Intestinal mucin is a chaperone of multivalent copper.

Mucus protects the epithelial cells of the digestive and respiratory tracts from pathogens and other hazards. Progress in determining the molecular mechanisms of mucus barrier function has been limited by the lack of high-resolution structural information on mucins, the giant, secreted, gel-forming glycoproteins that are the major constituents of mucus. Here, we report how mucin structures we determined enabled the discovery of an unanticipated protective role of mucus: managing the toxic transition metal copper. Using two juxtaposed copper binding sites, one for Cu2+ and the other for Cu1+, the intestinal mucin, MUC2, prevents copper toxicity by blocking futile redox cycling and the squandering of dietary antioxidants, while nevertheless permitting uptake of this important trace metal into cells. These findings emphasize the value of molecular structure in advancing mucosal biology, while introducing mucins, produced in massive quantities to guard extensive mucosal surfaces, as extracellular copper chaperones.

Assembly Mechanism of Mucin and von Willebrand Factor Polymers.

The respiratory and intestinal tracts are exposed to physical and biological hazards accompanying the intake of air and food. Likewise, the vasculature is threatened by inflammation and trauma. Mucin glycoproteins and the related von Willebrand factor guard the vulnerable cell layers in these diverse systems. Colon mucins additionally house and feed the gut microbiome. Here, we present an integrated structural analysis of the intestinal mucin MUC2. Our findings reveal the shared mechanism by which complex macromolecules responsible for blood clotting, mucociliary clearance, and the intestinal mucosal barrier form protective polymers and hydrogels. Specifically, cryo-electron microscopy and crystal structures show how disulfide-rich bridges and pH-tunable interfaces control successive assembly steps in the endoplasmic reticulum and Golgi apparatus. Remarkably, a densely O-glycosylated mucin domain performs an organizational role in MUC2. The mucin assembly mechanism and its adaptation for hemostasis provide the foundation for rational manipulation of barrier function and coagulation.

A unique chaperoning mechanism in class A JDPs recognizes and stabilizes mutant p53.

J-domain proteins (JDPs) constitute a large family of molecular chaperones that bind a broad spectrum of substrates, targeting them to Hsp70, thus determining the specificity of and activating the entire chaperone functional cycle. The malfunction of JDPs is therefore inextricably linked to myriad human disorders. Here, we uncover a unique mechanism by which chaperones recognize misfolded clients, present in human class A JDPs. Through a newly identified ß-hairpin site, these chaperones detect changes in protein dynamics at the initial stages of misfolding, prior to exposure of hydrophobic regions or large structural rearrangements. The JDPs then sequester misfolding-prone proteins into large oligomeric assemblies, protecting them from aggregation. Through this mechanism, class A JDPs bind destabilized p53 mutants, preventing clearance of these oncoproteins by Hsp70-mediated degradation, thus promoting cancer progression. Removal of the ß-hairpin abrogates this protective activity while minimally affecting other chaperoning functions. This suggests the class A JDP ß-hairpin as a highly specific target for cancer therapeutics.

The disulfide catalyst QSOX1 maintains the colon mucosal barrier by regulating Golgi glycosyltransferases.

Mucus is made of enormous mucin glycoproteins that polymerize by disulfide crosslinking in the Golgi apparatus. QSOX1 is a catalyst of disulfide bond formation localized to the Golgi. Both QSOX1 and mucins are highly expressed in goblet cells of mucosal tissues, leading to the hypothesis that QSOX1 catalyzes disulfide-mediated mucin polymerization. We found that knockout mice lacking QSOX1 had impaired mucus barrier function due to production of defective mucus. However, an investigation on the molecular level revealed normal disulfide-mediated polymerization of mucins and related glycoproteins. Instead, we detected a drastic decrease in sialic acid in the gut mucus glycome of the QSOX1 knockout mice, leading to the discovery that QSOX1 forms regulatory disulfides in Golgi glycosyltransferases. Sialylation defects in the colon are known to cause colitis in humans. Here we show that QSOX1 redox control of sialylation is essential for maintaining mucosal function.

T cell antigen receptor signaling and immunological synapse stability require myosin IIA.

Immunological synapses are initiated by signaling in discrete T cell antigen receptor microclusters and are important for the differentiation and effector functions of T cells. Synapse formation involves the orchestrated movement of microclusters toward the center of the contact area with the antigen-presenting cell. Microcluster movement is associated with centripetal actin flow, but the function of motor proteins is unknown. Here we show that myosin IIA was necessary for complete assembly and movement of T cell antigen receptor microclusters. In the absence of myosin IIA or its ATPase activity, T cell signaling was interrupted 'downstream' of the kinase Lck and the synapse was destabilized. Thus, T cell antigen receptor signaling and the subsequent formation of immunological synapses are active processes dependent on myosin IIA.

Now at the Met: fine art of reversible sulfoxidation.

In this issue, Lee et al. (2013) exhibit methionine sulfoxidation in a new light. By bringing together two antagonistic enzymes affecting methionine redox state, the authors demonstrate that methionine oxidation constitutes a reversible, posttranslational regulatory mechanism, akin to protein phosphorylation.

Quiescin sulfhydryl oxidase 1 (QSOX1) glycosite mutation perturbs secretion but not Golgi localization.

Quiescin sulfhydryl oxidase 1 (QSOX1) catalyzes the formation of disulfide bonds in protein substrates. Unlike other enzymes with related activities, which are commonly found in the endoplasmic reticulum, QSOX1 is localized to the Golgi apparatus or secreted. QSOX1 is upregulated in quiescent fibroblast cells and secreted into the extracellular environment, where it contributes to extracellular matrix assembly. QSOX1 is also upregulated in adenocarcinomas, though the extent to which it is secreted in this context is currently unknown. To achieve a better understanding of factors that dictate QSOX1 localization and function, we aimed to determine how post-translational modifications affect QSOX1 trafficking and activity. We found a highly conserved N-linked glycosylation site to be required for QSOX1 secretion from fibroblasts and other cell types. Notably, QSOX1 lacking a glycan at this site arrives at the Golgi, suggesting that it passes endoplasmic reticulum quality control but is not further transported to the cell surface for secretion. The QSOX1 transmembrane segment is dispensable for Golgi localization and secretion, as fully luminal and transmembrane variants displayed the same trafficking behavior. This study provides a key example of the effect of glycosylation on Golgi exit and contributes to an understanding of late secretory sorting and quality control.

Protein-Protein Communication and Enzyme Activation Mediated by a Synthetic Chemical Transducer.

The design and function of a synthetic "chemical transducer" that can generate an unnatural communication channel between two proteins is described. Specifically, we show how this transducer enables platelet-derived growth factor to trigger (in vitro) the catalytic activity of glutathione-s-transferase (GST), which is not its natural enzyme partner. GST activity can be further controlled by adding specific oligonucleotides that switch the enzymatic reaction on and off. We also demonstrate that a molecular machine, which can regulate the function of an enzyme, could be used to change the way a prodrug is activated in a "programmable" manner.

Occupancy of lymphocyte LFA-1 by surface-immobilized ICAM-1 is critical for TCR- but not for chemokine-triggered LFA-1 conversion to an open headpiece high-affinity state.

Lymphocyte arrest and spreading on ICAM-1-expressing APCs require activation of lymphocyte LFA-1 by TCR signals, but the conformational switches of this integrin during these critical processes are still elusive. Using Ab probes that distinguish between different LFA-1 conformations, we found that, unlike strong chemokine signals, potent TCR stimuli were insufficient to trigger LFA-1 extension or headpiece opening in primary human lymphocytes. Nevertheless, LFA-1 in these TCR-stimulated T cells became highly adhesive to both anchored and mobile surface-bound ICAM-1, although it failed to bind soluble ICAM-1 with measurable affinity. Rapid rearrangement of LFA-1 by immobilized ICAM-1 switched the integrin to an open headpiece conformation within numerous scattered submicron focal dots that did not readily collapse into a peripheral LFA-1 ring. Headpiece-activated LFA-1 microclusters were enriched with talin but were devoid of TCR and CD45. Notably, LFA-1 activation by TCR signals as well as subsequent T cell spreading on ICAM-1 took place independently of cytosolic Ca(2+). In contrast to LFA-1-activating chemokine signals, TCR activation of LFA-1 readily took place in the absence of external shear forces. LFA-1 activation by TCR signals also did not require internal myosin II forces but depended on intact actin cytoskeleton. Our results suggest that potent TCR signals fail to trigger LFA-1 headpiece activation unless the integrin first gets stabilized by surface-bound ICAM-1 within evenly scattered actin-dependent LFA-1 focal dots, the quantal units of TCR-stimulated T cell arrest and spreading on ICAM-1.

Correction: Inhibition of fibroblast secreted QSOX1 perturbs extracellular matrix in the tumor microenvironment and decreases tumor growth and metastasis in murine cancer models.

[This corrects the article DOI: 10.18632/oncotarget.27438.].

Inhibition of fibroblast secreted QSOX1 perturbs extracellular matrix in the tumor microenvironment and decreases tumor growth and metastasis in murine cancer models.

Extracellular matrix (ECM) plays an important role in tumor development and dissemination, but few points of therapeutic intervention targeting ECM of the tumor microenvironment have been exploited to date. Recent observations suggest that the enzymatic introduction of disulfide bond cross-links into the ECM may be modulated to affect cancer progression. Specifically, the disulfide bond-forming activity of the enzyme Quiescin sulfhydryl oxidase 1 (QSOX1) is required by fibroblasts to assemble ECM components for adhesion and migration of cancer cells. Based on this finding and the increased QSOX1 expression in the stroma of aggressive breast carcinomas, we developed monoclonal antibody inhibitors with the aim of preventing QSOX1 from participating in pro-metastatic ECM remodeling. Here we show that QSOX1 inhibitory antibodies decreased tumor growth and metastasis in murine cancer models and had added benefits when provided together with chemotherapy. Mechanistically, the inhibitors dampened stromal participation in tumor development, as the tumors of treated animals showed fewer myofibroblasts and poorer ECM organization. Thus, our findings demonstrate that specifically targeting excess stromal QSOX1 secreted in response to tumor-cell signaling provides a means to modulate the tumor microenvironment and may complement other therapeutic approaches in cancer.

cis-Proline mutants of quiescin sulfhydryl oxidase 1 with altered redox properties undermine extracellular matrix integrity and cell adhesion in fibroblast cultures.

The thioredoxin superfamily has expanded and diverged extensively throughout evolution such that distant members no longer show appreciable sequence homology. Nevertheless, redox-active thioredoxin-fold proteins functioning in diverse physiological contexts often share canonical amino acids near the active-site (di-)cysteine motif. Quiescin sulfhydryl oxidase 1 (QSOX1), a catalyst of disulfide bond formation secreted by fibroblasts, is a multi-domain thioredoxin superfamily enzyme with certain similarities to the protein disulfide isomerase (PDI) enzymes. Among other potential functions, QSOX1 supports extracellular matrix assembly in fibroblast cultures. We introduced mutations at a cis-proline in QSOX1 that is conserved across the thioredoxin superfamily and was previously observed to modulate redox interactions of the bacterial enzyme DsbA. The resulting QSOX1 variants showed a striking detrimental effect when added exogenously to fibroblasts: they severely disrupted the extracellular matrix and cell adhesion, even in the presence of naturally secreted, wild-type QSOX1. The specificity of this phenomenon for particular QSOX1 mutants inspired an investigation of the effects of mutation on catalytic and redox properties. For a series of QSOX1 mutants, the detrimental effect correlated with the redox potential of the first redox-active site, and an X-ray crystal structure of one of the mutants revealed the reorganization of the cis-proline loop caused by the mutations. Due to the conservation of the mutated residues across the PDI family and beyond, insights obtained in this study may be broadly applicable to a variety of physiologically important redox-active enzymes. IMPACT STATEMENT: We show that mutation of a conserved cis-proline amino acid, analogous to a mutation used to trap substrates of a bacterial disulfide catalyst, has a dramatic effect on the physiological function of the mammalian disulfide catalyst QSOX1. As the active-site region of QSOX1 is shared with the large family of protein disulfide isomerases in humans, the effects of such mutations on redox properties, enzymatic activity, and biological targeting may be relevant across the family.

Intestinal Gel-Forming Mucins Polymerize by Disulfide-Mediated Dimerization of D3 Domains.

The mucin 2 glycoprotein assembles into a complex hydrogel that protects intestinal epithelia and houses the gut microbiome. A major step in mucin 2 assembly is further multimerization of preformed mucin dimers, thought to produce a honeycomb-like arrangement upon hydrogel expansion. Important open questions are how multiple mucin 2 dimers become covalently linked to one another and how mucin 2 multimerization compares with analogous processes in related polymers such as respiratory tract mucins and the hemostasis protein von Willebrand factor. Here we report the x-ray crystal structure of the mucin 2 multimerization module, found to form a dimer linked by two intersubunit disulfide bonds. The dimer structure calls into question the current model for intestinal mucin assembly, which proposes disulfide-mediated trimerization of the same module. Key residues making interactions across the dimer interface are highly conserved in intestinal mucin orthologs, supporting the physiological relevance of the observed quaternary structure. With knowledge of the interface residues, it can be demonstrated that many of these amino acids are also present in other mucins and in von Willebrand factor, further indicating that the stable dimer arrangement reported herein is likely to be shared across this functionally broad protein family. The mucin 2 module structure thus reveals the manner by which both mucins and von Willebrand factor polymerize, drawing deep structural parallels between macromolecular assemblies critical to mucosal epithelia and the vasculature.

3D mapping of native extracellular matrix reveals cellular responses to the microenvironment.

Cells and extracellular matrix (ECM) are mutually interdependent: cells guide self-assembly of ECM precursors, and the resulting ECM architecture supports and instructs cells. Though bidirectional signaling between ECM and cells is fundamental to cell biology, it is challenging to gain high-resolution structural information on cellular responses to the matrix microenvironment. Here we used cryo-scanning transmission electron tomography (CSTET) to reveal the nanometer- to micron-scale organization of major fibroblast ECM components in a native-like context, while simultaneously visualizing internal cell ultrastructure including organelles and cytoskeleton. In addition to extending current models for collagen VI fibril organization, three-dimensional views of thick cell regions and surrounding matrix showed how ECM networks impact the structures and dynamics of intracellular organelles and how cells remodel ECM. Collagen VI and fibronectin were seen to distribute in fundamentally different ways in the cell microenvironment and perform distinct roles in supporting and interacting with cells. This work demonstrates that CSTET provides a new perspective for the study of ECM in cell biology, highlighting labeled extracellular elements against a backdrop of unlabeled but morphologically identifiable cellular features with nanometer resolution detail.

3D visualization of mitochondrial solid-phase calcium stores in whole cells.

The entry of calcium into mitochondria is central to metabolism, inter-organelle communication, and cell life/death decisions. Long-sought transporters involved in mitochondrial calcium influx and efflux have recently been identified. To obtain a unified picture of mitochondrial calcium utilization, a parallel advance in understanding the forms and quantities of mitochondrial calcium stores is needed. We present here the direct 3D visualization of mitochondrial calcium in intact mammalian cells using cryo-scanning transmission electron tomography (CSTET). Amorphous solid granules containing calcium and phosphorus were pervasive in the mitochondrial matrices of a variety of mammalian cell types. Analysis based on quantitative electron scattering revealed that these repositories are equivalent to molar concentrations of dissolved ions. These results demonstrate conclusively that calcium buffering in the mitochondrial matrix in live cells occurs by phase separation, and that solid-phase stores provide a major ion reservoir that can be mobilized for bioenergetics and signaling.

Protein recognition by a pattern-generating fluorescent molecular probe.

Fluorescent molecular probes have become valuable tools in protein research; however, the current methods for using these probes are less suitable for analysing specific populations of proteins in their native environment. In this study, we address this gap by developing a unimolecular fluorescent probe that combines the properties of small-molecule-based probes and cross-reactive sensor arrays (the so-called chemical 'noses/tongues'). On the one hand, the probe can detect different proteins by generating unique identification (ID) patterns, akin to cross-reactive arrays. On the other hand, its unimolecular scaffold and selective binding enable this ID-generating probe to identify combinations of specific protein families within complex mixtures and to discriminate among isoforms in living cells, where macroscopic arrays cannot access. The ability to recycle the molecular device and use it to track several binding interactions simultaneously further demonstrates how this approach could expand the fluorescent toolbox currently used to detect and image proteins.

Overcoming a species-specificity barrier in development of an inhibitory antibody targeting a modulator of tumor stroma.

The secreted disulfide catalyst Quiescin sulfhydryl oxidase-1 (QSOX1) affects extracellular matrix organization and is overexpressed in various adenocarcinomas and associated stroma. Inhibition of extracellular human QSOX1 by a monoclonal antibody decreased tumor cell migration in a cell co-culture model and hence may have therapeutic potential. However, the species specificity of the QSOX1 monoclonal antibody has been a setback in assessing its utility as an anti-metastatic agent in vivo, a common problem in the antibody therapy industry. We therefore used structurally guided engineering to expand the antibody species specificity, improving its affinity toward mouse QSOX1 by at least four orders of magnitude. A crystal structure of the re-engineered variant, complexed with its mouse antigen, revealed that the antibody accomplishes dual-species targeting through altered contacts between its heavy and light chains, plus replacement of bulky aromatics by flexible side chains and versatile water-bridged polar interactions. In parallel, we produced a surrogate antibody targeting mouse QSOX1 that exhibits a new QSOX1 inhibition mode. This set of three QSOX1 inhibitory antibodies is compatible with various mouse models for pre-clinical trials and biotechnological applications. In this study we provide insights into structural blocks to cross-reactivity and set up guideposts for successful antibody design and re-engineering.

Serum thioredoxin reductase is highly increased in mice with hepatocellular carcinoma and its activity is restrained by several mechanisms.

Increased thioredoxin reductase (TrxR) levels in serum were recently identified as possible prognostic markers for human prostate cancer or hepatocellular carcinoma. We had earlier shown that serum levels of TrxR protein are very low in healthy mice, but can in close correlation to alanine aminotransferase (ALT) increase more than 200-fold upon chemically induced liver damage. We also found that enzymatic TrxR activity in serum is counteracted by a yet unidentified oxidase activity in serum. In the present study we found that mice carrying H22 hepatocellular carcinoma tumors present highly increased levels of TrxR in serum, similarly to that reported in human patients. In this case ALT levels did not parallel those of TrxR. We also discovered here that the TrxR-antagonistic oxidase activity in serum is due to the presence of quiescin Q6 sulfhydryl oxidase 1 (QSOX1). We furthermore found that the chemotherapeutic agents cisplatin or auranofin, when given systemically to H22 tumor bearing mice, can further inhibit TrxR activities in serum. The TrxR serum activity was also inhibited by endogenous electrophilic inhibitors, found to increase in tumor-bearing mice and to include protoporphyrin IX (PpIX) and 4-hydroxynonenal (HNE). Thus, hepatocellular carcinoma triggers high levels of serum TrxR that are not paralleled by ALT, and TrxR enzyme activity in serum is counteracted by several different mechanisms. The physiological role of TrxR in serum, if any, as well as its potential value as a prognostic marker for tumor progression, needs to be studied further.

Dopaminergic regulation of immune cells via D3 dopamine receptor: a pathway mediated by activated T cells.

Neuro-immune interactions enable mutual regulation of the nervous and immune systems. To date, evidence exists for manipulations of immune cells by neurotransmitters in the periphery. In this study, we suggest the existence of a pathway by which the brain affects immune cells. The pathway we describe here is mediated by dopamine receptors expressed on activated T cells, termed blasts. Blasts can cross the blood brain barrier regardless of antigen specificity and can therefore encounter neurotransmitters in the brain. We show that blasts have a unique response to dopaminergic activation, which has no counterpart in resting T cells. Dopaminergic activation of blasts induces a Th1 bias in their cytokine profile and causes changes in surface marker expression. We further suggest that these changes can subsequently be transferred to peripheral T cells. We have tested this pathway in two in vivo systems: in rats exogenously administered with L-dopa, and in schizophrenia, which is characterized by a central nervous system-restricted increase in dopamine. In both models, peripheral T cells exhibit similar features to those of dopaminergically activated blasts. The existence of such a pathway by which the brain can regulate immune cells opens a conceptually new direction in neuro-immune interactions.

The alpha7 nicotinic acetylcholine receptor in schizophrenia: decreased mRNA levels in peripheral blood lymphocytes.

Recent studies have suggested that the alpha7 nicotinic acetylcholine receptor (alpha7 AChR) may play a role in the pathogenesis of schizophrenia. In search for peripheral biological markers for schizophrenia we have investigated alpha7 mRNA levels in peripheral blood lymphocytes (PBLs) of schizophrenic patients and healthy controls. Peripheral blood samples were collected from medicated and non-medicated (drug naive) schizophrenic patients as well as from healthy (non-mentally ill) smokers and non-smokers. RNA was prepared from isolated lymphocytes. Polymerase chain reaction products specific for human alpha7 AChR were quantified by densitometry using Scion image-analysis (shared NIH software). We observed a significant decrease of alpha7 mRNA levels on PBLs of schizophrenic patients compared with controls. The decrease in alpha7 mRNA levels was not a result of medication management, because non-medicated schizophrenic patients displayed the same level of reduction in alpha7 mRNA as did patients receiving medication. In addition, we exclude the possibility that the observed decrease in alpha7 mRNA levels resulted from nicotine consumption in smoking, because healthy smokers exhibited the same levels of alpha7 mRNA as non-smokers. We propose that alpha7 AChR may be involved in the pathophysiology of the disease and may serve as a reliable peripheral biological marker in schizophrenia.