The ubiquitin-specific protease USP36 SUMOylates EXOSC10 and promotes the nucleolar RNA exosome function in rRNA processing.

The RNA exosome is an essential 3' to 5' exoribonuclease complex that mediates degradation, processing and quality control of virtually all eukaryotic RNAs. The nucleolar RNA exosome, consisting of a nine-subunit core and a distributive 3' to 5' exonuclease EXOSC10, plays a critical role in processing and degrading nucleolar RNAs, including pre-rRNA. However, how the RNA exosome is regulated in the nucleolus is poorly understood. Here, we report that the nucleolar ubiquitin-specific protease USP36 is a novel regulator of the nucleolar RNA exosome. USP36 binds to the RNA exosome through direct interaction with EXOSC10 in the nucleolus. Interestingly, USP36 does not significantly regulate the levels of EXOSC10 and other tested exosome subunits. Instead, it mediates EXOSC10 SUMOylation at lysine (K) 583. Mutating K583 impaired the binding of EXOSC10 to pre-rRNAs, and the K583R mutant failed to rescue the defects in rRNA processing and cell growth inhibition caused by knockdown of endogenous EXOSC10. Furthermore, EXOSC10 SUMOylation is markedly reduced in cells in response to perturbation of ribosomal biogenesis. Together, these results suggest that USP36 acts as a SUMO ligase to promote EXOSC10 SUMOylation critical for the RNA exosome function in ribosome biogenesis.

Measurement of absolute abundance of crystallins in human and αA N101D transgenic mouse lenses using 15N-labeled crystallin standards.

Stable isotope labeled standards of all major human lens crystallins were created to measure the abundance of lens endogenous crystallins from birth to adulthood. All major human crystallins (αA, αB, ßA2, ßA3/A1, ßA4, ßB1, ßB2, ßB3, γA, γB, γC, γD, γS) were cloned with N-terminal 6 x His tagged SUMO for ease of purification and the ability to generate natural N-termini by SUMO protease cleavage when producing crystallins for structure/function studies. They were then expressed in 15N-enriched media, quantified by mass spectrometry, and mixed in proportions found in young human lens to act as an artificial lens standard. The absolute quantification method was tested using soluble protein from 5-day, 23-day, 18-month, and 18-year-old human lenses spiked with the 15N artificial lens standard. Proteins were trypsinized, relative ratios of light and heavy labeled peptides determined using high-resolution precursor and data independent MS2 scans, and data analysis performed using Skyline software. Crystallin abundances were measured in both human donor lenses and in transgenic mouse αA N101D cataract lenses. Technical replicates of human crystallin abundance measurements were performed with average coefficients of variation of approximately 2% across all 13 crystallins. αA crystallin comprised 27% of the soluble protein of 5-day-old lens and decreased to 16% by 18-years of age. Over this time period αB increased from 6% to 9% and the αA/αB ratio decreased from 4.5/1 to 2/1. γS-crystallin also increased nearly 2-fold from 7% to 12%, becoming the 3rd most abundant protein in adult lens, while ßB1 increased from 14% to 20%, becoming the most abundant crystallin of adult lens. Minor crystallins ßA2, ßB3, and γA comprised only about 1% each of the newborn lens soluble protein, and their abundance dropped precipitously by adulthood. While 9 of the SUMO tagged crystallins were useful for purification of crystallins for structural studies, γA, γB, γC, and γD were resistant to cleavage by SUMO protease. The abundance of WT and N101D human αA in transgenic mouse lenses was approximately 40-fold lower than endogenous mouse αA, but the deamidation mimic human αA N101D was less soluble than human WT αA. The high content of αA and the transient abundance of ßA2, ßB3, and γA in young lens suggest these crystallins play a role in early lens development and growth. ßB1 becoming the most abundant crystallin may result from its role in promoting higher order ß-crystallin oligomerization in mature lens. The full set of human crystallin expression vectors in the Addgene repository should be a useful resource for future crystallin studies. 15N labeling of these crystallins will be useful to accurately quantify crystallins in lens anatomic regions, as well as measure the composition of insoluble light scattering crystallin aggregates. The standards will also be useful to measure the abundance of crystallins expressed in transgenic animal models.

Fatiguing exercise reduces cellular passive Young's modulus in human vastus lateralis muscle.

Previous studies demonstrated that acute fatiguing exercise transiently reduces whole-muscle stiffness, which might contribute to increased risk of injury and impaired contractile performance. We sought to elucidate potential intracellular mechanisms underlying these reductions. To that end, the cellular passive Young's modulus was measured in muscle fibres from healthy, young males and females. Eight volunteers (four male and four female) completed unilateral, repeated maximal voluntary knee extensions until task failure, immediately followed by bilateral percutaneous needle muscle biopsy of the post-fatigued followed by the non-fatigued control vastus lateralis. Muscle samples were processed for mechanical assessment and separately for imaging and phosphoproteomics. Fibres were passively (pCa 8.0) stretched incrementally to 156% of initial sarcomere length to assess Young's modulus, calculated as the slope of the resulting stress-strain curve at short (sarcomere length = 2.4-3.0 µm) and long (sarcomere length = 3.2-3.8 µm) lengths. Titin phosphorylation was assessed by liquid chromatography followed by high-resolution mass spectrometry. The passive modulus was significantly reduced in post-fatigued versus control fibres from male, but not female, participants. Post-fatigued samples showed altered phosphorylation of five serine residues (four located within the elastic region of titin) but did not exhibit altered active tension or sarcomere ultrastructure. Collectively, these results suggest that acute fatigue is sufficient to alter phosphorylation of skeletal titin in multiple locations. We also found reductions in the passive modulus, consistent with prior reports in the literature investigating striated muscle stiffness. These results provide mechanistic insight contributing to the understanding of dynamic regulation of whole-muscle tissue mechanics in vivo. HIGHLIGHTS: What is the central question of this study? Previous studies have shown that skeletal muscle stiffness is reduced following a single bout of fatiguing exercise in whole muscle, but it is not known whether these changes manifest at the cellular level, and their potential mechanisms remain unexplored. What is the main finding and its importance? Fatiguing exercise reduces cellular stiffness in skeletal muscle from males but not females, suggesting that fatigue alters tissue compliance in a sex-dependent manner. The phosphorylation status of titin, a potential mediator of skeletal muscle cellular stiffness, is modified by fatiguing exercise. Previous studies have shown that passive skeletal muscle stiffness is reduced following a single bout of fatiguing exercise. Lower muscle passive stiffness following fatiguing exercise might increase risk for soft-tissue injury; however, the underlying mechanisms of this change are unclear. Our findings show that fatiguing exercise reduces the passive Young's modulus in skeletal muscle cells from males but not females, suggesting that intracellular proteins contribute to reduced muscle stiffness following repeated loading to task failure in a sex-dependent manner. The phosphorylation status of the intracellular protein titin is modified by fatiguing exercise in a way that might contribute to altered muscle stiffness after fatiguing exercise. These results provide important mechanistic insight that might help to explain why biological sex impacts the risk for soft-tissue injury with repeated or high-intensity mechanical loading in athletes and the risk of falls in older adults.

Protein arginine methyltransferase 5 is essential for oncogene product EWSR1-ATF1-mediated gene transcription in clear cell sarcoma.

Transcription dysregulation is common in sarcomas driven by oncogenic transcription factors. Clear cell sarcoma of soft tissue (CCSST) is a rare sarcoma with poor prognosis presently with no therapy. It is characterized by a balanced t(12;22) (q13;q12) chromosomal translocation, resulting in a fusion of the Ewing's sarcoma gene EWSR1 with activating transcription factor 1 (ATF1) to give an oncogene EWSR1-ATF1. Unlike normal ATF1, whose transcription activity is dependent on phosphorylation, EWSR1-ATF1 is constitutively active to drive ATF1-dependent gene transcription to cause tumorigenesis. No EWSR1-ATF1-targeted therapies have been identified due to the challenges in targeting intracellular transcription factors. Through proteomics screening to identify potential druggable targets for CCSST, we discovered protein arginine methyltransferase 5 (PRMT5) as a novel protein to interact with EWSR1-ATF1. PRMT5 is a type II protein arginine methyltransferase to symmetrically dimethylate arginine residues in substrate proteins to regulate a diverse range of activities including gene transcription, RNA splicing, and DNA repair. We found that PRMT5 enhances EWSR1-ATF1-mediated gene transcription to sustain CCSST cell proliferation. Genetic silencing of PRMT5 in CCSST cells resulted in severely impaired cell proliferation and EWSR1-ATF1-driven transcription. Furthermore, we demonstrate that the clinical-stage PRMT5 inhibitor JNJ-64619178 potently and efficaciously inhibited CCSST cell growth in vitro and in vivo. These results provide new insights into PRMT5 as a transcription regulator and warrant JNJ-64619178 for further clinical development to treat CCSST patients.

Proteomic profiling of retina and retinal pigment epithelium combined embryonic tissue to facilitate ocular disease gene discovery.

To expedite gene discovery in eye development and its associated defects, we previously developed a bioinformatics resource-tool iSyTE (integrated Systems Tool for Eye gene discovery). However, iSyTE is presently limited to lens tissue and is predominantly based on transcriptomics datasets. Therefore, to extend iSyTE to other eye tissues on the proteome level, we performed high-throughput tandem mass spectrometry (MS/MS) on mouse embryonic day (E)14.5 retina and retinal pigment epithelium combined tissue and identified an average of 3300 proteins per sample (n = 5). High-throughput expression profiling-based gene discovery approaches-involving either transcriptomics or proteomics-pose a key challenge of prioritizing candidates from thousands of RNA/proteins expressed. To address this, we used MS/MS proteome data from mouse whole embryonic body (WB) as a reference dataset and performed comparative analysis-termed "in silico WB-subtraction"-with the retina proteome dataset. In silico WB-subtraction identified 90 high-priority proteins with retina-enriched expression at stringency criteria of ≥ 2.5 average spectral counts, ≥ 2.0 fold-enrichment, false discovery rate < 0.01. These top candidates represent a pool of retina-enriched proteins, several of which are associated with retinal biology and/or defects (e.g., Aldh1a1, Ank2, Ank3, Dcn, Dync2h1, Egfr, Ephb2, Fbln5, Fbn2, Hras, Igf2bp1, Msi1, Rbp1, Rlbp1, Tenm3, Yap1, etc.), indicating the effectiveness of this approach. Importantly, in silico WB-subtraction also identified several new high-priority candidates with potential regulatory function in retina development. Finally, proteins exhibiting expression or enriched-expression in the retina are made accessible in a user-friendly manner at iSyTE ( https://research.bioinformatics.udel.edu/iSyTE/ ), to allow effective visualization of this information and facilitate eye gene discovery.

Impact of α-crystallin protein loss on zebrafish lens development.

The α-crystallin small heat shock proteins contribute to the transparency and refractive properties of the vertebrate eye lens and prevent the protein aggregation that would otherwise produce lens cataracts, the leading cause of human blindness. There are conflicting data in the literature as to what role the α-crystallins may play in early lens development. In this study, we used CRISPR gene editing to produce zebrafish lines with mutations in each of the three α-crystallin genes (cryaa, cryaba and cryabb) to prevent protein production. The absence of each α-crystallin protein was analyzed by mass spectrometry, and lens phenotypes were assessed with differential interference contrast microscopy and histology. Loss of αA-crystallin produced a variety of lens defects with varying severity in larvae at 3 and 4 dpf but little substantial change in normal fiber cell denucleation. Loss of αBa-crystallin produced no substantial lens defects. Our cryabb mutant produced a truncated αBb-crystallin protein and showed no substantial change in lens development. Mutation of each α-crystallin gene did not alter the mRNA levels of the remaining two, suggesting a lack of genetic compensation. These data suggest that αA-crystallin plays some role in lens development, but the range of phenotype severity in null mutants indicates its loss simply increases the chance for defects and that the protein is not essential. Our finding that cryaba and cryabb mutants lack noticeable lens defects is congruent with insubstantial transcript levels for these genes in lens epithelial and fiber cells through five days of development. Future experiments can explore the molecular mechanisms leading to lens defects in cryaa null mutants and the impact of αA-crystallin loss during zebrafish lens aging.

The deubiquitinase USP36 promotes snoRNP group SUMOylation and is essential for ribosome biogenesis.

SUMOylation plays a crucial role in regulating diverse cellular processes including ribosome biogenesis. Proteomic analyses and experimental evidence showed that a number of nucleolar proteins involved in ribosome biogenesis are modified by SUMO. However, how these proteins are SUMOylated in cells is less understood. Here, we report that USP36, a nucleolar deubiquitinating enzyme (DUB), promotes nucleolar SUMOylation. Overexpression of USP36 enhances nucleolar SUMOylation, whereas its knockdown or genetic deletion reduces the levels of SUMOylation. USP36 interacts with SUMO2 and Ubc9 and directly mediates SUMOylation in cells and in vitro. We show that USP36 promotes the SUMOylation of the small nucleolar ribonucleoprotein (snoRNP) components Nop58 and Nhp2 in cells and in vitro and their binding to snoRNAs. It also promotes the SUMOylation of snoRNP components Nop56 and DKC1. Functionally, we show that knockdown of USP36 markedly impairs rRNA processing and translation. Thus, USP36 promotes snoRNP group SUMOylation and is critical for ribosome biogenesis and protein translation.

Cotranslational stabilization of Sec62/63 within the ER Sec61 translocon is controlled by distinct substrate-driven translocation events.

The ER Sec61 translocon is a large macromolecular machine responsible for partitioning secretory and membrane polypeptides into the lumen, cytosol, and lipid bilayer. Because the Sec61 protein-conducting channel has been isolated in multiple membrane-derived complexes, we determined how the nascent polypeptide modulates translocon component associations during defined cotranslational translocation events. The model substrate preprolactin (pPL) was isolated principally with Sec61αßγ upon membrane targeting, whereas higher-order complexes containing OST, TRAP, and TRAM were stabilized following substrate translocation. Blocking pPL translocation by passenger domain folding favored stabilization of an alternate complex that contained Sec61, Sec62, and Sec63. Moreover, Sec62/63 stabilization within the translocon occurred for native endogenous substrates, such as the prion protein, and correlated with a delay in translocation initiation. These data show that cotranslational translocon contacts are ultimately controlled by the engaged nascent chain and the resultant substrate-driven translocation events.

Phosphoproteomic quantitation and causal analysis reveal pathways in GPVI/ITAM-mediated platelet activation programs.

Platelets engage cues of pending vascular injury through coordinated adhesion, secretion, and aggregation responses. These rapid, progressive changes in platelet form and function are orchestrated downstream of specific receptors on the platelet surface and through intracellular signaling mechanisms that remain systematically undefined. This study brings together cell physiological and phosphoproteomics methods to profile signaling mechanisms downstream of the immunotyrosine activation motif (ITAM) platelet collagen receptor GPVI. Peptide tandem mass tag (TMT) labeling, sample multiplexing, synchronous precursor selection (SPS), and triple stage tandem mass spectrometry (MS3) detected >3000 significant (false discovery rate < 0.05) phosphorylation events on >1300 proteins over conditions initiating and progressing GPVI-mediated platelet activation. With literature-guided causal inference tools, >300 site-specific signaling relations were mapped from phosphoproteomics data among key and emerging GPVI effectors (ie, FcRγ, Syk, PLCγ2, PKCδ, DAPP1). Through signaling validation studies and functional screening, other less-characterized targets were also considered within the context of GPVI/ITAM pathways, including Ras/MAPK axis proteins (ie, KSR1, SOS1, STAT1, Hsp27). Highly regulated GPVI/ITAM targets out of context of curated knowledge were also illuminated, including a system of >40 Rab GTPases and associated regulatory proteins, where GPVI-mediated Rab7 S72 phosphorylation and endolysosomal maturation were blocked by TAK1 inhibition. In addition to serving as a model for generating and testing hypotheses from omics datasets, this study puts forth a means to identify hemostatic effectors, biomarkers, and therapeutic targets relevant to thrombosis, vascular inflammation, and other platelet-associated disease states.

Insights into the kinetics and dynamics of the furin-cleaved form of PCSK9.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates cholesterol metabolism by inducing the degradation of hepatic low density lipoprotein receptors (LDLRs). Plasma PCSK9 has 2 main molecular forms: a 62 kDa mature form (PCSK9_62) and a 55 kDa, furin-cleaved form (PCSK9_55). PCSK9_55 is considered less active than PCSK9_62 in degrading LDLRs. We aimed to identify the site of PCSK9_55 formation (intracellular vs. extracellular) and to further characterize the LDLR-degradative function of PCSK9_55 relative to PCSK9_62. Coexpressing PCSK9_62 with furin in cell culture induced formation of PCSK9_55, most of which was found in the extracellular space. Under the same conditions, we found that i) adding a cell-permeable furin inhibitor preferentially decreased the formation of PCSK9_55 extracellularly; ii) using pulse-chase analysis, we observed the formation of PCSK9_55 exclusively extracellularly in a time-dependent manner. A recombinant form of PCSK9_55 was efficiently produced but displayed impaired secretion that resulted in its intracellular trapping. However, the nonsecreted PCSK9_55 was able to induce degradation of LDLR, though with 50% lower efficiency than PCSK9_62. Collectively, our data show that 1) PCSK9_55 is formed extracellularly; 2) PCSK9_55 has a shorter half-life; 3) there is a small intracellular pool of PCSK9_55 that is not secreted; and 4) PCSK9_55 retained within the cell maintains a reduced efficiency to cause LDLR degradation.

Platelet proteome dynamics in hibernating 13-lined ground squirrels.

Hibernating mammals undergo a dramatic drop in temperature and blood flow during torpor, yet avoid stasis blood clotting through mechanisms that remain unspecified. The effects of hibernation on hemostasis are especially complex, as cold temperatures generally activate platelets, resulting in platelet clearance and cold storage lesions in the context of blood transfusion. With a hibernating body temperature of 4°C-8°C, 13-lined ground squirrels (Ictidomys tridecemlineatus) provide a model to study hemostasis as well as platelet cold storage lesion resistance during hibernation. Here, we quantified and systematically compared proteomes of platelets collected from ground squirrels at summer (active), fall (entrance), and winter (topor) to elucidate how molecular-level changes in platelets may support hemostatic adaptations in torpor. Platelets were isolated from a total of 11 squirrels in June, October, and January. Platelet lysates from each animal were digested with trypsin prior to 11-plex tandem mass tag (TMT) labeling, followed by LC-MS/MS analysis for relative protein quantification. We measured >700 proteins with significant variations in abundance in platelets over the course of entrance, torpor, and activity-including systems of proteins regulating translation, secretion, metabolism, complement, and coagulation cascades. We also noted species-specific differences in levels of hemostatic, secretory, and inflammatory regulators in ground squirrel platelets relative to human platelets. Altogether, we provide the first ever proteomic characterization of platelets from hibernating animals, where systematic changes in metabolic, hemostatic, and other proteins may account for physiological adaptations in torpor and also inform translational effort to improve cold storage of human platelets for transfusion.

Chemical Proteomics Approach for Profiling the NAD Interactome.

Nicotinamide adenine dinucleotide (NAD+) is a multifunctional molecule. Beyond redox metabolism, NAD+ has an equally important function as a substrate for post-translational modification enzymes, the largest family being the poly-ADP-ribose polymerases (PARPs, 17 family members in humans). The recent surprising discoveries of noncanonical NAD (NAD+/NADH)-binding proteins suggests that the NAD interactome is likely larger than previously thought; yet, broadly useful chemical tools for profiling and discovering NAD-binding proteins do not exist. Here, we describe the design, synthesis, and validation of clickable, photoaffinity labeling (PAL) probes, 2- and 6-ad-BAD, for interrogating the NAD interactome. We found that 2-ad-BAD efficiently labels PARPs in a UV-dependent manner. Chemical proteomics experiments with 2- and 6-ad-BAD identified known and unknown NAD+/NADH-binding proteins. Together, our study shows the utility of 2- and 6-ad-BAD as clickable PAL NAD probes.

The potential of tear proteomics for diagnosis and management of orbital inflammatory disorders including Graves' ophthalmopathy.

BACKGROUND: Orbital compartments harbor a variety of tissues that can be independently targeted in a plethora of disorders resulting in sight-threatening risks. Orbital inflammatory disorders (OID) including Graves' ophthalmopathy, sarcoidosis, IgG4 disease, granulomatosis with polyangiitis, and nonspecific orbital inflammation constitute an important cause of pain, diplopia and vision loss. Physical examination, laboratory tests, imaging, and even biopsy are not always adequate to classify orbital inflammation which is frequently deemed "nonspecific". Tear sampling and testing provide a potential "window" to the orbital disease process through a non-invasive technique that allows longitudinal sampling as the disease evolves. Using PubMed/Medline, we identified potentially relevant articles on tear proteomics published in the English language between 1988 and 2021. Of 303 citations obtained, 225 contained empirical data on tear proteins, including 33 publications on inflammatory conditions, 15 in glaucoma, 15 in thyroid eye disease, 1 in sarcoidosis (75) and 2 in uveitis (77,78). Review articles were used to identify an additional 56 relevant articles through citation search. In this review, we provide a short introduction to the potential use of tears as a diagnostic fluid and tool to investigate the mechanism of ocular diseases. A general review of previous tear proteomics studies is also provided, with a focus on Graves' ophthalmopathy (GO), and a discussion of unmet needs in the diagnosis and treatment of orbital inflammatory disease (OID). The review concludes by pointing out current limitations of mass spectrometric analysis of tear proteins and summarizes future needs in the field.

MS/MS in silico subtraction-based proteomic profiling as an approach to facilitate disease gene discovery: application to lens development and cataract.

While the bioinformatics resource-tool iSyTE (integrated Systems Tool for Eye gene discovery) effectively identifies human cataract-associated genes, it is currently based on just transcriptome data, and thus, it is necessary to include protein-level information to gain greater confidence in gene prioritization. Here, we expand iSyTE through development of a novel proteome-based resource on the lens and demonstrate its utility in cataract gene discovery. We applied high-throughput tandem mass spectrometry (MS/MS) to generate a global protein expression profile of mouse lens at embryonic day (E)14.5, which identified 2371 lens-expressed proteins. A major challenge of high-throughput expression profiling is identification of high-priority candidates among the thousands of expressed proteins. To address this problem, we generated new MS/MS proteome data on mouse whole embryonic body (WB). WB proteome was then used as a reference dataset for performing "in silico WB-subtraction" comparative analysis with the lens proteome, which effectively identified 422 proteins with lens-enriched expression at ≥ 2.5 average spectral counts, ≥ 2.0 fold enrichment (FDR < 0.01) cut-off. These top 20% candidates represent a rich pool of high-priority proteins in the lens including known human cataract-linked genes and many new potential regulators of lens development and homeostasis. This rich information is made publicly accessible through iSyTE (https://research.bioinformatics.udel.edu/iSyTE/), which enables user-friendly visualization of promising candidates, thus making iSyTE a comprehensive tool for cataract gene discovery.

Identification, Quantification, and System Analysis of Protein N-ε Lysine Methylation in Anucleate Blood Platelets.

Protein posttranslational modifications critically regulate a range of physiological and disease processes. In addition to tyrosine, serine, and threonine phosphorylation, reversible N-ε acylation and alkylation of protein lysine residues also modulate diverse aspects of cellular function. Studies of lysine acyl and alkyl modifications have focused on nuclear proteins in epigenetic regulation; however, lysine modifications are also prevalent on cytosolic proteins to serve increasingly apparent, although less understood roles in cell regulation. Here, the methyl-lysine (meK) proteome of anucleate blood platelets is characterized. With high-resolution, multiplex MS methods, 190 mono-, di-, and tri-meK modifications are identified on 150 different platelet proteins-including 28 meK modifications quantified by tandem mass tag (TMT) labeling. In addition to identifying meK modifications on calmodulin (CaM), GRP78 (HSPA5, BiP), and EF1A1 that have been previously characterized in other cell types, more novel modifications are also uncovered on cofilin, drebin-like protein (DBNL, Hip-55), DOCK8, TRIM25, and numerous other cytoplasmic proteins. Together, the results and analyses support roles for lysine methylation in mediating cytoskeletal, translational, secretory, and other cellular processes. MS data for this study have been deposited into the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD012217.

ELMOD1 Stimulates ARF6-GTP Hydrolysis to Stabilize Apical Structures in Developing Vestibular Hair Cells.

Sensory hair cells require control of physical properties of their apical plasma membranes for normal development and function. Members of the ADP-ribosylation factor (ARF) small GTPase family regulate membrane trafficking and cytoskeletal assembly in many cells. We identified ELMO domain-containing protein 1 (ELMOD1), a guanine nucleoside triphosphatase activating protein (GAP) for ARF6, as the most highly enriched ARF regulator in hair cells. To characterize ELMOD1 control of trafficking, we analyzed mice of both sexes from a strain lacking functional ELMOD1 [roundabout (rda)]. In rda/rda mice, cuticular plates of utricle hair cells initially formed normally, then degenerated after postnatal day 5; large numbers of vesicles invaded the compromised cuticular plate. Hair bundles initially developed normally, but the cell's apical membrane lifted away from the cuticular plate, and stereocilia elongated and fused. Membrane trafficking in type I hair cells, measured by FM1-43 dye labeling, was altered in rda/rda mice. Consistent with the proposed GAP role for ELMOD1, the ARF6 GTP/GDP ratio was significantly elevated in rda/rda utricles compared with controls, and the level of ARF6-GTP was correlated with the severity of the rda/rda phenotype. These results suggest that conversion of ARF6 to its GDP-bound form is necessary for final stabilization of the hair bundle.SIGNIFICANCE STATEMENT Assembly of the mechanically sensitive hair bundle of sensory hair cells requires growth and reorganization of apical actin and membrane structures. Hair bundles and apical membranes in mice with mutations in the Elmod1 gene degenerate after formation, suggesting that the ELMOD1 protein stabilizes these structures. We show that ELMOD1 is a GTPase-activating protein in hair cells for the small GTP-binding protein ARF6, known to participate in actin assembly and membrane trafficking. We propose that conversion of ARF6 into the GDP-bound form in the apical domain of hair cells is essential for stabilizing apical actin structures like the hair bundle and ensuring that the apical membrane forms appropriately around the stereocilia.

Altered Protein Dynamics and Increased Aggregation of Human γS-Crystallin Due to Cataract-Associated Deamidations.

Deamidation is a major age-related modification in the human lens that is highly prevalent in crystallins isolated from the insoluble fraction of cataractous lenses and also causes protein aggregation in vitro. However, the mechanism by which deamidation causes proteins to become insoluble is not known because only subtle structural changes were observed in vitro. We have identified Asn14 and Asn76 of γS-crystallin as highly deamidated in insoluble proteins isolated from aged lenses. These sites are on the surface of the N-terminal domain and were mimicked by replacing the Asn with Asp residues in order to generate recombinant human γS and deamidated mutants. Both N14D and N76D had increased light scattering compared to wild-type γS (WT) and increased aggregation during thermal-induced denaturation. Aggregation was enhanced by oxidized glutathione, suggesting deamidation may increase susceptibility to form disulfide bonds. These changes were correlated to changes in protein dynamics determined by NMR spectroscopy. Heteronuclear NMR spectroscopy was used to measure amide hydrogen exchange and 15N relaxation dynamics to identify regions with increased dynamics compared to γS WT. Residue-specific changes in solvent accessibility and dynamics were both near and distant from the sites of deamidation, suggesting that deamidation had both local and global effects on the protein structure at slow (ms to s) and fast (µs to ps) time scales. Thus, a potential mechanism for γS deamidation-induced insolubilization in cataractous lenses is altered dynamics due to local regions of unfolding and increased flexibility in both the N- and C-terminal domains particularly at surface helices. This conformational flexibility increases the likelihood of aggregation, which would be enhanced in the oxidizing cytoplasm of the aged and cataractous lens. The NMR data combined with the in vivo insolubility and in vitro aggregation findings support a model that deamidation drives changes in protein dynamics that facilitate protein aggregation associated with cataracts.

Proteome-transcriptome analysis and proteome remodeling in mouse lens epithelium and fibers.

Epithelial cells and differentiated fiber cells represent distinct compartments in the ocular lens. While previous studies have revealed proteins that are preferentially expressed in epithelial vs. fiber cells, a comprehensive proteomics library comparing the molecular compositions of epithelial vs. fiber cells is essential for understanding lens formation, function, disease and regenerative potential, and for efficient differentiation of pluripotent stem cells for modeling of lens development and pathology in vitro. To compare protein compositions between the lens epithelium and fibers, we employed tandem mass spectrometry (2D-LC/MS) analysis of microdissected mouse P0.5 lenses. Functional classifications of the top 525 identified proteins into gene ontology categories by molecular processes and subcellular localizations, were adapted for the lens. Expression levels of both epithelial and fiber proteomes were compared with whole lens proteome and mRNA levels using E14.5, E16.5, E18.5, and P0.5 RNA-Seq data sets. During this developmental time window, multiple complex biosynthetic and catabolic processes generate the molecular and structural foundation for lens transparency. As expected, crystallins showed a high correlation between their mRNA and protein levels. Comprehensive data analysis confirmed and/or predicted roles for transcription factors (TFs), RNA-binding proteins (e.g. Carhsp1), translational apparatus including ribosomal heterogeneity and initiation factors, microtubules, cytoskeletal [e.g. non-muscle myosin IIA heavy chain (Myh9) and ßB2-spectrin (Sptbn2)] and membrane proteins in lens formation and maturation. Our data highlighted many proteins with unknown functions in the lens that were preferentially enriched in epithelium or fibers, setting the stage for future studies to further dissect the roles of these proteins in fiber cell differentiation vs. epithelial cell maintenance. In conclusion, the present proteomic datasets represent the first mouse lens epithelium and fiber cell proteomes, establish comparative analyses of protein and RNA-Seq data, and characterize the major proteome remodeling required to form the mature lens fiber cells.

Extended Multiplexing of Tandem Mass Tags (TMT) Labeling Reveals Age and High Fat Diet Specific Proteome Changes in Mouse Epididymal Adipose Tissue.

The lack of high-throughput methods to analyze the adipose tissue protein composition limits our understanding of the protein networks responsible for age and diet related metabolic response. We have developed an approach using multiple-dimension liquid chromatography tandem mass spectrometry and extended multiplexing (24 biological samples) with tandem mass tags (TMT) labeling to analyze proteomes of epididymal adipose tissues isolated from mice fed either low or high fat diet for a short or a long-term, and from mice that aged on low versus high fat diets. The peripheral metabolic health (as measured by body weight, adiposity, plasma fasting glucose, insulin, triglycerides, total cholesterol levels, and glucose and insulin tolerance tests) deteriorated with diet and advancing age, with long-term high fat diet exposure being the worst. In response to short-term high fat diet, 43 proteins representing lipid metabolism (e.g. AACS, ACOX1, ACLY) and red-ox pathways (e.g. CPD2, CYP2E, SOD3) were significantly altered (FDR < 10%). Long-term high fat diet significantly altered 55 proteins associated with immune response (e.g. IGTB2, IFIT3, LGALS1) and rennin angiotensin system (e.g. ENPEP, CMA1, CPA3, ANPEP). Age-related changes on low fat diet significantly altered only 18 proteins representing mainly urea cycle (e.g. OTC, ARG1, CPS1), and amino acid biosynthesis (e.g. GMT, AKR1C6). Surprisingly, high fat diet driven age-related changes culminated with alterations in 155 proteins involving primarily the urea cycle (e.g. ARG1, CPS1), immune response/complement activation (e.g. C3, C4b, C8, C9, CFB, CFH, FGA), extracellular remodeling (e.g. EFEMP1, FBN1, FBN2, LTBP4, FERMT2, ECM1, EMILIN2, ITIH3) and apoptosis (e.g. YAP1, HIP1, NDRG1, PRKCD, MUL1) pathways. Using our adipose tissue tailored approach we have identified both age-related and high fat diet specific proteomic signatures highlighting a pronounced involvement of arginine metabolism in response to advancing age, and branched chain amino acid metabolism in early response to high fat feeding. Data are available via ProteomeXchange with identifier PXD005953.

Platelet procoagulant phenotype is modulated by a p38-MK2 axis that regulates RTN4/Nogo proximal to the endoplasmic reticulum: utility of pathway analysis.

Upon encountering physiological cues associated with damaged or inflamed endothelium, blood platelets set forth intracellular responses to ultimately support hemostatic plug formation and vascular repair. To gain insights into the molecular events underlying platelet function, we used a combination of interactome, pathway analysis, and other systems biology tools to analyze associations among proteins functionally modified by reversible phosphorylation upon platelet activation. While an interaction analysis mapped out a relative organization of intracellular mediators in platelet signaling, pathway analysis revealed directional signaling relations around protein kinase C (PKC) isoforms and mitogen-activated protein kinases (MAPKs) associated with platelet cytoskeletal dynamics, inflammatory responses, and hemostatic function. Pathway and causality analysis further suggested that platelets activate a specific p38-MK2 axis to phosphorylate RTN4 (reticulon-4, also known as Nogo), a Bcl-xl sequestration protein and critical regulator of endoplasmic reticulum (ER) physiology. In vitro, we find that platelets drive a p38-MK2-RTN4-Bcl-xl pathway associated with the regulation of the ER and platelet phosphatidylserine exposure. Together, our results support the use of pathway tools in the analysis of omics data sets as a means to help generate novel, mechanistic, and testable hypotheses for platelet studies while uncovering RTN4 as a putative regulator of platelet cell physiological responses.