The complex of Fas-associated factor 1 with Hsp70 stabilizes the adherens junction integrity by suppressing RhoA activation.

Fas-associated factor 1 (FAF1) is a scaffolding protein that plays multiple functions, and dysregulation of FAF1 is associated with many types of diseases such as cancers. FAF1 contains multiple ubiquitin-related domains (UBA, UBL1, UBL2, UAS, and UBX), each domain interacting with a specific partner. In particular, the interaction of UBL1 with heat shock protein 70 (Hsp70) is associated with tumor formation, although the molecular understanding remains unknown. In this study, the structural analysis revealed that His160 of FAF1 is important for its interaction with Hsp70. The association of Hsp70 with FAF1 is required for the interaction with IQGAP1. FAF1 negatively regulates RhoA activation by FAF1-Hsp70 complex formation, which then interacts with IQGAP1. These steps play a key role in maintaining the stability of cell-to-cell junction. We conclude that FAF1 plays a critical role in the structure and function of adherens junction during tissue homeostasis and morphogenesis by suppressing RhoA activation, which induces the activation of Rho-associated protein kinase, phosphorylation of myosin light chain, formation of actin stress fiber, and disruption of adherens junction. In addition, depletion of FAF1 increased collective invasion in a 3D spheroid cell culture. These results provide insight into how the FAF1-Hsp70 complex acts as a novel regulator of the adherens junction integrity. The complex can be a potential therapeutic target to inhibit tumorigenesis and metastasis.

Building a Korean morphological analyzer using two Korean BERT models.

A morphological analyzer plays an essential role in identifying functional suffixes of Korean words. The analyzer input and output differ from each other in their length and strings, which can be dealt with by an encoder-decoder architecture. We adopt a Transformer architecture, which is an encoder-decoder architecture with self-attention rather than a recurrent connection, to implement a Korean morphological analyzer. Bidirectional Encoder Representations from Transformers (BERT) is one of the most popular pretrained representation models; it can present an encoded sequence of input words, considering contextual information. We initialize both the Transformer encoder and decoder with two types of Korean BERT, one of which is pretrained with a raw corpus, and the other is pretrained with a morphologically analyzed dataset. Therefore, implementing a Korean morphological analyzer based on Transformer is a fine-tuning process with a relatively small corpus. A series of experiments proved that parameter initialization using pretrained models can alleviate the chronic problem of a lack of training data and reduce the time required for training. In addition, we can determine the number of layers required for the encoder and decoder to optimize the performance of a Korean morphological analyzer.

Nm23-H1 activator phenylbutenoid dimer exerts cytotoxic effects on metastatic breast cancer cells by inducing mitochondrial dysfunction only under glucose starvation.

Mitochondrial oxidative phosphorylation (OXPHOS) has become an attractive target in anti-cancer studies in recent years. In this study, we found that a small molecule phenylbutenoid dimer NMac1 (Nm23-H1 activator 1), (±)-trans-3-(3,4-dimethoxyphenyl)-4-[(E)-3,4-dimethoxystyryl]cyclohex-1-ene, a previously identified anti-metastatic agent, has novel anti-proliferative effect only under glucose starvation in metastatic breast cancer cells. NMac1 causes significant activation of AMPK by decreasing ATP synthesis, lowers mitochondrial membrane potential (MMP, ΔΨm), and inhibits oxygen consumption rate (OCR) under glucose starvation. These effects of NMac1 are provoked by a consequence of OXPHOS complex I inhibition. Through the structure-activity relationship (SAR) study of NMac1 derivatives, NMac24 was identified as the most effective compound in anti-proliferation. NMac1 and NMac24 effectively suppress cancer cell proliferation in 3D-spheroid in vivo-like models only under glucose starvation. These results suggest that NMac1 and NMac24 have the potential as anti-cancer agents having cytotoxic effects selectively in glucose restricted cells.

Stepwise oxidations play key roles in the structural and functional regulations of DJ-1.

DJ-1 is known to play neuroprotective roles by eliminating reactive oxygen species (ROS) as an antioxidant protein. However, the molecular mechanism of DJ-1 function has not been well elucidated. This study explored the structural and functional changes of DJ-1 in response to oxidative stress. Human DJ-1 has three cysteine residues (Cys46, Cys53 and Cys106). We found that, in addition to Cys106, Cys46 is the most reactive cysteine residue in DJ-1, which was identified employing an NPSB-B chemical probe (Ctag) that selectively reacts with redox-sensitive cysteine sulfhydryl. Peroxidatic Cys46 readily formed an intra-disulfide bond with adjacent resolving Cys53, which was identified with nanoUPLC-ESI-q-TOF tandem mass spectrometry (MS/MS) employing DBond algorithm under the non-reducing condition. Mutants (C46A and C53A), not forming Cys46-Cys53 disulfide cross-linking, increased oxidation of Cys106 to sulfinic and sulfonic acids. Furthermore, we found that DJ-1 C46A mutant has distorted unstable structure identified by biochemical assay and employing hydrogen/deuterium exchange-mass spectrometry (HDX-MS) analysis. All three Cys mutants lost antioxidant activities in SN4741 cell, a dopaminergic neuronal cell, unlike WT DJ-1. These findings suggest that all three Cys residues including Cys46-Cys53 disulfide cross-linking are required for maintaining the structural integrity, the regulation process and cellular function as an antioxidant protein. These studies broaden the understanding of regulatory mechanisms of DJ-1 that operate under oxidative conditions.

Activation of Nm23-H1 to suppress breast cancer metastasis via redox regulation.

Non-metastatic protein 23 H1 (Nm23-H1), a housekeeping enzyme, is a nucleoside diphosphate kinase-A (NDPK-A). It was the first identified metastasis suppressor protein. Nm23-H1 prolongs disease-free survival and is associated with a good prognosis in breast cancer patients. However, the molecular mechanisms underlying the role of Nm23-H1 in biological processes are still not well understood. This is a review of recent studies focusing on controlling NDPK activity based on the redox regulation of Nm23-H1, structural, and functional changes associated with the oxidation of cysteine residues, and the relationship between NDPK activity and cancer metastasis. Further understanding of the redox regulation of the NDPK function will likely provide a new perspective for developing new strategies for the activation of NDPK-A in suppressing cancer metastasis.

Cataract-Associated New Mutants S175G/H181Q of ßΒ2-Crystallin and P24S/S31G of γD-Crystallin Are Involved in Protein Aggregation by Structural Changes.

ß/γ-Crystallins, the main structural protein in human lenses, have highly stable structure for keeping the lens transparent. Their mutations have been linked to cataracts. In this study, we identified 10 new mutations of ß/γ-crystallins in lens proteomic dataset of cataract patients using bioinformatics tools. Of these, two double mutants, S175G/H181Q of ßΒ2-crystallin and P24S/S31G of γD-crystallin, were found mutations occurred in the largest loop linking the distant ß-sheets in the Greek key motif. We selected these double mutants for identifying the properties of these mutations, employing biochemical assay, the identification of protein modifications with nanoUPLC-ESI-TOF tandem MS and examining their structural dynamics with hydrogen/deuterium exchange-mass spectrometry (HDX-MS). We found that both double mutations decrease protein stability and induce the aggregation of ß/γ-crystallin, possibly causing cataracts. This finding suggests that both the double mutants can serve as biomarkers of cataracts.

deMix: Decoding Deuterated Distributions from Heterogeneous Protein States via HDX-MS.

Characterization of protein structural changes in response to protein modifications, ligand or chemical binding, or protein-protein interactions is essential for understanding protein function and its regulation. Amide hydrogen/deuterium exchange (HDX) coupled with mass spectrometry (MS) is one of the most favorable tools for characterizing the protein dynamics and changes of protein conformation. However, currently the analysis of HDX-MS data is not up to its full power as it still requires manual validation by mass spectrometry experts. Especially, with the advent of high throughput technologies, the data size grows everyday and an automated tool is essential for the analysis. Here, we introduce a fully automated software, referred to as 'deMix', for the HDX-MS data analysis. deMix deals directly with the deuterated isotopic distributions, but not considering their centroid masses and is designed to be robust over random noises. In addition, unlike the existing approaches that can only determine a single state from an isotopic distribution, deMix can also detect a bimodal deuterated distribution, arising from EX1 behavior or heterogeneous peptides in conformational isomer proteins. Furthermore, deMix comes with visualization software to facilitate validation and representation of the analysis results.

Activity-Regulated Cytoskeleton-Associated Protein (Arc/Arg3.1) is Transiently Expressed after Heat Shock Stress and Suppresses Heat Shock Factor 1.

Heat shock proteins are induced by activation of heat shock factor 1 (HSF1) in response to heat shock and protect against heat stress. However, the molecular mechanisms underlying the downstream signal of heat shock have not been fully elucidated. We found that similarly to canonical Hsps, Arc/Arg3.1 is also markedly induced by heat shock and by other cellular stress inducers, including diamide, sodium arsenite and H2O2 in various cells. We noted that heat stress-induced Arc/Arg3.1 protein is short lived, with a half-life of <30 min, and is readily degraded by the ubiquitin-proteasome system. Arc/Arg3.1 overexpression inhibited the up-regulation of heat shock-induced Hsp70 and Hsp27, suggesting that Arc/Arg3.1 is a negative regulator of heat shock response (HSR). Studying the effect of Arc/Arg3.1 on HSF1, a major transcription factor in HSR, we found that Arc/Arg3.1 binds to HSF1 and inhibits its binding to the heat shock element in gene promoters, resulting in reduced induction of Hsp27 and Hsp70 mRNAs, without affecting HSF1's phosphorylation-dependent activation, or nuclear localization. Arc/Arg3.1 overexpression decreased cell survival in response to heat shock. We conclude that Arc/Arg3.1 is transiently expressed after heat shock and negatively regulates HSF1 in the feedback loop of HSR.

Network-based integrated analysis of omics data reveal novel players of TGF-ß1-induced EMT in human peritoneal mesothelial cells.

Long-term peritoneal dialysis is associated with progressive fibrosis of the peritoneum. Epithelial-mesenchymal transition (EMT) of mesothelial cells is an important mechanism involved in peritoneal fibrosis, and TGF-ß1 is considered central in this process. However, targeting currently known TGF-ß1-associated pathways has not proven effective to date. Therefore, there are still gaps in understanding the mechanisms underlying TGF-ß1-associated EMT and peritoneal fibrosis. We conducted network-based integrated analysis of transcriptomic and proteomic data to systemically characterize the molecular signature of TGF-ß1-stimulated human peritoneal mesothelial cells (HPMCs). To increase the power of the data, multiple expression datasets of TGF-ß1-stimulated human cells were employed, and extended based on a human functional gene network. Dense network sub-modules enriched with differentially expressed genes by TGF-ß1 stimulation were prioritized and genes of interest were selected for functional analysis in HPMCs. Through integrated analysis, ECM constituents and oxidative stress-related genes were shown to be the top-ranked genes as expected. Among top-ranked sub-modules, TNFAIP6, ZC3H12A, and NNT were validated in HPMCs to be involved in regulation of E-cadherin, ZO-1, fibronectin, and αSMA expression. The present data shows the validity of network-based integrated analysis in discovery of novel players in TGF-ß1-induced EMT in peritoneal mesothelial cells, which may serve as new prognostic markers and therapeutic targets for peritoneal dialysis patients.

Small molecule activator of Nm23/NDPK as an inhibitor of metastasis.

Nm23-H1/NDPK-A is a tumor metastasis suppressor having NDP kinase (NDPK) activity. Nm23-H1 is positively associated with prolonged disease-free survival and good prognosis of cancer patients. Approaches to increasing the cellular levels of Nm23-H1 therefore have significance in the therapy of metastatic cancers. We found a small molecule, (±)-trans-3-(3,4-dimethoxyphenyl)-4-[(E)-3,4-dimethoxystyryl]cyclohex-1-ene, that activates Nm23, hereafter called NMac1. NMac1 directly binds to Nm23-H1 and increases its NDPK activity. Employing various NMac1 derivatives and hydrogen/deuterium mass spectrometry (HDX-MS), we identified the pharmacophore and mode of action of NMac1. We found that NMac1 binds to the C-terminal of Nm23-H1 and induces the NDPK activation through its allosteric conformational changes. NMac1-treated MDA-MB-231 breast cancer cells showed dramatic changes in morphology and actin-cytoskeletal organization following inhibition of Rac1 activation. NMac1 also suppressed invasion and migration in vitro, and metastasis in vivo, in a breast cancer mouse model. NMac1 as an activator of NDPK has potential as an anti-metastatic agent.

Proteomic Analysis of Hippocampus in a Mouse Model of Depression Reveals Neuroprotective Function of Ubiquitin C-terminal Hydrolase L1 (UCH-L1) via Stress-induced Cysteine Oxidative Modifications.

Chronic physical restraint stress increases oxidative stress in the brain, and dysregulation of oxidative stress can be one of the causes of major depressive disorder. To understand the underlying mechanisms, we undertook a systematic proteomic analysis of hippocampus in a chronic restraint stress mouse model of depression. Combining two-dimensional gel electrophoresis (2D-PAGE) for protein separation with nanoUPLC-ESI-q-TOF tandem mass spectrometry, we identified sixty-three protein spots that changed in the hippocampus of mice subjected to chronic restraint stress. We identified and classified the proteins that changed after chronic stress, into three groups respectively functioning in neural plasticity, metabolic processes and protein aggregation. Of these, 5 proteins including ubiquitin C-terminal hydrolase L1 (UCH-L1), dihydropyrimidinase-related protein 2 (DPYL2), haloacid dehalogenase-like hydrolase domain-containing protein 2 (HDHD2), actin-related protein 2/3 complex subunit 5 (ARPC5) and peroxiredoxin-2 (PRDX2), showed pI shifts attributable to post-translational modifications. Further analysis indicated that UCH-L1 underwent differential oxidations of 2 cysteine residues following chronic stress. We investigated whether the oxidized form of UCH-L1 plays a role in stressed hippocampus, by comparing the effects of UCH-L1 and its Cys mutants on hippocampal cell line HT-22 in response to oxidative stress. This study demonstrated that UCH-L1 wild-type and cysteine to aspartic acid mutants, but not its cysteine to serine mutants, afforded neuroprotective effects against oxidative stress; there were no discernible differences between wild-type UCH-L1 and its mutants in the absence of oxidative stress. These findings suggest that cysteine oxidative modifications of UCH-L1 in the hippocampus play key roles in neuroprotection against oxidative stress caused in major depressive disorder.

Ubiquitin C-terminal hydrolase-L1 plays a key role in angiogenesis by regulating hydrogen peroxide generated by NADPH oxidase 4.

Ubiquitin C-terminal hydrolase-L1 (UCH-L1), which catalyzes the hydrolysis of ubiquitin esters and amides, is highly expressed in brain. Recently, UCH-L1 has been found to increase cancer cell migration and invasion by modulating hydrogen peroxide generated by NADPH oxidase 4 (NOX4). Because angiogenesis is also mediated by hydrogen peroxide, we explored the role of UCH-L1 in angiogenesis in human umbilical vein endothelial cells (HUVECs). Silencing UCH-L1 suppressed tubule formation in HUVECs, indicating that UCH-L1 promotes angiogenesis in vitro. This was confirmed using in vivo Matrigel plug studies of HUVECs, after overexpressing or silencing UCH-L1. Silencing UCH-L1 significantly suppressed VEGF-induced ROS levels as well as activation of VEGFR, both of which are required for angiogenesis. This study also showed that UCH-L1 promotes angiogenesis of HUVECs, as well as invasion in cancer cells, by up-regulating ROS by deubiquitination of NOX4, suggesting that UCH-L1 plays a key role in angiogenesis of HUVECS by regulating ROS levels by deubiquitination of NOX4.

Calcium Ion Induced Structural Changes Promote Dimerization of Secretagogin, Which Is Required for Its Insulin Secretory Function.

Secretagogin (SCGN), a hexa EF-hand calcium binding protein, plays key roles in insulin secretion in pancreatic ß-cells. It is not yet understood how the binding of Ca2+ to human SCGN (hSCGN) promotes secretion. Here we have addressed this question, using mass spectrometry combined with a disulfide searching algorithm DBond. We found that the binding of Ca2+ to hSCGN promotes the dimerization of hSCGN via the formation of a Cys193-Cys193 disulfide bond. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics studies revealed that Ca2+ binding to the EF-hands of hSCGN induces significant structural changes that affect the solvent exposure of N-terminal region, and hence the redox sensitivity of the Cys193 residue. These redox sensitivity changes were confirmed using biotinylated methyl-3-nitro-4-(piperidin-1-ylsulfonyl) benzoate (NPSB-B), a chemical probe that specifically labels reactive cysteine sulfhydryls. Furthermore, we found that wild type hSCGN overexpression promotes insulin secretion in pancreatic ß cells, while C193S-hSCGN inhibits it. These findings suggest that insulin secretion in pancreatic cells is regulated by Ca2+ and ROS signaling through Ca2+-induced structural changes promoting dimerization of hSCGN.

Heterogeneous nuclear ribonucleoprotein K inhibits heat shock-induced transcriptional activity of heat shock factor 1.

When cells are exposed to heat shock and various other stresses, heat shock factor 1 (HSF1) is activated, and the heat shock response (HSR) is elicited. To better understand the molecular regulation of the HSR, we used 2D-PAGE-based proteome analysis to screen for heat shock-induced post-translationally modified cellular proteins. Our analysis revealed that two protein spots typically present on 2D-PAGE gels and containing heterogeneous nuclear ribonucleoprotein K (hnRNP K) with trioxidized Cys132 disappeared after the heat shock treatment and reappeared during recovery, but the total amount of hnRNP K protein remained unchanged. We next tested whether hnRNP K plays a role in HSR by regulating HSF1 and found that hnRNP K inhibits HSF1 activity, resulting in reduced expression of hsp70 and hsp27 mRNAs. hnRNP K also reduced binding affinity of HSF1 to the heat shock element by directly interacting with HSF1 but did not affect HSF1 phosphorylation-dependent activation or nuclear localization. hnRNP K lost its ability to induce these effects when its Cys132 was substituted with Ser, Asp, or Glu. These findings suggest that hnRNP K inhibits transcriptional activity of HSF1 by inhibiting its binding to heat shock element and that the oxidation status of Cys132 in hnRNP K is critical for this inhibition.

ARD1-mediated Hsp70 acetylation balances stress-induced protein refolding and degradation.

Heat shock protein (Hsp)70 is a molecular chaperone that maintains protein homoeostasis during cellular stress through two opposing mechanisms: protein refolding and degradation. However, the mechanisms by which Hsp70 balances these opposing functions under stress conditions remain unknown. Here, we demonstrate that Hsp70 preferentially facilitates protein refolding after stress, gradually switching to protein degradation via a mechanism dependent on ARD1-mediated Hsp70 acetylation. During the early stress response, Hsp70 is immediately acetylated by ARD1 at K77, and the acetylated Hsp70 binds to the co-chaperone Hop to allow protein refolding. Thereafter, Hsp70 is deacetylated and binds to the ubiquitin ligase protein CHIP to complete protein degradation during later stages. This switch is required for the maintenance of protein homoeostasis and ultimately rescues cells from stress-induced cell death in vitro and in vivo. Therefore, ARD1-mediated Hsp70 acetylation is a regulatory mechanism that temporally balances protein refolding/degradation in response to stress.

Degradation of Redox-Sensitive Proteins including Peroxiredoxins and DJ-1 is Promoted by Oxidation-induced Conformational Changes and Ubiquitination.

Reactive oxygen species (ROS) are key molecules regulating various cellular processes. However, what the cellular targets of ROS are and how their functions are regulated is unclear. This study explored the cellular proteomic changes in response to oxidative stress using H2O2 in dose- and recovery time-dependent ways. We found discernible changes in 76 proteins appearing as 103 spots on 2D-PAGE. Of these, Prxs, DJ-1, UCH-L3 and Rla0 are readily oxidized in response to mild H2O2 stress, and then degraded and active proteins are newly synthesized during recovery. In studies designed to understand the degradation process, multiple cellular modifications of redox-sensitive proteins were identified by peptide sequencing with nanoUPLC-ESI-q-TOF tandem mass spectrometry and the oxidative structural changes of Prx2 explored employing hydrogen/deuterium exchange-mass spectrometry (HDX-MS). We found that hydrogen/deuterium exchange rate increased in C-terminal region of oxidized Prx2, suggesting the exposure of this region to solvent under oxidation. We also found that Lys191 residue in this exposed C-terminal region of oxidized Prx2 is polyubiquitinated and the ubiquitinated Prx2 is readily degraded in proteasome and autophagy. These findings suggest that oxidation-induced ubiquitination and degradation can be a quality control mechanism of oxidized redox-sensitive proteins including Prxs and DJ-1.

Secretagogin affects insulin secretion in pancreatic ß-cells by regulating actin dynamics and focal adhesion.

Secretagogin (SCGN), a Ca(2+)-binding protein having six EF-hands, is selectively expressed in pancreatic ß-cells and neuroendocrine cells. Previous studies suggested that SCGN enhances insulin secretion by functioning as a Ca(2+)-sensor protein, but the underlying mechanism has not been elucidated. The present study explored the mechanism by which SCGN enhances glucose-induced insulin secretion in NIT-1 insulinoma cells. To determine whether SCGN influences the first or second phase of insulin secretion, we examined how SCGN affects the kinetics of insulin secretion in NIT-1 cells. We found that silencing SCGN suppressed the second phase of insulin secretion induced by glucose and H2O2, but not the first phase induced by KCl stimulation. Recruitment of insulin granules in the second phase of insulin secretion was significantly impaired by knocking down SCGN in NIT-1 cells. In addition, we found that SCGN interacts with the actin cytoskeleton in the plasma membrane and regulates actin remodelling in a glucose-dependent manner. Since actin dynamics are known to regulate focal adhesion, a critical step in the second phase of insulin secretion, we examined the effect of silencing SCGN on focal adhesion molecules, including FAK (focal adhesion kinase) and paxillin, and the cell survival molecules ERK1/2 (extracellular-signal-regulated kinase 1/2) and Akt. We found that glucose- and H2O2-induced activation of FAK, paxillin, ERK1/2 and Akt was significantly blocked by silencing SCGN. We conclude that SCGN controls glucose-stimulated insulin secretion and thus may be useful in the therapy of Type 2 diabetes.

Fas-Associated Factor 1 Negatively Regulates the Antiviral Immune Response by Inhibiting Translocation of Interferon Regulatory Factor 3 to the Nucleus.

This study is designed to examine the cellular functions of human Fas-associated factor 1 (FAF1) containing multiple ubiquitin-related domains. Microarray analyses revealed that interferon-stimulated genes related to the antiviral response are significantly increased in FAF1-knockdown HeLa cells. Silencing FAF1 enhanced the poly(I·C)- and respiratory syncytial virus (RSV)-induced production of type I interferons (IFNs), the target genes of interferon regulator factor 3 (IRF3). IRF3 is a key transcription factor in IFN-ß signaling responsible for the host innate immune response. This study also found that FAF1 and IRF3 physically associate with IPO5/importin-ß3 and that overexpression of FAF1 reduces the interaction between IRF3 and IPO5/importin-ß3. These findings suggest that FAF1 negatively regulates IRF3-mediated IFN-ß production and the antiviral innate immune response by regulating nuclear translocation of IRF3. We conclude that FAF1 plays a novel role in negatively regulating virus-induced IFN-ß production and the antiviral response by inhibiting the translocation of active, phosphorylated IRF3 from the cytosol to the nucleus.

Mechanistic elucidation guided by covalent inhibitors for the development of anti-diabetic PPARγ ligands.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-regulated transcription factor that plays crucial roles in adipogenesis, lipid metabolism, and glucose homeostasis. Several PPARγ ligands possess anti-diabetic activity and they commonly inhibit the phosphorylation of PPARγ at serine 273 (Ser273). The recently reported PPARγ ligand SR1664, which selectively blocks the phosphorylation of PPARγ without classical agonism, has potent anti-diabetic activity, indicating that the inhibition of Ser273 phosphorylation is sufficient to provoke anti-diabetic effects. In this study, we revealed the X-ray structure of PPARγ co-crystallized with SR1664 bound to the alternate binding site of PPARγ and confirmed that the alternate site binding of SR1664 blocks the phosphorylation of Ser273. Furthermore, using covalent inhibitors as chemical tools, we demonstrated that the inhibition of phosphorylation is attributed to the occupation of a specific site which is a hydrophobic region between helix 3 and ß3-ß4 at the binding pocket of PPARγ. In high-fat diet-induced obese mice, we confirmed the anti-diabetic activity of our covalent inhibitor SB1453 that was designed to bind at the specific site in PPARγ for blocking the phosphorylation of Ser273. Lastly, the target selectivity of SB1453 was demonstrated by fluorescence-based visualization of target proteins complexed with the covalent probe 11 containing a bioorthogonal functional group.

Evidence for the dimerization-mediated catalysis of methionine sulfoxide reductase A from Clostridium oremlandii.

Clostridium oremlandii MsrA (CoMsrA) is a natively selenocysteine-containing methionine-S-sulfoxide reductase and classified into a 1-Cys type MsrA. CoMsrA exists as a monomer in solution. Herein, we report evidence that CoMsrA can undergo homodimerization during catalysis. The monomeric CoMsrA dimerizes in the presence of its substrate methionine sulfoxide via an intermolecular disulfide bond between catalytic Cys16 residues. The dimeric CoMsrA is resolved by the reductant glutaredoxin, suggesting the relevance of dimerization in catalysis. The dimerization reaction occurs in a concentration- and time-dependent manner. In addition, the occurrence of homodimer formation in the native selenoprotein CoMsrA is confirmed. We also determine the crystal structure of the dimeric CoMsrA, having the dimer interface around the two catalytic Cys16 residues. A central cone-shaped hole is present in the surface model of dimeric structure, and the two Cys16 residues constitute the base of the hole. Collectively, our biochemical and structural analyses suggest a novel dimerization-mediated mechanism for CoMsrA catalysis that is additionally involved in CoMsrA regeneration by glutaredoxin.