Optimization of quenched fluorescent peptide substrates of SARS-CoV-2 3CLpro main protease (Mpro) from proteomic identification of P6-P6' active site specificity.

SARS-CoV-2 3C-like main protease (3CLpro) is essential for protein excision from the viral polyprotein. 3CLpro inhibitor drug development to block SARS-CoV-2 replication focuses on the catalytic non-prime (P) side for specificity and potency, but the importance of the prime (P') side in substrate specificity and for drug development remains underappreciated. We determined the P6-P6' specificity for 3CLpro from >800 cleavage sites that we identified using Proteomic Identification of Cleavage site Specificity (PICS). Cleavage occurred after the canonical P1-Gln and non-canonical P1-His and P1-Met residues. Moreover, P3 showed a preference for Arg/Lys and P3' for His. Essential H-bonds between the N-terminal Ser1 of protomer-B in 3CLpro dimers form with P1-His, but not with P1-Met. Nonetheless, cleavage occurs at P1-Met456 in native MAP4K5. Elevated reactive oxygen species in SARS-CoV-2 infection oxidize methionines. Molecular simulations revealed P1-MetOX forms an H-bond with Ser1 and notably, strong positive cooperativity between P1-Met with P3'-His was revealed, which enhanced peptide-cleavage rates. The highly plastic S3' subsite accommodates P3'-His that displays stabilizing backbone H-bonds with Thr25 lying central in a "'threonine trio" (Thr24-Thr25-Thr26) in the P'-binding domain I. Molecular docking simulations unveiled structure-activity relationships impacting 3CLpro-substrate interactions, and the role of these structural determinants was confirmed by MALDI-TOF-MS cleavage assays of P1'- and P3'-positional scanning peptide libraries carrying a 2nd optimal cut-site as an internal positive control. These data informed the design of two new and highly soluble 3CLproquenched-fluorescent peptide substrates for improved FRET monitoring of 3CLpro activity with 15× improved sensitivity over current assays.IMPORTANCEFrom global proteomics identification of >800 cleavage sites, we characterized the P6-P6' active site specificity of SARS-CoV-2 3CLpro using proteome-derived peptide library screens, molecular modeling simulations, and focussed positional peptide libraries. In P1', we show that alanine and serine are cleaved 3× faster than glycine and the hydrophobic small amino acids Leu, Ile, or Val prevent cleavage of otherwise optimal non-prime sequences. In characterizing non-canonical non-prime P1 specificity, we explored the unusual P1-Met specificity, discovering enhanced cleavage when in the oxidized state (P1-MetOX). We unveiled unexpected amino acid cooperativity at P1-Met with P3'-His and noncanonical P1-His with P2-Phe, and the importance of the threonine trio (Thr24-Thr25-Thr26) in the prime side binding domain I in defining prime side binding in SARS-CoV-2 3CLpro. From these analyses, we rationally designed quenched-fluorescence natural amino acid peptide substrates with >15× improved sensitivity and high peptide solubility, facilitating handling and application for screening of new antiviral drugs.

No Substrate Left behind-Mining of Shotgun Proteomics Datasets Rescues Evidence of Proteolysis by SARS-CoV-2 3CLpro Main Protease.

Proteolytic processing is the most ubiquitous post-translational modification and regulator of protein function. To identify protease substrates, and hence the function of proteases, terminomics workflows have been developed to enrich and detect proteolytically generated protein termini from mass spectrometry data. The mining of shotgun proteomics datasets for such 'neo'-termini, to increase the understanding of proteolytic processing, is an underutilized opportunity. However, to date, this approach has been hindered by the lack of software with sufficient speed to make searching for the relatively low numbers of protease-generated semi-tryptic peptides present in non-enriched samples viable. We reanalyzed published shotgun proteomics datasets for evidence of proteolytic processing in COVID-19 using the recently upgraded MSFragger/FragPipe software, which searches data with a speed that is an order of magnitude greater than many equivalent tools. The number of protein termini identified was higher than expected and constituted around half the number of termini detected by two different N-terminomics methods. We identified neo-N- and C-termini generated during SARS-CoV-2 infection that were indicative of proteolysis and were mediated by both viral and host proteases-a number of which had been recently validated by in vitro assays. Thus, re-analyzing existing shotgun proteomics data is a valuable adjunct for terminomics research that can be readily tapped (for example, in the next pandemic where data would be scarce) to increase the understanding of protease function and virus-host interactions, or other diverse biological processes.

Integrating knowledge of protein sequence with protein function for the prediction and validation of new MALT1 substrates.

We developed a bioinformatics-led substrate discovery workflow to expand the known substrate repertoire of MALT1. Our approach, termed GO-2-Substrates, integrates protein function information, including GO terms from known substrates, with protein sequences to rank substrate candidates by similarity. We applied GO-2-Substrates to MALT1, a paracaspase and master regulator of NF-κB signalling in adaptive immune responses. With only 12 known substrates, the evolutionarily conserved paracaspase functions and phenotypes of Malt1 -/- mice strongly implicate the existence of undiscovered substrates. We tested the ranked predictions from GO-2-Substrates of new MALT1 human substrates by co-expression of candidates transfected with the oncogenic constitutively active cIAP2-MALT1 fusion protein or CARD11/BCL10/MALT1 active signalosome. We identified seven new MALT1 substrates by the co-transfection screen: TANK, TAB3, CASP10, ZC3H12D, ZC3H12B, CILK1 and ILDR2. Using catalytically inactive cIAP2-MALT1 (Cys464Ala), a MALT1 inhibitor, MLT-748, and noncleavable P1-Arg to Ala mutant versions of each substrate in dual transfections, we validated the seven new substrates in vitro. We confirmed the cleavage of endogenous TANK and the RNase ZC3H12D in B cells by Western blotting and mining TAILS N-terminomics datasets, where we also uncovered evidence for these and 12 other candidate substrates by endogenous MALT1. Thus, protein function information improves substrate predictions. The new substrates and other high-ranked MALT1 candidate substrates should open new biological frontiers for further validation and exploration of the function of MALT1 within and beyond NF-κB regulation.

Mechanistic insights into COVID-19 by global analysis of the SARS-CoV-2 3CLpro substrate degradome.

The main viral protease (3CLpro) is indispensable for SARS-CoV-2 replication. We delineate the human protein substrate landscape of 3CLpro by TAILS substrate-targeted N-terminomics. We identify more than 100 substrates in human lung and kidney cells supported by analyses of SARS-CoV-2-infected cells. Enzyme kinetics and molecular docking simulations of 3CLpro engaging substrates reveal how noncanonical cleavage sites, which diverge from SARS-CoV, guide substrate specificity. Cleaving the interactors of essential effector proteins, effectively stranding them from their binding partners, amplifies the consequences of proteolysis. We show that 3CLpro targets the Hippo pathway, including inactivation of MAP4K5, and key effectors of transcription, mRNA processing, and translation. We demonstrate that Spike glycoprotein directly binds galectin-8, with galectin-8 cleavage disengaging CALCOCO2/NDP52 to decouple antiviral-autophagy. Indeed, in post-mortem COVID-19 lung samples, NDP52 rarely colocalizes with galectin-8, unlike in healthy lungs. The 3CLpro substrate degradome establishes a foundational substrate atlas to accelerate exploration of SARS-CoV-2 pathology and drug design.

Mechanistic understanding of the combined immunodeficiency in complete human CARD11 deficiency.

BACKGROUND: Germline pathogenic variants impairing the caspase recruitment domain family member 11 (CARD11)-B cell chronic lymphocytic leukemia/lymphoma 10 (BCL10)-MALT1 paracaspase (MALT1) (CBM) complex are associated with diverse human diseases including combined immunodeficiency (CID), atopy, and lymphoproliferation. However, the impact of CARD11 deficiency on human B-cell development, signaling, and function is incompletely understood. OBJECTIVES: This study sought to determine the cellular, immunological, and biochemical basis of disease for 2 unrelated patients who presented with profound CID associated with viral and fungal respiratory infections, interstitial lung disease, and severe colitis. METHODS: Patients underwent next-generation sequencing, immunophenotyping by flow cytometry, signaling assays by immunoblot, and transcriptome profiling by RNA-sequencing. RESULTS: Both patients carried identical novel pathogenic biallelic loss-of-function variants in CARD11 (c.2509C>T; p.Arg837∗) leading to undetectable protein expression. This variant prevented CBM complex formation, severely impairing the activation of nuclear factor-κB, c-Jun N-terminal kinase, and MALT1 paracaspase activity in B and T cells. This functional defect resulted in a developmental block in B cells at the naive and type 1 transitional B-cell stage and impaired circulating T follicular helper cell (cTFH) development, which was associated with impaired antibody responses and absent germinal center structures on lymph node histology. Transcriptomics indicated that CARD11-dependent signaling is essential for immune signaling pathways involved in the development of these cells. Both patients underwent hematopoietic stem cell transplantations, which led to functional normalization. CONCLUSIONS: Complete human CARD11 deficiency causes profound CID by impairing naive/type 1 B-cell and cTFH cell development and abolishing activation of MALT1 paracaspase, NF-κB, and JNK activity. Hematopoietic stem cell transplantation functionally restores impaired signaling pathways.

Moonlighting matrix metalloproteinase substrates: Enhancement of proinflammatory functions of extracellular tyrosyl-tRNA synthetase upon cleavage.

Tyrosyl-tRNA synthetase ligates tyrosine to its cognate tRNA in the cytoplasm, but it can also be secreted through a noncanonical pathway. We found that extracellular tyrosyl-tRNA synthetase (YRS) exhibited proinflammatory activities. In addition to acting as a monocyte/macrophage chemoattractant, YRS initiated signaling through Toll-like receptor 2 (TLR2) resulting in NF-κB activation and release of tumor necrosis factor α (TNFα) and multiple chemokines, including MIP-1α/ß, CXCL8 (IL8), and CXCL1 (KC) from THP1 monocyte and peripheral blood mononuclear cell-derived macrophages. Furthermore, YRS up-regulated matrix metalloproteinase (MMP) activity in a TNFα-dependent manner in M0 macrophages. Because MMPs process a variety of intracellular proteins that also exhibit extracellular moonlighting functions, we profiled 10 MMPs for YRS cleavage and identified 55 cleavage sites by amino-terminal oriented mass spectrometry of substrates (ATOMS) positional proteomics and Edman degradation. Stable proteoforms resulted from cleavages near the start of the YRS C-terminal EMAPII domain. All of the MMPs tested cleaved at ADS386↓387LYV and VSG405↓406LVQ, generating 43- and 45-kDa fragments. The highest catalytic efficiency for YRS was demonstrated by MMP7, which is highly expressed by monocytes and macrophages, and by neutrophil-specific MMP8. MMP-cleaved YRS enhanced TLR2 signaling, increased TNFα secretion from macrophages, and amplified monocyte/macrophage chemotaxis compared with unprocessed YRS. The cleavage of YRS by MMP8, but not MMP7, was inhibited by tyrosine, a substrate of the YRS aminoacylation reaction. Overall, the proinflammatory activity of YRS is enhanced by MMP cleavage, which we suggest forms a feed-forward mechanism to promote inflammation.

Proteomic and N-Terminomic TAILS Analyses of Human Alveolar Bone Proteins: Improved Protein Extraction Methodology and LysargiNase Digestion Strategies Increase Proteome Coverage and Missing Protein Identification.

With 2129 proteins still classified by the Human Proteome Organisation Human Proteome Project (HPP) as "missing" without compelling evidence of protein existence (PE) in humans, we hypothesized that in-depth proteomic characterization of tissues that are technically challenging to access and extract would yield evidence for tissue-specific missing proteins. Paradoxically, although the skeleton is the most massive tissue system in humans, as one of the poorest characterized by proteomics, bone falls under the HPP umbrella term as a "rare tissue". Therefore, we aimed to optimize mineralized tissue protein extraction methodology and workflows for proteomic and data analyses of small quantities of healthy young adult human alveolar bone. Osteoid was solubilized by GuHCl extraction, with hydroxyapatite-bound proteins then released by ethylenediaminetetraacetic acid demineralization. A subsequent GuHCl solubilization extraction was followed by solid-phase digestion of the remaining insoluble cross-linked protein using trypsin and then 6 M urea dissolution incorporating LysC digestion. Bone extracts were digested in parallel using trypsin, LysargiNase, AspN, or GluC prior to liquid chromatography-mass spectrometry analysis. Terminal Amine Isotopic Labeling of Substrates was used to purify semitryptic peptides, identifying natural and proteolytic-cleaved neo N-termini of bone proteins. Our strategy enabled complete solubilization of the organic bone matrix leading to extensive categorization of bone proteins in different bone matrix extracts, and hence matrix compartments, for the first time. Moreover, this led to the high confidence identification of pannexin-3, a "missing protein", found only in the insoluble collagenous matrix and revealed for the first time by trypsin solid-phase digestion. We also found a singleton proteotypic peptide of another missing protein, meiosis inhibitor protein 1. We also identified 17 proteins classified in neXtprot as PE1 based on evidence other than from MS, termed non-MS PE1 proteins, including ≥9-mer proteotypic peptides of four proteins.

Matrix metalloproteinases inactivate the proinflammatory functions of secreted moonlighting tryptophanyl-tRNA synthetase.

Tryptophanyl-tRNA synthetase (WRS) is a cytosolic aminoacyl-tRNA synthetase essential for protein synthesis. WRS is also one of a growing number of intracellular proteins that are attributed distinct noncanonical "moonlighting" functions in the extracellular milieu. Moonlighting aminoacyl-tRNA synthetases regulate processes such as inflammation, but how these multifunctional enzymes are themselves regulated remains unclear. Here, we demonstrate that WRS is secreted from human macrophages, fibroblasts, and endothelial cells in response to the proinflammatory cytokine interferon γ (IFNγ). WRS signaled primarily through Toll-like receptor 2 (TLR2) in macrophages, leading to phosphorylation of the p65 subunit of NF-κB with associated loss of NF-κB inhibitor α (IκB-α) protein. This signaling initiated secretion of tumor necrosis factor α (TNFα) and CXCL8 (IL8) from macrophages. We also demonstrated that WRS is a potent monocyte chemoattractant. Of note, WRS increased matrix metalloproteinase (MMP) activity in the conditioned medium of macrophages in a TNFα-dependent manner. Using purified recombinant proteins and LC-MS/MS to identify proteolytic cleavage sites, we demonstrated that multiple MMPs, but primarily macrophage MMP7 and neutrophil MMP8, cleave secreted WRS at several sites. Loss of the WHEP domain following cleavage at Met48 generated a WRS proteoform that also results from alternative splicing, designated Δ1-47 WRS. The MMP-cleaved WRS lacked TLR signaling and proinflammatory activities. Thus, our results suggest that moonlighting WRS promotes IFNγ proinflammatory activities, and these responses can be dampened by MMPs.

New therapeutic targets in rare genetic skeletal diseases.

Introduction: Genetic skeletal diseases (GSDs) are a diverse and complex group of rare genetic conditions that affect the development and homeostasis of the skeleton. Although individually rare, as a group of related diseases, GSDs have an overall prevalence of at least 1 per 4,000 children. There are currently very few specific therapeutic interventions to prevent, halt or modify skeletal disease progression and therefore the generation of new and effective treatments requires novel and innovative research that can identify tractable therapeutic targets and biomarkers of these diseases. Areas covered: Remarkable progress has been made in identifying the genetic basis of the majority of GSDs and in developing relevant model systems that have delivered new knowledge on disease mechanisms and are now starting to identify novel therapeutic targets. This review will provide an overview of disease mechanisms that are shared amongst groups of different GSDs and describe potential therapeutic approaches that are under investigation. Expert opinion: The extensive clinical variability and genetic heterogeneity of GSDs renders this broad group of rare diseases a bench to bedside challenge. However, the evolving hypothesis that clinically different diseases might share common disease mechanisms is a powerful concept that will generate critical mass for the identification and validation of novel therapeutic targets and biomarkers.

The utility of mouse models to provide information regarding the pathomolecular mechanisms in human genetic skeletal diseases: The emerging role of endoplasmic reticulum stress (Review).

Genetic skeletal diseases (GSDs) are an extremely diverse and complex group of rare genetic diseases that primarily affect the development and homeostasis of the osseous skeleton. There are more than 450 unique and well-characterised phenotypes that range in severity from relatively mild to severe and lethal forms. Although individually rare, as a group of related genetic diseases, GSDs have an overall prevalence of at least 1 per 4,000 children. Qualitative defects in cartilage structural proteins result in a broad spectrum of both recessive and dominant GSDs. This review focused on a disease spectrum resulting from mutations in the non-collagenous glycoproteins, cartilage oligomeric matrix protein (COMP) and matrilin-3, which together cause a continuum of phenotypes that are amongst the most common autosomal dominant GSDs. Pseudoachondroplasia (PSACH) and autosomal dominant multiple epiphyseal dysplasia (MED) comprise a disease spectrum characterised by varying degrees of disproportionate short stature, joint pain and stiffness and early-onset osteoarthritis. Over the past decade, the generation and deep phenotyping of a range of genetic mouse models of the PSACH and MED disease spectrum has allowed the disease mechanisms to be characterised in detail. Moreover, the generation of novel phenocopies to model specific disease mechanisms has confirmed the importance of endoplasmic reticulum (ER) stress and reduced chondrocyte proliferation as key modulators of growth plate dysplasia and reduced bone growth. Finally, new insight into related musculoskeletal complications (such as myopathy and tendinopathy) has also been gained through the in-depth analysis of targeted mouse models of the PSACH-MED disease spectrum.

Genotype to phenotype correlations in cartilage oligomeric matrix protein associated chondrodysplasias.

Pseudoachondroplasia (PSACH) and autosomal dominant multiple epiphyseal dysplasia (MED) are chondrodysplasias resulting in short-limbed dwarfism, joint pain and stiffness and early onset osteoarthritis. All PSACH, and the largest proportion of MED, result from mutations in cartilage oligomeric matrix protein (COMP). The first mutations in COMP were identified in 1995 in patients with both PSACH and MED and subsequently there has been over 30 publications describing COMP mutations in at least 250 PSACH-MED patients. However, despite these discoveries, a methodical analysis of the relationship between COMP mutations and phenotypes has not been undertaken. In particular, there has, to date, been little correlation between the type and location of a COMP mutation and the resulting phenotype of PSACH or MED. To determine if genotype to phenotype correlations could be derived for COMP, we collated 300 COMP mutations, including 25 recently identified novel mutations. The results of this analysis demonstrate that mutations in specific residues and/or regions of the type III repeats of COMP are significantly associated with either PSACH or MED. This newly derived genotype to phenotype correlation may aid in determining the prognosis of PSACH and MED, including the prediction of disease severity, and in the long term guide genetic counselling and contribute to the clinical management of patients with these diseases.

Abnormal chondrocyte apoptosis in the cartilage growth plate is influenced by genetic background and deletion of CHOP in a targeted mouse model of pseudoachondroplasia.

Pseudoachondroplasia (PSACH) is an autosomal dominant skeletal dysplasia caused by mutations in cartilage oligomeric matrix protein (COMP) and characterised by short limbed dwarfism and early onset osteoarthritis. Mouse models of PSACH show variable retention of mutant COMP in the ER of chondrocytes, however, in each case a different stress pathway is activated and the underlying disease mechanisms remain largely unknown. T585M COMP mutant mice are a model of moderate PSACH and demonstrate a mild ER stress response. Although mutant COMP is not retained in significant quantities within the ER of chondrocytes, both BiP and the pro-apoptotic ER stress-related transcription factor CHOP are mildly elevated, whilst bcl-2 levels are decreased, resulting in increased and spatially dysregulated chondrocyte apoptosis. To determine whether the abnormal chondrocyte apoptosis observed in the growth plate of mutant mice is CHOP-mediated, we bred T585M COMP mutant mice with CHOP-null mice to homozygosity, and analysed the resulting phenotype. Although abnormal apoptosis was alleviated in the resting zone following CHOP deletion, the mutant growth plates were generally more disorganised. Furthermore, the bone lengths of COMP mutant CHOP null mice were significantly shorter at 9 weeks of age when compared to the COMP mutant mice, including a significant difference in the skull length. Overall, these data demonstrate that CHOP-mediated apoptosis is an early event in the pathobiology of PSACH and suggest that the lack of CHOP, in conjunction with a COMP mutation, may lead to aggravation of the skeletal phenotype via a potentially synergistic effect on endochondral ossification.

A novel transgenic mouse model of growth plate dysplasia reveals that decreased chondrocyte proliferation due to chronic ER stress is a key factor in reduced bone growth.

Disease mechanisms leading to different forms of chondrodysplasia include extracellular matrix (ECM) alterations and intracellular stress resulting in abnormal changes to chondrocyte proliferation and survival. Delineating the relative contribution of these two disease mechanisms is a major challenge in understanding disease pathophysiology in genetic skeletal diseases and a prerequisite for developing effective therapies. To determine the influence of intracellular stress and changes in chondrocyte phenotype to the development of chondrodysplasia, we targeted the expression of the G2320R mutant form of thyroglobulin to the endoplasmic reticulum (ER) of resting and proliferating chondrocytes. Previous studies on this mutant protein have shown that it induces intracellular aggregates and causes cell stress and death in the thyroid gland. The expression and retention of this exogenous mutant protein in resting and proliferating chondrocytes resulted in a chronic cell stress response, growth plate dysplasia and reduced bone growth, without inducing any alterations to the architecture and organization of the cartilage ECM. More significantly, the decreased bone growth seemed to be the direct result of reduced chondrocyte proliferation in the proliferative zone of growth plates in transgenic mice, without transcriptional activation of a classical unfolded protein response (UPR) or apoptosis. Overall, these data show that mutant protein retention in the ER of resting and proliferative zone chondrocytes is sufficient to cause disrupted bone growth. The specific disease pathways triggered by mutant protein retention do not necessarily involve a prototypic UPR, but all pathways impact upon chondrocyte proliferation in the cartilage growth plate.

Armet/Manf and Creld2 are components of a specialized ER stress response provoked by inappropriate formation of disulphide bonds: implications for genetic skeletal diseases.

Mutant matrilin-3 (V194D) forms non-native disulphide bonded aggregates in the rER of chondrocytes from cell and mouse models of multiple epiphyseal dysplasia (MED). Intracellular retention of mutant matrilin-3 causes endoplasmic reticulum (ER) stress and induces an unfolded protein response (UPR) including the upregulation of two genes recently implicated in ER stress: Armet and Creld2. Nothing is known about the role of Armet and Creld2 in human genetic diseases. In this study, we used a variety of cell and mouse models of chondrodysplasia to determine the genotype-specific expression profiles of Armet and Creld2. We also studied their interactions with various mutant proteins and investigated their potential roles as protein disulphide isomerases (PDIs). Armet and Creld2 were up-regulated in cell and/or mouse models of chondrodysplasias caused by mutations in Matn3 and Col10a1, but not Comp. Intriguingly, both Armet and Creld2 were also secreted into the ECM of these disease models following ER stress. Armet and Creld2 interacted with mutant matrilin-3, but not with COMP, thereby validating the genotype-specific expression. Substrate-trapping experiments confirmed Creld2 processed PDI-like activity, thus identifying a putative functional role. Finally, alanine substitution of the two terminal cysteine residues from the A-domain of V194D matrilin-3 prevented aggregation, promoted mutant protein secretion and reduced the levels of Armet and Creld2 in a cell culture model. We demonstrate that Armet and Creld2 are genotype-specific ER stress response proteins with substrate specificities, and that aggregation of mutant matrilin-3 is a key disease trigger in MED that could be exploited as a potential therapeutic target.

Analysis of the cartilage proteome from three different mouse models of genetic skeletal diseases reveals common and discrete disease signatures.

Pseudoachondroplasia and multiple epiphyseal dysplasia are genetic skeletal diseases resulting from mutations in cartilage structural proteins. Electron microscopy and immunohistochemistry previously showed that the appearance of the cartilage extracellular matrix (ECM) in targeted mouse models of these diseases is disrupted; however, the precise changes in ECM organization and the pathological consequences remain unknown. Our aim was to determine the effects of matrilin-3 and COMP mutations on the composition and extractability of ECM components to inform how these detrimental changes might influence cartilage organization and degeneration. Cartilage was sequentially extracted using increasing denaturants and the extraction profiles of specific proteins determined using SDS-PAGE/Western blotting. Furthermore, the relative composition of protein pools was determined using mass spectrometry for a non-biased semi-quantitative analysis. Western blotting revealed changes in the extraction of matrilins, COMP and collagen IX in mutant cartilage. Mass spectrometry confirmed quantitative changes in the extraction of structural and non-structural ECM proteins, including proteins with roles in cellular processes such as protein folding and trafficking. In particular, genotype-specific differences in the extraction of collagens XII and XIV and tenascins C and X were identified; interestingly, increased expression of several of these genes has recently been implicated in susceptibility and/or progression of murine osteoarthritis. We demonstrated that mutation of matrilin-3 and COMP caused changes in the extractability of other cartilage proteins and that proteomic analyses of Matn3 V194D, Comp T585M and Comp DelD469 mouse models revealed both common and discrete disease signatures that provide novel insight into skeletal disease mechanisms and cartilage degradation.

Loss of matrilin 1 does not exacerbate the skeletal phenotype in a mouse model of multiple epiphyseal dysplasia caused by a Matn3 V194D mutation.

OBJECTIVE: Mutations in matrilin 3 can result in multiple epiphyseal dysplasia (MED), a disease characterized by delayed and irregular bone growth and early-onset osteoarthritis. Although intracellular retention of the majority of mutant matrilin 3 was previously observed in a murine model of MED caused by a Matn3 V194D mutation, some mutant protein was secreted into the extracellular matrix. Thus, it was proposed that secretion of mutant matrilin 3 may be dependent on the formation of hetero-oligomers with matrilin 1. The aim of this study was to investigate the hypothesis that deletion of matrilin 1 would abolish the formation of matrilin 1/matrilin 3 hetero-oligomers, eliminate the secretion of mutant matrilin 3, and influence disease severity. METHODS: Mice with a Matn3 V194D mutation were crossed with Matn1-null mice, generating mice that were homozygous for V194D and null for matrilin 1. This novel mouse was used for in-depth phenotyping, while cartilage and chondrocytes were studied both histochemically and biochemically. RESULTS: Endochondral ossification was not disrupted any further in mice with a double V194D mutation compared with mice with a single mutation. A similar proportion of mutant matrilin 3 was present in the extracellular matrix, and the amount of retained mutant matrilin 3 was not noticeably increased. Retained mutant matrilin 3 formed disulfide-bonded aggregates and caused the co-retention of matrilin 1. CONCLUSION: We showed that secretion of matrilin 3 V194D mutant protein is not dependent on hetero-oligomerization with matrilin 1, and that the total ablation of matrilin 1 expression has no impact on disease severity in mice with MED. Mutant matrilin 3 oligomers form non-native disulfide-bonded aggregates through the misfolded A domain.

An unfolded protein response is the initial cellular response to the expression of mutant matrilin-3 in a mouse model of multiple epiphyseal dysplasia.

Multiple epiphyseal dysplasia (MED) can result from mutations in matrilin-3, a structural protein of the cartilage extracellular matrix. We have previously shown that in a mouse model of MED the tibia growth plates were normal at birth but developed a progressive dysplasia characterised by the intracellular retention of mutant matrilin-3 and abnormal chondrocyte morphology. By 3 weeks of age, mutant mice displayed a significant decrease in chondrocyte proliferation and dysregulated apoptosis. The aim of this current study was to identify the initial post-natal stages of the disease. We confirmed that the disease phenotype is seen in rib and xiphoid cartilage and, like tibia growth plate cartilage is characterised by the intracellular retention of mutant matrilin-3. Gene expression profiling showed a significant activation of classical unfolded protein response (UPR) genes in mutant chondrocytes at 5 days of age, which was still maintained by 21 days of age. Interestingly, we also noted the upregulation of arginine-rich, mutated in early stage of tumours (ARMET) and cysteine-rich with EGF-like domain protein 2 (CRELD2) are two genes that have only recently been implicated in the UPR. This endoplasmic reticulum (ER) stress and UPR did not lead to increased chondrocyte apoptosis in mutant cartilage by 5 days of age. In an attempt to alleviate ER stress, mutant mice were fed with a chemical chaperone, 4-sodium phenylbutyrate (SPB). SPB at the dosage used had no effect on chaperone expression at 5 days of age but modestly decreased levels of chaperone proteins at 3 weeks. However, this did not lead to increased secretion of mutant matrilin-3 and in the long term did not improve the disease phenotype. We performed similar studies with a mouse model of Schmid metaphyseal chondrodysplasia, but again this treatment did not improve the phenotype.

Quantitation of proteins using a dye-metal-based colorimetric protein assay.

We describe a dye-metal (polyhydroxybenzenesulfonephthalein-type dye and a transition metal) complex-based total protein determination method. The binding of the complex to protein causes a shift in the absorption maximum of the dye-metal complex from 450 to 660 nm. The dye-metal complex has a reddish brown color that changes to green on binding to protein. The color produced from this reaction is stable and increases in a proportional manner over a broad range of protein concentrations. The new Pierce 660 nm Protein Assay is very reproducible, rapid, and more linear compared with the Coomassie dye-based Bradford assay. The assay reagent is room temperature stable, and the assay is a simple and convenient mix-and-read format. The assay has a moderate protein-to-protein variation and is compatible with most detergents, reducing agents, and other commonly used reagents. This is an added advantage for researchers needing to determine protein concentrations in samples containing both detergents and reducing agents.

Solid-phase biotinylation of antibodies.

Biotinylation is an established method of labeling antibody molecules for several applications in life science research. Antibody functional groups such as amines, cis hydroxyls in carbohydrates or sulfhydryls may be modified with a variety of biotinylation reagents. Solution-based biotinylation is accomplished by incubating antibody in an appropriate buffered solution with biotinylation reagent. Unreacted biotinylation reagent must be removed via dialysis, diafiltration or desalting. Disadvantages of the solution-based approach include dilution and loss of antibody during post-reaction purification steps, and difficulty in biotinylation and recovery of small amounts of antibody. Solid-phase antibody biotinylation exploits the affinity of mammalian IgG-class antibodies for nickel IMAC (immobilized metal affinity chromatography) supports. In this method, antibody is immobilized on a nickel-chelated chromatography support and derivitized on-column. Excess reagents are easily washed away following reaction, and biotinylated IgG molecule is recovered under mild elution conditions. Successful solid phase labeling of antibodies through both amine and sulfhydryl groups is reported, in both column and mini-spin column formats. Human or goat IgG was bound to a Ni-IDA support. For sulfhydryl labeling, native disulfide bonds were reduced with TCEP, and reduced IgG was biotinylated with maleimide-PEO(2) biotin. For amine labeling, immobilized human IgG was incubated with a solution of NHS-PEO(4) biotin. Biotinylated IgG was eluted from the columns using a buffered 0.2 M imidazole solution and characterized by ELISA, HABA/avidin assay, probing with a streptavidin-alkaline phosphatase conjugate, and binding to a monomeric avidin column. The solid phase protocol for sulfhydryl labeling is significantly shorter than the corresponding solution phase method. Biotinylation in solid phase is convenient, efficient and easily applicable to small amounts of antibody (e.g. 100 microg). Antibody biotinylated on-column was found to be equivalent in stability and antigen-recognition ability to antibody biotinylated in solution. Solid-phase methods utilizing Ni-IDA resin have potential for labeling nucleic acids, histidine-rich proteins and recombinant proteins containing polyhistidine purification tags, and may also be applicable for other affinity systems and labels.