=1 SV=1 40 >sp|P31930|QCR1_HUMAN Cytochrome b‐c1 complex subunit 1, mitochondrial OS=Homo sapiens GN=UQCRC1 PE=1 SV=3 4 0 >sp|P35520|CBS_HUMAN Cystathionine beta‐synthase OS=Homo sapiens GN=CBS PE=1 SV=2 40 >sp|P50570|DYN2_HUMAN Dynamin‐2 OS=Homo sapiens GN=DNM2 PE=1 SV=2 40 >sp|P55265|DSRAD_HUMAN Double‐stranded RNA‐specific adenosine deaminase OS=Homo sapiens GN=ADAR PE=1 SV=4 4 0 >sp|Q02218|ODO1_HUMAN 2‐oxoglutarate dehydrogenase, mitochondrial OS=Homo sapiens GN=OGDH PE=1 SV=3 4 0 >sp|Q06210|GFPT1_HUMAN Glutamine‐‐fructose‐6‐phosphate aminotransferase [isomerizing] 1 OS=Homo sapiens GN=GFPT1 PE=1 SV=3 4 0 >sp|Q14839|CHD4_HUMAN Chromodomain‐helicase‐DNA‐binding protein 4 OS=Homo sapiens GN=CHD4 PE=1 SV=2 4 0 >sp|Q15436|SC23A_HUMAN Protein transport protein Sec23A OS=Homo sapiens GN=SEC23A PE=1 SV=2 4 0 >sp|Q6NXE6|ARMC6_HUMAN Armadillo repeat‐containing protein 6 OS=Homo sapiens GN=ARMC6 PE=1 SV=2 4 0 >sp|Q6PI48|SYDM_HUMAN Aspartate‐‐tRNA ligase, mitochondrial OS=Homo sapiens GN=DARS2 PE=1 SV=1 4 0 >sp|Q8WVM8|SCFD1_HUMAN Sec1 family domain‐containing protein 1 OS=Homo sapiens GN=SCFD1 PE=1 SV=4 4 0 >sp|Q9BVG4|PBDC1_HUMAN Protein PBDC1 OS=Homo sapiens GN=PBDC1 PE=1 SV=1 40 >sp|Q9HCC0|MCCB_HUMAN Methylcrotonoyl‐CoA carboxylase beta chain, mitochondrial OS=Homo sapiens GN=MCCC2 PE=1 SV=1 4 0 >sp|Q9UL46|PSME2_HUMAN Proteasome activator complex subunit 2 OS=Homo sapiens GN=PSME2 PE=1 SV=4 4 0 >sp|O43765|SGTA_HUMAN Small glutamine‐rich tetratricopeptide repeat‐containing protein alpha OS=Homo sapiens GN=SGTA PE=1 SV=1 4 2 >sp|O60256|KPRB_HUMAN Phosphoribosyl pyrophosphate synthase‐associated protein 2 OS=Homo sapiens GN=PRPSAP2 PE=1 SV=1 4 2 >sp|O75083|WDR1_HUMAN WD repeat‐containing protein 1 OS=Homo sapiens GN=WDR1 PE=1 SV=4 42 >sp|O94776|MTA2_HUMAN Metastasis‐associated protein MTA2 OS=Homo sapiens GN=MTA2 PE=1 SV=1 4 2 >sp|P08240|SRPR_HUMAN Signal recognition particle receptor subunit alpha OS=Homo sapiens GN=SRPR PE=1 SV=2 4 2 >sp|P09936|UCHL1_HUMAN Ubiquitin carboxyl‐terminal hydrolase isozyme L1 OS=Homo sapiens GN=UCHL1 PE=1 SV=2 4 2 >sp|P15121|ALDR_HUMAN Aldose reductase OS=Homo sapiens GN=AKR1B1 PE=1 SV=3 42 >sp|P15170|ERF3A_HUMAN Eukaryotic peptide chain release factor GTP‐binding subunit ERF3A OS=Homo sapiens GN=GSPT1 PE=1 SV=1 4 2 >sp|P16435|NCPR_HUMAN NADPH‐‐cytochrome P450 reductase OS=Homo sapiens GN=POR PE=1 SV=2 42 >sp|P30085|KCY_HUMAN UMP‐CMP kinase OS=Homo sapiens GN=CMPK1 PE=1 SV=3 42 >sp|P33991|MCM4_HUMAN DNA replication licensing factor MCM4 OS=Homo sapiens GN=MCM4 PE=1 SV=5 4 2 >sp|P43246|MSH2_HUMAN DNA mismatch repair protein Msh2 OS=Homo sapiens GN=MSH2 PE=1 SV=1 4 2 >sp|P49419|AL7A1_HUMAN Alpha‐aminoadipic semialdehyde dehydrogenase OS=Homo sapiens GN=ALDH7A1 PE=1 SV=5 4 2 >sp|P62913|RL11_HUMAN 60S ribosomal protein L11 OS=Homo sapiens GN=RPL11 PE=1 SV=2 42 >sp|Q00325|MPCP_HUMAN Phosphate carrier protein, mitochondrial OS=Homo sapiens GN=SLC25A3 PE=1 SV=2 4 2 >sp|Q13404|UB2V1_HUMAN Ubiquitin‐conjugating enzyme E2 variant 1 OS=Homo sapiens GN=UBE2V1 PE=1 SV=2 4 2 >sp|Q15155|NOMO1_HUMAN Nodal modulator 1 OS=Homo sapiens GN=NOMO1 PE=1 SV=5 42 >sp|Q16555|DPYL2_HUMAN Dihydropyrimidinase‐related protein 2 OS=Homo sapiens GN=DPYSL2 PE=1 SV=1 4 2 >sp|Q8NBS9|TXND5_HUMAN Thioredoxin domain‐containing protein 5 OS=Homo sapiens GN=TXNDC5 PE=1 SV=2 4 2 >sp|Q8TCS8|PNPT1_HUMAN Polyribonucleotide nucleotidyltransferase 1, mitochondrial OS=Homo sapiens GN=PNPT1 PE=1 SV=2 4 2 >sp|Q92900|RENT1_HUMAN Regulator of nonsense transcripts 1 OS=Homo sapiens GN=UPF1 PE=1 SV=2 4 2 >sp|Q93009|UBP7_HUMAN Ubiquitin carboxyl‐terminal hydrolase 7 OS=Homo sapiens GN=USP7 PE=1 SV=2 4 2 >sp|Q96RP9|EFGM_HUMAN Elongation factor G, mitochondrial OS=Homo sapiens GN=GFM1 PE=1 SV=2 4 2 >sp|Q99615|DNJC7_HUMAN DnaJ homolog subfamily C member 7 OS=Homo sapiens GN=DNAJC7 PE=1 SV=2 4 2 >sp|Q9BY44|EIF2A_HUMAN Eukaryotic translation initiation factor 2A OS=Homo sapiens GN=EIF2A PE=1 SV=3 4 2 >sp|Q9NQW7|XPP1_HUMAN Xaa‐Pro aminopeptidase 1 OS=Homo sapiens GN=XPNPEP1 PE=1 SV=3 42 >sp|Q9NUU7|DD19A_HUMAN ATP‐dependent RNA helicase DDX19A OS=Homo sapiens GN=DDX19A PE=1 SV=1 4 2 >sp|Q9UMS4|PRP19_HUMAN Pre‐mRNA‐processing factor 19 OS=Homo sapiens GN=PRPF19 PE=1 SV=1 4 2 >sp|A6NHR9|SMHD1_HUMAN Structural maintenance of chromosomes flexible hinge domain‐containing protein 1 OS=Homo sapiens GN=SMCHD1 PE=1 SV=2 3 0 >sp|O14828|SCAM3_HUMAN Secretory carrier‐associated membrane protein 3 OS=Homo sapiens GN=SCAMP3 PE=1 SV=3 3 0 >sp|O14983|AT2A1_HUMAN Sarcoplasmic/endoplasmic reticulum calcium ATPase 1 OS=Homo sapiens GN=ATP2A1 PE=1 SV=1 3 0 >sp|O43447|PPIH_HUMAN Peptidyl‐prolyl cis‐trans isomerase H OS=Homo sapiens GN=PPIH PE=1 SV=1 30 >sp|O60343|TBCD4_HUMAN TBC1 domain family member 4 OS=Homo sapiens GN=TBC1D4 PE=1 SV=2 30 >sp|O75844|FACE1_HUMAN CAAX prenyl protease 1 homolog OS=Homo sapiens GN=ZMPSTE24 PE=1 SV=2 3 0 >sp|O95563|MPC2_HUMAN Mitochondrial pyruvate carrier 2 OS=Homo sapiens GN=MPC2 PE=1 SV=1 30 >sp|P05091|ALDH2_HUMAN Aldehyde dehydrogenase, mitochondrial OS=Homo sapiens GN=ALDH2 PE=1 SV=2 3 0 >sp|P11717|MPRI_HUMAN Cation‐independent mannose‐6‐phosphate receptor OS=Homo sapiens GN=IGF2R PE=1 SV=3 3 0 >sp|P13489|RINI_HUMAN Ribonuclease inhibitor OS=Homo sapiens GN=RNH1 PE=1 SV=2 30 >sp|P17858|K6PL_HUMAN 6‐phosphofructokinase, liver type OS=Homo sapiens GN=PFKL PE=1 SV=6 30 >sp|P23258|TBG1_HUMAN Tubulin gamma‐1 chain OS=Homo sapiens GN=TUBG1 PE=1 SV=2 30 >sp|P30566|PUR8_HUMAN Adenylosuccinate lyase OS=Homo sapiens GN=ADSL PE=1 SV=2 30 >sp|P31947|1433S_HUMAN 14‐3‐3 protein sigma OS=Homo sapiens GN=SFN PE=1 SV=1 30 >sp|P33176|KINH_HUMAN Kinesin‐1 heavy chain OS=Homo sapiens GN=KIF5B PE=1 SV=1 30 >sp|P35221|CTNA1_HUMAN Catenin alpha‐1 OS=Homo sapiens GN=CTNNA1 PE=1 SV=1 30 >sp|P42224|STAT1_HUMAN Signal transducer and activator of transcription 1‐alpha/beta OS=Homo sapiens GN=STAT1 PE=1 SV=2 3 0 >sp|P54707|AT12A_HUMAN Potassium‐transporting ATPase alpha chain 2 OS=Homo sapiens GN=ATP12A PE=1 SV=3 3 0 >sp|P63010|AP2B1_HUMAN AP‐2 complex subunit beta OS=Homo sapiens GN=AP2B1 PE=1 SV=1 30 >sp|Q02413|DSG1_HUMAN Desmoglein‐1 OS=Homo sapiens GN=DSG1 PE=1 SV=2 30 >sp|Q13838|DX39B_HUMAN Spliceosome RNA helicase DDX39B OS=Homo sapiens GN=DDX39B PE=1 SV=1 3 0 >sp|Q15813|TBCE_HUMAN Tubulin‐specific chaperone E OS=Homo sapiens GN=TBCE PE=1 SV=1 30 >sp|Q66K14|TBC9B_HUMAN TBC1 domain family member 9B OS=Homo sapiens GN=TBC1D9B PE=1 SV=3 3 0 >sp|Q69YN4|VIR_HUMAN Protein virilizer homolog OS=Homo sapiens GN=KIAA1429 PE=1 SV=2 30 >sp|Q7L576|CYFP1_HUMAN Cytoplasmic FMR1‐interacting protein 1 OS=Homo sapiens GN=CYFIP1 PE=1 SV=1 3 0 >sp|Q7Z4Q2|HEAT3_HUMAN HEAT repeat‐containing protein 3 OS=Homo sapiens GN=HEATR3 PE=1 SV=2 3 0 >sp|Q86Y56|HEAT2_HUMAN HEAT repeat‐containing protein 2 OS=Homo sapiens GN=HEATR2 PE=1 SV=4 3 0 >sp|Q8N163|K1967_HUMAN DBIRD complex subunit KIAA1967 OS=Homo sapiens GN=KIAA1967 PE=1 SV=2 3 0 >sp|Q8NFH4|NUP37_HUMAN Nucleoporin Nup37 OS=Homo sapiens GN=NUP37 PE=1 SV=1 30 >sp|Q93008|USP9X_HUMAN Probable ubiquitin carboxyl‐terminal hydrolase FAF‐X OS=Homo sapiens GN=USP9X PE=1 SV=3 3 0 >sp|Q9BT78|CSN4_HUMAN COP9 signalosome complex subunit 4 OS=Homo sapiens GN=COPS4 PE=1 SV=1 3 0 >sp|Q9BTE7|DCNL5_HUMAN DCN1‐like protein 5 OS=Homo sapiens GN=DCUN1D5 PE=1 SV=1 30 >sp|Q9C0B1|FTO_HUMAN Alpha‐ketoglutarate‐dependent dioxygenase FTO OS=Homo sapiens GN=FTO PE=1 SV=3 3 0 >sp|Q9HCE1|MOV10_HUMAN Putative helicase MOV‐10 OS=Homo sapiens GN=MOV10 PE=1 SV=2 30 >sp|Q9UBB6|NCDN_HUMAN Neurochondrin OS=Homo sapiens GN=NCDN PE=1 SV=1 30 >sp|Q9UN86|G3BP2_HUMAN Ras GTPase‐activating protein‐binding protein 2 OS=Homo sapiens GN=G3BP2 PE=1 SV=2 3 0 >sp|Q9Y314|NOSIP_HUMAN Nitric oxide synthase‐interacting protein OS=Homo sapiens GN=NOSIP PE=1 SV=1 3 0 >sp|Q9Y4W2|LAS1L_HUMAN Ribosomal biogenesis protein LAS1L OS=Homo sapiens GN=LAS1L PE=1 SV=2 3 0 >sp|Q9Y5K6|CD2AP_HUMAN CD2‐associated protein OS=Homo sapiens GN=CD2AP PE=1 SV=1 30 >sp|Q9Y606|TRUA_HUMAN tRNA pseudouridine synthase A, mitochondrial OS=Homo sapiens GN=PUS1 PE=1 SV=3 3 0 >sp|Q9Y696|CLIC4_HUMAN Chloride intracellular channel protein 4 OS=Homo sapiens GN=CLIC4 PE=1 SV=4 3 0 >sp|A5YKK6|CNOT1_HUMAN CCR4‐NOT transcription complex subunit 1 OS=Homo sapiens GN=CNOT1 PE=1 SV=2 2 0 >sp|O00159|MYO1C_HUMAN Unconventional myosin‐Ic OS=Homo sapiens GN=MYO1C PE=1 SV=4 20 >sp|O00442|RTCA_HUMAN RNA 3'‐terminal phosphate cyclase OS=Homo sapiens GN=RTCA PE=1 SV=1 20 >sp|O00764|PDXK_HUMAN Pyridoxal kinase OS=Homo sapiens GN=PDXK PE=1 SV=1 20 >sp|O14617|AP3D1_HUMAN AP‐3 complex subunit delta‐1 OS=Homo sapiens GN=AP3D1 PE=1 SV=1 20 >sp|O14949|QCR8_HUMAN Cytochrome b‐c1 complex subunit 8 OS=Homo sapiens GN=UQCRQ PE=1 SV=4 2 0 >sp|O15372|EIF3H_HUMAN Eukaryotic translation initiation factor 3 subunit H OS=Homo sapiens GN=EIF3H PE=1 SV=1 2 0 >sp|O43156|TTI1_HUMAN TELO2‐interacting protein 1 homolog OS=Homo sapiens GN=TTI1 PE=1 SV=3 20 >sp|O43542|XRCC3_HUMAN DNA repair protein XRCC3 OS=Homo sapiens GN=XRCC3 PE=1 SV=1 20 >sp|O43660|PLRG1_HUMAN Pleiotropic regulator 1 OS=Homo sapiens GN=PLRG1 PE=1 SV=1 20 >sp|O43719|HTSF1_HUMAN HIV Tat‐specific factor 1 OS=Homo sapiens GN=HTATSF1 PE=1 SV=1 20 >sp|O60231|DHX16_HUMAN Putative pre‐mRNA‐splicing factor ATP‐dependent RNA helicase DHX16 OS=Homo sapiens GN=DHX16 PE=1 SV=2 2 0 >sp|O60610|DIAP1_HUMAN Protein diaphanous homolog 1 OS=Homo sapiens GN=DIAPH1 PE=1 SV=2 20 >sp|O75150|BRE1B_HUMAN E3 ubiquitin‐protein ligase BRE1B OS=Homo sapiens GN=RNF40 PE=1 SV=4 20 >sp|O75153|CLU_HUMAN Clustered mitochondria protein homolog OS=Homo sapiens GN=CLUH PE=1 SV=2 2 0 >sp|O75306|NDUS2_HUMAN NADH dehydrogenase [ubiquinone] iron‐sulfur protein 2, mitochondrial OS=Homo sapiens GN=NDUFS2 PE=1 SV=2 2 0 >sp|O76094|SRP72_HUMAN Signal recognition particle subunit SRP72 OS=Homo sapiens GN=SRP72 PE=1 SV=3 2 0 >sp|O94906|PRP6_HUMAN Pre‐mRNA‐processing factor 6 OS=Homo sapiens GN=PRPF6 PE=1 SV=1 20 >sp|O96000|NDUBA_HUMAN NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 10 OS=Homo sapiens GN=NDUFB10 PE=1 SV=3 2 0 >sp|P06702|S10A9_HUMAN Protein S100‐A9 OS=Homo sapiens GN=S100A9 PE=1 SV=1 20 >sp|P09525|ANXA4_HUMAN Annexin A4 OS=Homo sapiens GN=ANXA4 PE=1 SV=4 20 >sp|P10768|ESTD_HUMAN S‐formylglutathione hydrolase OS=Homo sapiens GN=ESD PE=1 SV=2 20 >sp|P11216|PYGB_HUMAN Glycogen phosphorylase, brain form OS=Homo sapiens GN=PYGB PE=1 SV=5 2 0 >sp|P13807|GYS1_HUMAN Glycogen [starch] synthase, muscle OS=Homo sapiens GN=GYS1 PE=1 SV=2 20 >sp|P14174|MIF_HUMAN Macrophage migration inhibitory factor OS=Homo sapiens GN=MIF PE=1 SV=4 2 0 >sp|P18754|RCC1_HUMAN Regulator of chromosome condensation OS=Homo sapiens GN=RCC1 PE=1 SV=1 2 0 >sp|P21266|GSTM3_HUMAN Glutathione S‐transferase Mu 3 OS=Homo sapiens GN=GSTM3 PE=1 SV=3 20 >sp|P21399|ACOC_HUMAN Cytoplasmic aconitate hydratase OS=Homo sapiens GN=ACO1 PE=1 SV=3 20 >sp|P23786|CPT2_HUMAN Carnitine O‐palmitoyltransferase 2, mitochondrial OS=Homo sapiens GN=CPT2 PE=1 SV=2 2 0 >sp|P28331|NDUS1_HUMAN NADH‐ubiquinone oxidoreductase 75 kDa subunit, mitochondrial OS=Homo sapiens GN=NDUFS1 PE=1 SV=3 2 0 >sp|P28340|DPOD1_HUMAN DNA polymerase delta catalytic subunit OS=Homo sapiens GN=POLD1 PE=1 SV=2 2 0 >sp|P36543|VATE1_HUMAN V‐type proton ATPase subunit E 1 OS=Homo sapiens GN=ATP6V1E1 PE=1 SV=1 2 0 >sp|P47929|LEG7_HUMAN Galectin‐7 OS=Homo sapiens GN=LGALS7 PE=1 SV=2 20 >sp|P49643|PRI2_HUMAN DNA primase large subunit OS=Homo sapiens GN=PRIM2 PE=1 SV=2 20 >sp|P49902|5NTC_HUMAN Cytosolic purine 5'‐nucleotidase OS=Homo sapiens GN=NT5C2 PE=1 SV=1 20 >sp|P49916|DNLI3_HUMAN DNA ligase 3 OS=Homo sapiens GN=LIG3 PE=1 SV=2 20 >sp|P50851|LRBA_HUMAN Lipopolysaccharide‐responsive and beige‐like anchor protein OS=Homo sapiens GN=LRBA PE=1 SV=4 2 0 >sp|P51153|RAB13_HUMAN Ras‐related protein Rab‐13 OS=Homo sapiens GN=RAB13 PE=1 SV=1 20 >sp|P51570|GALK1_HUMAN Galactokinase OS=Homo sapiens GN=GALK1 PE=1 SV=1 20 >sp|P51571|SSRD_HUMAN Translocon‐associated protein subunit delta OS=Homo sapiens GN=SSR4 PE=1 SV=1 2 0 >sp|P52907|CAZA1_HUMAN F‐actin‐capping protein subunit alpha‐1 OS=Homo sapiens GN=CAPZA1 PE=1 SV=3 2 0 >sp|P53004|BIEA_HUMAN Biliverdin reductase A OS=Homo sapiens GN=BLVRA PE=1 SV=2 20 >sp|P53350|PLK1_HUMAN Serine/threonine‐protein kinase PLK1 OS=Homo sapiens GN=PLK1 PE=1 SV=1 2 0 >sp|P53597|SUCA_HUMAN Succinyl‐CoA ligase [ADP/GDP‐forming] subunit alpha, mitochondrial OS=Homo sapiens GN=SUCLG1 PE=1 SV=4 2 0 >sp|P54578|UBP14_HUMAN Ubiquitin carboxyl‐terminal hydrolase 14 OS=Homo sapiens GN=USP14 PE=1 SV=3 2 0 >sp|P55210|CASP7_HUMAN Caspase‐7 OS=Homo sapiens GN=CASP7 PE=1 SV=1 20 >sp|P60520|GBRL2_HUMAN Gamma‐aminobutyric acid receptor‐associated protein‐like 2 OS=Homo sapiens GN=GABARAPL2 PE=1 SV=1 2 0 >sp|P61326|MGN_HUMAN Protein mago nashi homolog OS=Homo sapiens GN=MAGOH PE=1 SV=1 20 >sp|P82912|RT11_HUMAN 28S ribosomal protein S11, mitochondrial OS=Homo sapiens GN=MRPS11 PE=1 SV=2 2 0 >sp|P83881|RL36A_HUMAN 60S ribosomal protein L36a OS=Homo sapiens GN=RPL36A PE=1 SV=2 20 >sp|Q01081|U2AF1_HUMAN Splicing factor U2AF 35 kDa subunit OS=Homo sapiens GN=U2AF1 PE=1 SV=3 2 0 >sp|Q01813|K6PP_HUMAN 6‐phosphofructokinase type C OS=Homo sapiens GN=PFKP PE=1 SV=2 20 >sp|Q10570|CPSF1_HUMAN Cleavage and polyadenylation specificity factor subunit 1 OS=Homo sapiens GN=CPSF1 PE=1 SV=2 2 0 >sp|Q12840|KIF5A_HUMAN Kinesin heavy chain isoform 5A OS=Homo sapiens GN=KIF5A PE=1 SV=2 20 >sp|Q13011|ECH1_HUMAN Delta(3,5)‐Delta(2,4)‐dienoyl‐CoA isomerase, mitochondrial OS=Homo sapiens GN=ECH1 PE=1 SV=2 2 0 >sp|Q13057|COASY_HUMAN Bifunctional coenzyme A synthase OS=Homo sapiens GN=COASY PE=1 SV=4 2 0 >sp|Q13144|EI2BE_HUMAN Translation initiation factor eIF‐2B subunit epsilon OS=Homo sapiens GN=EIF2B5 PE=1 SV=3 2 0 >sp|Q13151|ROA0_HUMAN Heterogeneous nuclear ribonucleoprotein A0 OS=Homo sapiens GN=HNRNPA0 PE=1 SV=1 2 0 >sp|Q13630|FCL_HUMAN GDP‐L‐fucose synthase OS=Homo sapiens GN=TSTA3 PE=1 SV=1 20 >sp|Q14137|BOP1_HUMAN Ribosome biogenesis protein BOP1 OS=Homo sapiens GN=BOP1 PE=1 SV=2 20 >sp|Q14203|DCTN1_HUMAN Dynactin subunit 1 OS=Homo sapiens GN=DCTN1 PE=1 SV=3 20 >sp|Q14651|PLSI_HUMAN Plastin‐1 OS=Homo sapiens GN=PLS1 PE=1 SV=2 20 >sp|Q14789|GOGB1_HUMAN Golgin subfamily B member 1 OS=Homo sapiens GN=GOLGB1 PE=1 SV=2 20 >sp|Q16539|MK14_HUMAN Mitogen‐activated protein kinase 14 OS=Homo sapiens GN=MAPK14 PE=1 SV=3 2 0 >sp|Q16637|SMN_HUMAN Survival motor neuron protein OS=Homo sapiens GN=SMN1 PE=1 SV=1 20 >sp|Q29RF7|PDS5A_HUMAN Sister chromatid cohesion protein PDS5 homolog A OS=Homo sapiens GN=PDS5A PE=1 SV=1 2 0 >sp|Q2TAY7|SMU1_HUMAN WD40 repeat‐containing protein SMU1 OS=Homo sapiens GN=SMU1 PE=1 SV=2 2 0 >sp|Q52LJ0|FA98B_HUMAN Protein FAM98B OS=Homo sapiens GN=FAM98B PE=1 SV=1 20 >sp|Q53T59|H1BP3_HUMAN HCLS1‐binding protein 3 OS=Homo sapiens GN=HS1BP3 PE=1 SV=1 20 >sp|Q5C9Z4|NOM1_HUMAN Nucleolar MIF4G domain‐containing protein 1 OS=Homo sapiens GN=NOM1 PE=1 SV=1 2 0 >sp|Q5R3I4|TTC38_HUMAN Tetratricopeptide repeat protein 38 OS=Homo sapiens GN=TTC38 PE=1 SV=1 2 0 >sp|Q5T8P6|RBM26_HUMAN RNA‐binding protein 26 OS=Homo sapiens GN=RBM26 PE=1 SV=3 20 >sp|Q5TGY3|AHDC1_HUMAN AT‐hook DNA‐binding motif‐containing protein 1 OS=Homo sapiens GN=AHDC1 PE=1 SV=1 2 0 >sp|Q6DKJ4|NXN_HUMAN Nucleoredoxin OS=Homo sapiens GN=NXN PE=1 SV=2 20 >sp|Q6L8Q7|PDE12_HUMAN 2',5'‐phosphodiesterase 12 OS=Homo sapiens GN=PDE12 PE=1 SV=2 20 >sp|Q6P1N0|C2D1A_HUMAN Coiled‐coil and C2 domain‐containing protein 1A OS=Homo sapiens GN=CC2D1A PE=1 SV=1 2 0 >sp|Q7Z478|DHX29_HUMAN ATP‐dependent RNA helicase DHX29 OS=Homo sapiens GN=DHX29 PE=1 SV=2 2 0 >sp|Q7Z4G4|TRM11_HUMAN tRNA (guanine(10)‐N2)‐methyltransferase homolog OS=Homo sapiens GN=TRMT11 PE=1 SV=1 2 0 >sp|Q8IWZ3|ANKH1_HUMAN Ankyrin repeat and KH domain‐containing protein 1 OS=Homo sapiens GN=ANKHD1 PE=1 SV=1 2 0 >sp|Q8IXB1|DJC10_HUMAN DnaJ homolog subfamily C member 10 OS=Homo sapiens GN=DNAJC10 PE=1 SV=2 2 0 >sp|Q8IXH7|NELFD_HUMAN Negative elongation factor C/D OS=Homo sapiens GN=NELFCD PE=1 SV=2 20 >sp|Q8IZL8|PELP1_HUMAN Proline‐, glutamic acid‐ and leucine‐rich protein 1 OS=Homo sapiens GN=PELP1 PE=1 SV=2 2 0 >sp|Q8N543|OGFD1_HUMAN 2‐oxoglutarate and iron‐dependent oxygenase domain‐containing protein 1 OS=Homo sapiens GN=OGFOD1 PE=1 SV=1 2 0 >sp|Q8N9N8|EIF1A_HUMAN Probable RNA‐binding protein EIF1AD OS=Homo sapiens GN=EIF1AD PE=1 SV=1 2 0 >sp|Q8NI27|THOC2_HUMAN THO complex subunit 2 OS=Homo sapiens GN=THOC2 PE=1 SV=2 20 >sp|Q8TCD5|NT5C_HUMAN 5'(3')‐deoxyribonucleotidase, cytosolic type OS=Homo sapiens GN=NT5C PE=1 SV=2 2 0 >sp|Q92665|RT31_HUMAN 28S ribosomal protein S31, mitochondrial OS=Homo sapiens GN=MRPS31 PE=1 SV=3 2 0 >sp|Q96I24|FUBP3_HUMAN Far upstream element‐binding protein 3 OS=Homo sapiens GN=FUBP3 PE=1 SV=2 2 0 >sp|Q96IJ6|GMPPA_HUMAN Mannose‐1‐phosphate guanyltransferase alpha OS=Homo sapiens GN=GMPPA PE=1 SV=1 2 0 >sp|Q96JB5|CK5P3_HUMAN CDK5 regulatory subunit‐associated protein 3 OS=Homo sapiens GN=CDK5RAP3 PE=1 SV=2 2 0 >sp|Q96PK6|RBM14_HUMAN RNA‐binding protein 14 OS=Homo sapiens GN=RBM14 PE=1 SV=2 20 >sp|Q96PZ0|PUS7_HUMAN Pseudouridylate synthase 7 homolog OS=Homo sapiens GN=PUS7 PE=1 SV=2 2 0 >sp|Q96T76|MMS19_HUMAN MMS19 nucleotide excision repair protein homolog OS=Homo sapiens GN=MMS19 PE=1 SV=2 2 0 >sp|Q96TA2|YMEL1_HUMAN ATP‐dependent zinc metalloprotease YME1L1 OS=Homo sapiens GN=YME1L1 PE=1 SV=2 2 0 >sp|Q99471|PFD5_HUMAN Prefoldin subunit 5 OS=Homo sapiens GN=PFDN5 PE=1 SV=2 20 >sp|Q9BPX3|CND3_HUMAN Condensin complex subunit 3 OS=Homo sapiens GN=NCAPG PE=1 SV=1 20 >sp|Q9BVI4|NOC4L_HUMAN Nucleolar complex protein 4 homolog OS=Homo sapiens GN=NOC4L PE=1 SV=1 2 0 >sp|Q9H0D6|XRN2_HUMAN 5'‐3' exoribonuclease 2 OS=Homo sapiens GN=XRN2 PE=1 SV=1 20 >sp|Q9H3P2|NELFA_HUMAN Negative elongation factor A OS=Homo sapiens GN=NELFA PE=1 SV=3 20 >sp|Q9H3U1|UN45A_HUMAN Protein unc‐45 homolog A OS=Homo sapiens GN=UNC45A PE=1 SV=1 20 >sp|Q9HCN8|SDF2L_HUMAN Stromal cell‐derived factor 2‐like protein 1 OS=Homo sapiens GN=SDF2L1 PE=1 SV=2 2 0 >sp|Q9NPF4|OSGEP_HUMAN Probable tRNA threonylcarbamoyladenosine biosynthesis protein OSGEP OS=Homo sapiens GN=OSGEP PE=1 SV=1 2 0 >sp|Q9NQ88|TIGAR_HUMAN Fructose‐2,6‐bisphosphatase TIGAR OS=Homo sapiens GN=TIGAR PE=1 SV=1 2 0 >sp|Q9NRF8|PYRG2_HUMAN CTP synthase 2 OS=Homo sapiens GN=CTPS2 PE=1 SV=1 20 >sp|Q9NTZ6|RBM12_HUMAN RNA‐binding protein 12 OS=Homo sapiens GN=RBM12 PE=1 SV=1 20 >sp|Q9NU22|MDN1_HUMAN Midasin OS=Homo sapiens GN=MDN1 PE=1 SV=2 20 >sp|Q9P035|HACD3_HUMAN Very‐long‐chain (3R)‐3‐hydroxyacyl‐[acyl‐carrier protein] dehydratase 3 OS=Homo sapiens GN=PTPLAD1 PE=1 SV=2 2 0 >sp|Q9P266|JCAD_HUMAN Junctional protein associated with coronary artery disease OS=Homo sapiens GN=KIAA1462 PE=1 SV=3 2 0 >sp|Q9P2I0|CPSF2_HUMAN Cleavage and polyadenylation specificity factor subunit 2 OS=Homo sapiens GN=CPSF2 PE=1 SV=2 2 0 >sp|Q9P2W9|STX18_HUMAN Syntaxin‐18 OS=Homo sapiens GN=STX18 PE=1 SV=1 20 >sp|Q9UBC2|EP15R_HUMAN Epidermal growth factor receptor substrate 15‐like 1 OS=Homo sapiens GN=EPS15L1 PE=1 SV=1 2 0 >sp|Q9UH62|ARMX3_HUMAN Armadillo repeat‐containing X‐linked protein 3 OS=Homo sapiens GN=ARMCX3 PE=1 SV=1 2 0 >sp|Q9UHG3|PCYOX_HUMAN Prenylcysteine oxidase 1 OS=Homo sapiens GN=PCYOX1 PE=1 SV=3 20 >sp|Q9UKX7|NUP50_HUMAN Nuclear pore complex protein Nup50 OS=Homo sapiens GN=NUP50 PE=1 SV=2 2 0 >sp|Q9ULX3|NOB1_HUMAN RNA‐binding protein NOB1 OS=Homo sapiens GN=NOB1 PE=1 SV=1 20 >sp|Q9Y3B8|ORN_HUMAN Oligoribonuclease, mitochondrial OS=Homo sapiens GN=REXO2 PE=1 SV=3 20 >sp|Q9Y3E0|GOT1B_HUMAN Vesicle transport protein GOT1B OS=Homo sapiens GN=GOLT1B PE=1 SV=1 2 0 >sp|Q9Y4C2|F115A_HUMAN Protein FAM115A OS=Homo sapiens GN=FAM115A PE=1 SV=3 20 >sp|Q9Y5K5|UCHL5_HUMAN Ubiquitin carboxyl‐terminal hydrolase isozyme L5 OS=Homo sapiens GN=UCHL5 PE=1 SV=3 2 0 Supplemental Figure 1. Representative gating scheme used in flow cytometry analysis experiments. The data represented corresponds to a wild-type animal.