Multiple redox switches of the SARS-CoV-2 main protease in vitro provide opportunities for drug design.

Besides vaccines, the development of antiviral drugs targeting SARS-CoV-2 is critical for preventing future COVID outbreaks. The SARS-CoV-2 main protease (Mpro), a cysteine protease with essential functions in viral replication, has been validated as an effective drug target. Here, we show that Mpro is subject to redox regulation in vitro and reversibly switches between the enzymatically active dimer and the functionally dormant monomer through redox modifications of cysteine residues. These include a disulfide-dithiol switch between the catalytic cysteine C145 and cysteine C117, and generation of an allosteric cysteine-lysine-cysteine SONOS bridge that is required for structural stability under oxidative stress conditions, such as those exerted by the innate immune system. We identify homo- and heterobifunctional reagents that mimic the redox switching and inhibit Mpro activity. The discovered redox switches are conserved in main proteases from other coronaviruses, e.g. MERS-CoV and SARS-CoV, indicating their potential as common druggable sites.

Assessing the risk to develop a growing teratoma syndrome based on molecular and epigenetic subtyping as well as novel secreted biomarkers.

In germ cell tumors (GCT), a growing teratoma during chemotherapy with decreasing tumor markers was defined as 'growing teratoma syndrome' (GTS) by Logothetis et al. in 1982. So far, its pathogenesis and specific treatment options remain elusive. We aimed at updating the GTS definition based on molecular and epigenetic features as well as identifying circulating biomarkers. We selected 50 GTS patients for clinical characterization and subsequently 12 samples were molecularly analyzed. We further included 7 longitudinal samples of 2 GTS patients. Teratomas (TER) showing no features of GTS served as controls. GTS were stratified based on growth rates into a slow (<0.5 cm/month), medium (0.5-1.5) and rapid (>1.5) group. By analyzing DNA methylation, microRNA expression and the secretome, we identified putative epigenetic and secreted biomarkers for the GTS subgroups. We found that proteins enriched in the GTS groups compared to TER were involved in proliferation, DNA replication and the cell cycle, while proteins interacting with the immune system were depleted. Additionally, GTSrapid seem to interact more strongly with the surrounding microenvironment than GTSslow. Expression of pluripotency- and yolk-sac tumor-associated genes in GTS and formation of a yolk-sac tumor or somatic-type malignancy in the longitudinal GTS samples, pointed at an additional occult non-seminomatous component after chemotherapy. Thus, updating the Logothetis GTS definition is necessary, which we propose as follows: The GTS describes a continuously growing teratoma that might harbor occult non-seminomatous components considerably reduced during therapy but outgrowing over time again.

The Golgi stacking protein GRASP55 is targeted by the natural compound prodigiosin.

BACKGROUND: The bacterial secondary metabolite prodigiosin has been shown to exert anticancer, antimalarial, antibacterial and immunomodulatory properties. With regard to cancer, it has been reported to affect cancer cells but not non-malignant cells, rendering prodigiosin a promising lead compound for anticancer drug discovery. However, a direct protein target has not yet been experimentally identified. METHODS: We used mass spectrometry-based thermal proteome profiling in order to identify target proteins of prodigiosin. For target validation, we employed a genetic knockout approach and electron microscopy. RESULTS: We identified the Golgi stacking protein GRASP55 as target protein of prodigiosin. We show that prodigiosin treatment severely affects Golgi morphology and functionality, and that prodigiosin-dependent cytotoxicity is partially reduced in GRASP55 knockout cells. We also found that prodigiosin treatment results in decreased cathepsin activity and overall blocks autophagic flux, whereas co-localization of the autophagosomal marker LC3 and the lysosomal marker LAMP1 is clearly promoted. Finally, we observed that autophagosomes accumulate at GRASP55-positive structures, pointing towards an involvement of an altered Golgi function in the autophagy-inhibitory effect of this natural compound. CONCLUSION: Taken together, we propose that prodigiosin affects autophagy and Golgi apparatus integrity in an interlinked mode of action involving the regulation of organelle alkalization and the Golgi stacking protein GRASP55. Video Abstract.

Closer to the Reality-Proteome Changes Evoked by Endometrial Scratching in Fertile Females.

Endometrial scratching (ES) has been widely used in assisted reproductive technology to possibly improve pregnancy rates, but its exact mechanism is still not understood or investigated, and its benefits are controversially discussed. Hypothetically, ES may trigger a local immune response, leading to an improved endometrial receptivity. So far, it has been shown that ES affects the gene expression of cytokines, growth factors, and adhesive proteins, potentially modulating inflammatory pathways and adhesion molecule expression. Our pilot study applying proteomic analysis reveals that ES probably has an impact on the proteins involved in immune response pathways and cytoskeleton formation, which could potentially increase endometrial receptivity. Specifically, proteins that are involved in the immune response and cytoskeleton regulation showed a trend toward higher abundance after the first ES. On the other hand, proteins with a decreasing abundance after the first ES play roles in the regulation of the actin cytoskeleton and cellular processes such as intracellular transport, apoptosis, and autophagy. These trends in protein changes suggest that ES may affect endometrial tissue stiffness and extracellular matrix remodeling, potentially enhancing the embryos' implantation. To our knowledge, this pilot study provides, for the first time, data investigating potential changes in the endometrium due to the scratching procedure that might explain its possible benefit for patients in infertility treatment. Furthermore, the proteome of a group of patients suffering from repeated implantation failure was compared to that of the fertile group in order to transfer the basic science to clinical routine and application.

Hepatocytes reprogram liver macrophages involving control of TGF-ß activation, influencing liver regeneration and injury.

BACKGROUND: Macrophages play an important role in maintaining liver homeostasis and regeneration. However, it is not clear to what extent the different macrophage populations of the liver differ in terms of their activation state and which other liver cell populations may play a role in regulating the same. METHODS: Reverse transcription PCR, flow cytometry, transcriptome, proteome, secretome, single cell analysis, and immunohistochemical methods were used to study changes in gene expression as well as the activation state of macrophages in vitro and in vivo under homeostatic conditions and after partial hepatectomy. RESULTS: We show that F4/80+/CD11bhi/CD14hi macrophages of the liver are recruited in a C-C motif chemokine receptor (CCR2)-dependent manner and exhibit an activation state that differs substantially from that of the other liver macrophage populations, which can be distinguished on the basis of CD11b and CD14 expressions. Thereby, primary hepatocytes are capable of creating an environment in vitro that elicits the same specific activation state in bone marrow-derived macrophages as observed in F4/80+/CD11bhi/CD14hi liver macrophages in vivo. Subsequent analyses, including studies in mice with a myeloid cell-specific deletion of the TGF-ß type II receptor, suggest that the availability of activated TGF-ß and its downregulation by a hepatocyte-conditioned milieu are critical. Reduction of TGF-ßRII-mediated signal transduction in myeloid cells leads to upregulation of IL-6, IL-10, and SIGLEC1 expression, a hallmark of the activation state of F4/80+/CD11bhi/CD14hi macrophages, and enhances liver regeneration. CONCLUSIONS: The availability of activated TGF-ß determines the activation state of specific macrophage populations in the liver, and the observed rapid transient activation of TGF-ß may represent an important regulatory mechanism in the early phase of liver regeneration in this context.

Pseudomonas aeruginosa responds to altered membrane phospholipid composition by adjusting the production of two-component systems, proteases and iron uptake proteins.

Membrane protein and phospholipid (PL) composition changes in response to environmental cues and during infections. To achieve these, bacteria use adaptation mechanisms involving covalent modification and remodelling of the acyl chain length of PLs. However, little is known about bacterial pathways regulated by PLs. Here, we investigated proteomic changes in the biofilm of P. aeruginosa phospholipase mutant (∆plaF) with altered membrane PL composition. The results revealed profound alterations in the abundance of many biofilm-related two-component systems (TCSs), including accumulation of PprAB, a key regulator of the transition to biofilm. Furthermore, a unique phosphorylation pattern of transcriptional regulators, transporters and metabolic enzymes, as well as differential production of several proteases, in ∆plaF, indicate that PlaF-mediated virulence adaptation involves complex transcriptional and posttranscriptional response. Moreover, proteomics and biochemical assays revealed the depletion of pyoverdine-mediated iron uptake pathway proteins in ∆plaF, while proteins from alternative iron-uptake systems were accumulated. These suggest that PlaF may function as a switch between different iron-acquisition pathways. The observation that PL-acyl chain modifying and PL synthesis enzymes were overproduced in ∆plaF reveals the interconnection of degradation, synthesis and modification of PLs for proper membrane homeostasis. Although the precise mechanism by which PlaF simultaneously affects multiple pathways remains to be elucidated, we suggest that alteration of PL composition in ∆plaF plays a role for the global adaptive response in P. aeruginosa mediated by TCSs and proteases. Our study revealed the global regulation of virulence and biofilm by PlaF and suggests that targeting this enzyme may have therapeutic potential.

SEC14-GOLD protein PATELLIN2 binds IRON-REGULATED TRANSPORTER1 linking root iron uptake to vitamin E.

Organisms require micronutrients, and Arabidopsis (Arabidopsis thaliana) IRON-REGULATED TRANSPORTER1 (IRT1) is essential for iron (Fe2+) acquisition into root cells. Uptake of reactive Fe2+ exposes cells to the risk of membrane lipid peroxidation. Surprisingly little is known about how this is avoided. IRT1 activity is controlled by an intracellular variable region (IRT1vr) that acts as a regulatory protein interaction platform. Here, we describe that IRT1vr interacted with peripheral plasma membrane SEC14-Golgi dynamics (SEC14-GOLD) protein PATELLIN2 (PATL2). SEC14 proteins bind lipophilic substrates and transport or present them at the membrane. To date, no direct roles have been attributed to SEC14 proteins in Fe import. PATL2 affected root Fe acquisition responses, interacted with ROS response proteins in roots, and alleviated root lipid peroxidation. PATL2 had high affinity in vitro for the major lipophilic antioxidant vitamin E compound α-tocopherol. Molecular dynamics simulations provided insight into energetic constraints and the orientation and stability of the PATL2-ligand interaction in atomic detail. Hence, this work highlights a compelling mechanism connecting vitamin E with root metal ion transport at the plasma membrane with the participation of an IRT1-interacting and α-tocopherol-binding SEC14 protein.

Profiling the 3D interaction between germ cell tumors and microenvironmental cells at the transcriptome and secretome level.

The tumor microenvironment (TM), consisting of the extracellular matrix (ECM), fibroblasts, endothelial cells, and immune cells, might affect tumor invasiveness and the outcome of standard chemotherapy. This study investigated the cross talk between germ cell tumors (GCT) and surrounding TM cells (macrophages, T-lymphocytes, endothelial cells, and fibroblasts) at the transcriptome and secretome level. Using high-throughput approaches of three-dimensional (3D) co-cultured cellular aggregates, this study offers newly identified pathways to be studied with regard to sensitivity toward cisplatin-based chemotherapy or tumor invasiveness as a consequence of the cross talk between tumor cells and TM components. Mass-spectrometry-based secretome analyses revealed that TM cells secreted factors involved in ECM organization, cell adhesion, angiogenesis, and regulation of insulin-like growth factor (IGF) transport. To evaluate direct cell-cell contacts, green fluorescent protein (GFP)-expressing GCT cells and mCherry-expressing TM cells were co-cultured in 3D. Afterward, cell populations were separated by flow cytometry and analyzed by RNA sequencing. Correlating the secretome with transcriptome data indicated molecular processes such as cell adhesion and components of the ECM being enriched in most cell populations. Re-analyses of secretome data with regard to lysine- and proline-hydroxylated peptides revealed a gain in proteins, such as collagens and fibronectin. Cultivation of GCT cells on collagen I/IV- or fibronectin-coated plates significantly elevated adhesive and migratory capacity, while decreasing cisplatin sensitivity of GCT cells. Correspondingly, cisplatin sensitivity was significantly reduced in GCT cells under the influence of conditioned medium from fibroblasts and endothelial cells. This study sheds light on the cross talk between GCT cells and their circumjacent TM, which results in deposition of the ECM and eventually promotes a pro-tumorigenic environment through enhanced migratory and adhesive capacity, as well as decreased cisplatin sensitivity. Hence, our observations indicate that targeting the ECM and its cellular components might be a novel therapeutic option in combination with cisplatin-based chemotherapy for GCT patients.

Proteomic and transcriptomic profiles of human urothelial cancer cells with histone deacetylase 5 overexpression.

Urothelial carcinoma (UC) of the urinary bladder is a prevalent cancer worldwide. Because histone deacetylases (HDACs) are important factors in cancer, targeting these epigenetic regulators is considered an attractive strategy to develop novel anticancer drugs. Whereas HDAC1 and HDAC2 promote UC, HDAC5 is often downregulated and only weakly expressed in UC cell lines, suggesting a tumor-suppressive function. We studied the effect of stable lentiviral-mediated HDAC5 overexpression in four UC cell lines with different phenotypes (RT112, VM-Cub-1, SW1710, and UM-UC-3, each with vector controls). In particular, comprehensive proteomics and RNA-seq transcriptomics analyses were performed on the four cell line pairs, which are described here. For comparison, the immortalized benign urothelial cell line HBLAK was included. These datasets will be a useful resource for researchers studying UC, and especially the influence of HDAC5 on epithelial-mesenchymal transition (EMT). Moreover, these data will inform studies on HDAC5 as a less studied member of the HDAC family in other cell types and diseases, especially fibrosis.

Glutaredoxin 2 promotes SP-1-dependent CSPG4 transcription and migration of wound healing NG2 glia and glioma cells: Enzymatic Taoism.

Redox regulation of specific cysteines via oxidoreductases of the thioredoxin family is increasingly being recognized as an important signaling pathway. Here, we demonstrate that the cytosolic isoform of the vertebrate-specific oxidoreductase Glutaredoxin 2 (Grx2c) regulates the redox state of the transcription factor SP-1 and thereby its binding affinity to both the promoter and an enhancer region of the CSPG4 gene encoding chondroitin sulfate proteoglycan nerve/glial antigen 2 (NG2). This leads to an increased number of NG2 glia during in vitro oligodendroglial differentiation and promotes migration of these wound healing cells. On the other hand, we found that the same mechanism also leads to increased invasion of glioma tumor cells. Using in vitro (human cell lines), ex vivo (mouse primary cells), and in vivo models (zebrafish), as well as glioblastoma patient tissue samples we provide experimental data highlighting the Yin and Yang of redox signaling in the central nervous system and the enzymatic Taoism of Grx2c.

PGRMC1 Promotes Progestin-Dependent Proliferation of Breast Cancer Cells by Binding Prohibitins Resulting in Activation of ERα Signaling.

In previous studies, we reported that progesterone receptor membrane component 1 (PGRMC1) is implicated in progestin signaling and possibly associated with increased breast cancer risk upon combined hormone replacement therapy. To gain mechanistic insight, we searched for potential PGRMC1 interaction partners upon progestin treatment by co-immunoprecipitation and mass spectrometry. The interactions with the identified partners were further characterized with respect to PGRMC1 phosphorylation status and with emphasis on the crosstalk between PGRMC1 and estrogen receptor α (ERα). We report that PGRMC1 overexpression resulted in increased proliferation of hormone receptor positive breast cancer cell lines upon treatment with a subgroup of progestins including norethisterone and dydrogesterone that promote PGRMC1-phosphorylation on S181. The ERα modulators prohibitin-1 (PHB1) and prohibitin-2 (PHB2) interact with PGRMC1 in dependency on S181-phosphorylation upon treatment with the same progestins. Moreover, increased interaction between PGRMC1 and PHBs correlated with decreased binding of PHBs to ERα and subsequent ERα activation. Inhibition of either PGRMC1 or ERα abolished this effect. In summary, we provide strong evidence that activated PGRMC1 associates with PHBs, competitively removing them from ERα, which then can develop its transcriptional activities on target genes. This study emphasizes the role of PGRMC1 in a key breast cancer signaling pathway which may provide a new avenue to target hormone-dependent breast cancer.

Quantitative MS Workflow for a High-Quality Secretome Analysis by a Quantitative Secretome-Proteome Comparison.

Cells secrete proteins to communicate with their environment. Therefore, it is interesting to characterize the proteins which are released from cells under certain experimental conditions the so-called secretome. Here, often proteins from conditioned medium of cultured cells are analyzed, but these additionally might include also contaminating proteins of serum that have not been sufficiently removed or proteins from dying cells. To provide high-quality secretome data and minimize potential contaminants, we describe a quantitative comparison of conditioned medium and the cellular proteome. The described workflow comprises cell cultivation, sample preparation, and final data analysis which is based on the comparison of data from label-free mass spectrometric quantification of proteins from the conditioned medium with corresponding cellular proteomes enabling the detection of bona fide secreted proteins.

The migration behavior of human glioblastoma cells is influenced by the redox-sensitive human macrophage capping protein CAPG.

The macrophage capping protein CAPG belongs to the gelsolin superfamily which modulates actin dynamics by capping the growing end of actin filaments in a Ca2+- and PIP2-dependent manner resulting in polymerization inhibition of actin filaments. In the last years, additional functions for CAPG in transcription regulation were described and higher CAPG amounts have been linked to increased invasiveness and migration behavior in different human tumor entities like e.g. glioblastoma. Nevertheless, there is a lack of knowledge how additional functions of CAPG are regulated. As CAPG contains several cysteine residues which may be accessible to oxidation we were especially interested to investigate how alterations in the cysteine oxidation state may influence the function, localization, and regulation of CAPG. In the present study, we provide strong evidence that CAPG is a redox-sensitive protein and identified two cysteines: C282 and C290 as reversibly oxidized in glioblastoma cell lines. Whereas no evidence could be found that the canonical actin capping function of CAPG is redox-regulated, our results point to a novel role of the identified cysteines in the regulation of cell migration. Along with this, we found a localization shift out of the nucleus of CAPG and RAVER1, a potential interaction partner identified in our study which might explain the observed altered cell migration properties. The newly identified redox sensitive cysteines of CAPG could perspectively be considered as new targets for controlling tumor invasive properties.

An Integrative Omics Approach Reveals Involvement of BRCA1 in Hepatic Metastatic Progression of Colorectal Cancer.

(1) Background & Aims: The roles of different cells in the tumor microenvironment (TME) are critical to the metastatic process. The phenotypic transformation of the liver cells is one of the most important stages of the hepatic metastasis progression of colorectal cancer (CRC). Our aim was to identify the major molecules (i.e., genes, miRNAs and proteins) involved in this process. (2) Methods: We isolated and performed whole-genome analysis of gene, miRNA, and protein expression in three types of liver cells (Ito cells, Kupffer cells, and liver sinusoidal endothelial cells) from the TME of a murine model of CRC liver metastasis. We selected the statistically significant differentially expressed molecules using the Student's t-test with Benjamini-Hochberg correction and performed functional statistically-significant enrichment analysis of differentially expressed molecules with hypergeometric distribution using the curated collection of molecular signatures, MSigDB. To build a gene-miRNA-protein network centered in Brca1, we developed a software package (miRDiana) that collects miRNA targets from the union of the TargetScan, MicroCosm, mirTarBase, and miRWalk databases. This was used to search for miRNAs targeting Brca1. We validated the most relevant miRNAs with real-time quantitative PCR. To investigate BRCA1 protein expression, we built tissue microarrays (TMAs) from hepatic metastases of 34 CRC patients. (3) Results: Using integrated omics analyses, we observed that the Brca1 gene is among the twenty transcripts simultaneously up-regulated in all three types of TME liver cells during metastasis. Further analysis revealed that Brca1 is the last BRCA1-associated genome surveillance complex (BASC) gene activated in the TME. We confirmed this finding in human reanalyzing transcriptomics datasets from 184 patients from non-tumor colorectal tissue, primary colorectal tumor and colorectal liver metastasis of the GEO database. We found that the most probable sequence of cell activation during metastasis is Endothelial→Ito→Kupffer. Immunohistochemical analysis of human liver metastases showed the BRCA1 protein was co-localized in Ito, Kupffer, and endothelial cells in 81.8% of early or synchronous metastases. However, in the greater part of the metachronous liver metastases, this protein was not expressed in any of these TME cells. (4) Conclusions: These results suggest a possible role of the co-expression of BRCA1 in Ito, Kupffer, and sinusoidal endothelial cells in the early occurrence of CRC liver metastases, and point to BRCA1 as a potential TME biomarker.

A Multiplex Assay for the Stratification of Patients with Primary Central Nervous System Lymphoma Using Targeted Mass Spectrometry.

Primary central nervous system lymphomas (PCNSL) account for approximately 2% to 3% of all primary brain tumors. Until now, neuropathological tumor tissue analysis, most frequently gained by stereotactic biopsy, is still the diagnostic gold standard. Here, we rigorously analyzed two independent patient cohorts comprising the clinical entities PCNSL (n = 47), secondary central nervous system lymphomas (SCNSL; n = 13), multiple sclerosis (MS, n = 23), glioma (n = 10), other tumors (n = 17) and tumor-free controls (n = 21) by proteomic approaches. In total, we identified more than 1220 proteins in the cerebrospinal fluid (CSF) and validated eight candidate biomarkers by a peptide-centric approach in an independent patient cohort (n = 63). Thus, we obtained excellent diagnostic accuracy for the stratification between PCNSL, MS and glioma patients as well as tumor-free controls for three peptides originating from the three proteins VSIG4, GPNMB4 and APOC2. The combination of all three biomarker candidates resulted in diagnostic accuracy with an area under the curve (AUC) of 0.901 (PCNSL vs. MS), AUC of 0.953 (PCNSL vs. glioma) and AUC 0.850 (PCNSL vs. tumor-free control). In summary, the determination of VSIG4, GPNMB4 and APOC2 in CSF as novel biomarkers for supporting the diagnosis of PCNSL is suggested.

Secretome Analysis of Mesenchymal Stem Cell Factors Fostering Oligodendroglial Differentiation of Neural Stem Cells In Vivo.

Mesenchymal stem cell (MSC)-secreted factors have been shown to significantly promote oligodendrogenesis from cultured primary adult neural stem cells (aNSCs) and oligodendroglial precursor cells (OPCs). Revealing underlying mechanisms of how aNSCs can be fostered to differentiate into a specific cell lineage could provide important insights for the establishment of novel neuroregenerative treatment approaches aiming at myelin repair. However, the nature of MSC-derived differentiation and maturation factors acting on the oligodendroglial lineage has not been identified thus far. In addition to missing information on active ingredients, the degree to which MSC-dependent lineage instruction is functional in vivo also remains to be established. We here demonstrate that MSC-derived factors can indeed stimulate oligodendrogenesis and myelin sheath generation of aNSCs transplanted into different rodent central nervous system (CNS) regions, and furthermore, we provide insights into the underlying mechanism on the basis of a comparative mass spectrometry secretome analysis. We identified a number of secreted proteins known to act on oligodendroglia lineage differentiation. Among them, the tissue inhibitor of metalloproteinase type 1 (TIMP-1) was revealed to be an active component of the MSC-conditioned medium, thus validating our chosen secretome approach.

ARID1A Regulates Transcription and the Epigenetic Landscape via POLE and DMAP1 while ARID1A Deficiency or Pharmacological Inhibition Sensitizes Germ Cell Tumor Cells to ATR Inhibition.

Germ cell tumors (GCTs) are the most common solid malignancies found in young men. Although they generally have high cure rates, metastases, resistance to cisplatin-based therapy, and late toxicities still represent a lethal threat, arguing for the need of new therapeutic options. In a previous study, we identified downregulation of the chromatin-remodeling SWI/SNF complex member ARID1A as a key event in the mode of action of the histone deacetylase inhibitor romidepsin. Additionally, the loss-of-function mutations re-sensitize different tumor types to various drugs, like EZH2-, PARP-, HDAC-, HSP90- or ATR-inhibitors. Thus, ARID1A presents as a promising target for synthetic lethality and combination therapy. In this study, we deciphered the molecular function of ARID1A and screened for the potential of two pharmacological ARID1A inhibitors as a new therapeutic strategy to treat GCTs. By CRISPR/Cas9, we generated ARID1A-deficient GCT cells and demonstrate by mass spectrometry that ARID1A is putatively involved in regulating transcription, DNA repair and the epigenetic landscape via DNA Polymerase POLE and the DNA methyltransferase 1-associated protein DMAP1. Additionally, ARID1A/ARID1A deficiency or pharmacological inhibition increased the efficacy of romidepsin and considerably sensitized GCT cells, including cisplatin-resistant subclones, towards ATR inhibition. Thus, targeting ARID1A in combination with romidepsin and ATR inhibitors presents as a new putative option to treat GCTs.

Extracellular Redox Regulation of α7ß Integrin-Mediated Cell Migration Is Signaled via a Dominant Thiol-Switch.

While adhering to extracellular matrix (ECM) proteins, such as laminin-111, cells temporarily produce hydrogen peroxide at adhesion sites. To study the redox regulation of α7ß1 integrin-mediated cell adhesion to laminin-111, a conserved cysteine pair within the α-subunit hinge region was replaced for alanines. The molecular and cellular effects were analyzed by electron and atomic force microscopy, impedance-based migration assays, flow cytometry and live cell imaging. This cysteine pair constitutes a thiol-switch, which redox-dependently governs the equilibrium between an extended and a bent integrin conformation with high and low ligand binding activity, respectively. Hydrogen peroxide oxidizes the cysteines to a disulfide bond, increases ligand binding and promotes cell migration toward laminin-111. Inversely, extracellular thioredoxin-1 reduces the disulfide, thereby decreasing laminin binding. Mutation of this cysteine pair into the non-oxidizable hinge-mutant shows molecular and cellular effects similar to the reduced wild-type integrin, but lacks redox regulation. This proves the existence of a dominant thiol-switch within the α subunit hinge of α7ß1 integrin, which is sufficient to implement activity regulation by extracellular redox agents in a redox-regulatory circuit. Our data reveal a novel and physiologically relevant thiol-based regulatory mechanism of integrin-mediated cell-ECM interactions, which employs short-lived hydrogen peroxide and extracellular thioredoxin-1 as signaling mediators.

The Proteomic Landscape of Cysteine Oxidation That Underpins Retinoic Acid-Induced Neuronal Differentiation.

The initial phases of neuronal differentiation are key to neuronal function. A particularly informative model to study these initial phases are retinoic acid-stimulated SH-SY5Y cells. Although these progressions are associated with redox-sensitive processes, it is largely undefined how the cellular proteome underpins redox dynamics and the management of reactive oxygen species. Here, we map the global cysteine-based redox landscape of SH-SY5Y cells using quantitative redox proteomics. We find evidence that redox alterations occurred early in differentiation and affect the expression of neuronal marker proteins and the extension of neurites. The spatiotemporal analysis of reactive oxygen species suggests a NOX2-dependent peak in cytoplasmic superoxide anions/hydrogen peroxide generation 2 h after retinoic acid stimulation. At the same time point, 241 out of 275 proteins with an altered cysteine redox state are reversibly oxidized in response to retinoic acid. Our analyses pinpoint redox alterations of proteins involved in the retinoic acid homeostasis and cytoskeletal dynamics.

Protein complexes including PGRMC1 and actin-associated proteins are disrupted by AG-205.

PGRMC1 is a protein from the MAPR family with a range of cellular functions. PGRMC1 has been described to play a role in fertility, neuroprotection, steroidogenesis, membrane trafficking and in cancer cell biology. PGRMC1 represents a likely key regulator of cell metabolism and proliferation, as well as a potential target for anti-cancer therapies. To further understand the functions of PGRMC1 and the mechanism of the small molecule inhibitor of PGRMC1, AG-205, proteins differentially bound to PGRMC1 were identified following AG-205 treatment of MIA PaCa-2 cells. Our results suggest that AG-205 influences PGRMC1 interactions with the actin cytoskeleton. The binding of two PGRMC1-associated proteins that support this, RACK1 and alpha-Actinin-1, was reduced following AG-205 treatment. The biology associated with PGRMC1 binding partners identified here merits further investigation.