Context:α-Klotho has emerged as a powerful regulator of the aging process. To date, the expression profile of α-Klotho in human tissues is unknown, and its existence in some human tissue types is subject to much controversy.Objective:This is the first study to characterize systemwide tissue expression of transmembrane α-Klotho in humans. We have employed next-generation targeted proteomic analysis using parallel reaction monitoring in parallel with conventional antibody-based methods to determine the expression and spatial distribution of human α-Klotho expression in health.Results:The distribution of α-Klotho in human tissues from various organ systems, including arterial, epithelial, endocrine, reproductive, and neuronal tissues, was first identified by immunohistochemistry. Kidney tissues showed strong α-Klotho expression, whereas liver did not reveal a detectable signal. These results were next confirmed by Western blotting of both whole tissues and primary cells. To validate our antibody-based results, α-Klotho-expressing tissues were subjected to parallel reaction monitoring mass spectrometry (data deposited at ProteomeXchange, PXD002775) identifying peptides specific for the full-length, transmembrane α-Klotho isoform.Conclusions:The data presented confirm α-Klotho expression in the kidney tubule and in the artery and provide evidence of α-Klotho expression across organ systems and cell types that has not previously been described in humans.

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Protein tyrosine phosphatases (PTPs) constitute a large enzyme family with important biological functions. Inhibition of PTP activity through reversible oxidation of the active-site cysteine residue is emerging as a general, yet poorly characterized, regulatory mechanism. In this study, we describe a generic antibody-based method for detection of oxidation-inactivated PTPs. Previous observations of oxidation of receptor-like PTP (RPTP) ␣ after treatment of cells with H2O2 were confirmed. Platelet-derived growth factor (PDGF)-induced oxidation of endogenous SHP-2, sensitive to treatment with the phosphatidylinositol 3-kinase inhibitor LY294002, was demonstrated. Furthermore, oxidation of RPTP␣ was shown after UV-irradiation. Interestingly, the catalytically inactive second PTP domain of RPTP␣ demonstrated higher susceptibility to oxidation. The experiments thus demonstrate previously unrecognized intrinsic differences between PTP domains to susceptibility to oxidation and suggest mechanisms for regulation of RPTPs with tandem PTP domains. The antibody strategy for detection of reversible oxidation is likely to facilitate further studies on regulation of PTPs and might be applicable to analysis of redox regulation of other enzyme families with active-site cysteine residues.

Quantitative mass-spectrometry-based spatial proteomics involves elaborate, expensive, and time-consuming experimental procedures, and considerable effort is invested in the generation of such data. Multiple research groups have described a variety of approaches for establishing high-quality proteome-wide datasets. However, data analysis is as critical as data production for reliable and insightful biological interpretation, and no consistent and robust solutions have been offered to the community so far. Here, we introduce the requirements for rigorous spatial proteomics data analysis, as well as the statistical machine learning methodologies needed to address them, including supervised and semi-supervised machine learning, clustering, and novelty detection. We present freely available software solutions that implement innovative state-of-the-art analysis pipelines and illustrate the use of these tools through several case studies involving multiple organisms, experimental designs, mass spectrometry platforms, and quantitation techniques. We also propose sound analysis strategies for identifying dynamic changes in subcellular localization by comparing and contrasting data describing different biological conditions. We conclude by discussing future needs and developments in spatial proteomics data analysis. Molecular &

Oxidation is emerging as an important regulatory mechanism of protein-tyrosine phosphatases (PTPs).Here we report that PTPs are differentially oxidized, and we provide evidence for the underlying mechanism. The membrane-proximal RPTP␣-D1 was catalytically active but not readily oxidized as assessed by immunoprobing with an antibody that recognized oxidized catalytic site cysteines in PTPs (oxPTPs). In contrast, the membrane-distal RPTP␣-D2, a poor PTP, was readily oxidized. Oxidized catalytic site cysteines in PTP immunoprobing and mass spectrometry demonstrated that mutation of two residues in the Tyr(P) loop and the WPD loop that reverse catalytic activity of RPTP␣-D1 and RPTP␣-D2 also reversed oxidizability, suggesting that oxidizability and catalytic activity are coupled. However, catalytically active PTP1B and LAR-D1 were readily oxidized. Oxidizability was strongly dependent on pH, indicating that the microenvironment of the catalytic cysteine has an important role. Crystal structures of PTP domains demonstrated that the orientation of the absolutely conserved PTP loop arginine correlates with oxidizability of PTPs, and consistently, RPTP-D1, with a similar conformation as RPTP␣-D1, was not readily oxidized. In conclusion, PTPs are differentially oxidized at physiological pH and H 2 O 2 concentrations, and the PTP loop arginine is an important determinant for susceptibility to oxidation.

Estrogen Receptor-β (ERβ) has been implicated in many cancers. In prostate and breast cancer its function is controversial, but genetic studies implicate a role in cancer progression. Much of the confusion around ERβ stems from antibodies that are inadequately validated, yet have become standard tools for deciphering its role. Using an ERβ-inducible cell system we assessed commonly utilized ERβ antibodies and show that one of the most commonly used antibodies, NCL-ER-BETA, is non-specific for ERβ. Other antibodies have limited ERβ specificity or are only specific in one experimental modality. ERβ is commonly studied in MCF-7 (breast) and LNCaP (prostate) cancer cell lines, but we found no ERβ expression in either, using validated antibodies and independent mass spectrometry-based approaches. Our findings question conclusions made about ERβ using the NCL-ER-BETA antibody, or LNCaP and MCF-7 cell lines. We describe robust reagents, which detect ERβ across multiple experimental approaches and in clinical samples.

Protein tyrosine phosphatases (PTPs) are fundamental to the regulation of cellular signalling cascades triggered by protein tyrosine kinases. Most receptor-like PTPs (RPTPs) comprise two tandem PTP domains, with only the membrane proximal domains (D1) having significant phosphatase activity; the membrane distal domains (D2) display little to no catalytic activity. Intriguingly, however, many RPTP D2s share the catalytically essential Cys and Arg residues of D1s. D2 of RPTPR may function as a redox sensor that mediates regulation of D1 via reactive oxygen species. Oxidation of Cys723 of RPTPR D2 (equivalent to PTP catalytic Cys residues) stabilizes RPTPR dimers, induces rotational coupling, and is required for inactivation of D1 phosphatase activity. Here, we investigated the structural consequences of RPTPR D2 oxidation. Exposure of RPTPR D2 to oxidants promotes formation of a cyclic sulfenamide species, a reversibly oxidized state of Cys723, accompanied by conformational changes of the D2 catalytic site. The cyclic sulfenamide is highly resistant to terminal oxidation to sulfinic and sulfonic acids. Conformational changes associated with RPTPR D2 oxidation have implications for RPTPR quaternary structure and allosteric regulation of D1 phosphatase activity.The reversible tyrosine phosphorylation of proteins, reciprocally controlled by tyrosine kinases and phosphatases, is crucial to the regulation of diverse cellular processes, including growth, proliferation, and differentiation (1). Redox reactions, mediated by reactive oxygen species (ROS), 1 control PTP activity in response to a variety of hormones and growth factors (2-6). Generation of ROS accompanies PTK activation (7,8), with oxidation-induced inhibition of PTP activity amplifying PTK-dependent signaling (9-11).PTP-catalyzed protein dephosphorylation proceeds via a nucleophilic displacement reaction utilizing an essential cysteine residue conserved within the PTP signature motif Cys(X) 5 Arg (12-15). The low pK a of the catalytic cysteine necessary for its function (16) renders PTPs susceptible to inactivation by ROS-induced cysteine oxidation. However, the reversibility of redox-mediated inactivation of PTPs requires that a reversibly oxidized state of the cysteine residue be stabilized at the PTP catalytic site and that formation of SO 2 and SO 3 , two irreversibly oxidized species of cysteine, be suppressed (17). Crystallographic studies of the tyrosine specific cytosolic PTP1B revealed that the reversibly oxidized form of the enzyme was not the expected sulfenic acid or disulfide bond, but an oxidation state of cysteine termed a cyclic sulfenamide, a species not previously observed in proteins (18,19). Generation of the cyclic sulfenamide inhibits catalytic activity by blocking the nucleophilic cysteine residue through a covalent bond of its thiol group with the neighboring main chain amide, a linkage that is accompanied by profound conformational transitions of the catalytic site PTP and pTyr loops. The oxidation properties of the catalyt...

The brassinosteroid receptor brassinosteroid insensitive 1 (BRI1) is a member of the leucine-rich repeat receptor-like kinase family. The intracellular kinase domain of BRI1 is an active kinase and also encapsulates a guanylate cyclase catalytic centre. Using liquid chromatography tandem mass spectrometry, we confirmed that the recombinant cytoplasmic domain of BRI1 generates pmol amounts of cGMP per μg protein with a preference for magnesium over manganese as a co-factor. Importantly, a functional BRI1 kinase is essential for optimal cGMP generation. Therefore, the guanylate cyclase activity of BRI1 is modulated by the kinase while cGMP, the product of the guanylate cyclase, in turn inhibits BRI1 kinase activity. Furthermore, we show using Arabidopsis root cell cultures that cGMP rapidly potentiates phosphorylation of the downstream substrate brassinosteroid signaling kinase 1 (BSK1). Taken together, our results suggest that cGMP acts as a modulator that enhances downstream signaling while dampening signal generation from the receptor.