Heterochromatin protein 1 (HP1) is a key component of constitutive heterochromatin in Drosophila and is required for stable epigenetic gene silencing classically observed as position effect variegation. Less is known of the family of mammalian HP1 proteins, which may be euchromatic, targeted to expressed loci by repressor-corepressor complexes, and retained there by Lys 9-methylated histone H3 (H3-MeK9). To characterize the physical properties of euchromatic loci bound by HP1, we developed a strategy for regulated recruitment of HP1 to an expressed transgene in mammalian cells by using a synthetic, hormone-regulated KRAB repression domain. We show that its obligate corepressor, KAP1, can coordinate all the machinery required for stable gene silencing. In the presence of hormone, the transgene is rapidly silenced, spatially recruited to HP1-rich nuclear regions, assumes a compact chromatin structure, and is physically associated with KAP1, HP1, and the H3 Lys 9-specific methyltransferase, SETDB1, over a highly localized region centered around the promoter. Remarkably, silencing established by a short pulse of hormone is stably maintained for >50 population doublings in the absence of hormone in clonal-cell populations, and the silent transgenes in these clones show promoter hypermethylation. Thus, like variegation in Drosophila, recruitment of mammalian HP1 to a euchromatic promoter can establish a silenced state that is epigenetically heritable. A recently emerging paradigm for the epigenetic control and propagation of gene expression states involves the role of chromatin structure. Though historically viewed as a passive packaging structure primarily used to assemble the enormous amount of DNA into a eukaryotic nucleus, the nucleosome with its complement of core histones has emerged as a key target for regulating gene expression (Wolffe and Hansen 2001). The dynamic regulation of chromatin organization appears to be accomplished by macromolecular protein complexes that contain enzymatic activities that modify the tails of the core histones. The constellation of these histone modifications, including acetylation, phosphorylation, ubiquitination, and methylation, create both synergistic and antagonistic signals that correlate with the transcriptional activity of a gene (Wu and Grunstein 2000). This emerging "histone code" is hypothesized to create functionally distinct subdomains in chromatin that define active versus transcriptionally silent genes (Jenuwein and Allis 2001). Histone modifications and the chromatin-associated proteins that interpret these signals may represent an epigenetic marking system responsible for setting and maintaining heritable programs of gene expression during development.The role of histone acetylation/deacetylation [mediated by histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively] in modulating gene activity is now well established (Kuo and Allis 1998). The role of histone methylation in the regulation of chromatin structure and gene transcription has been...

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s12192-010-0193-y) contains supplementary material, which is available to authorized users.

Single nucleotide polymorphism (SNP) genotyping is playing an increasing role in genome mapping, pharmacogenetic studies, and drug discovery. To date, genome-wide scans and studies involving thousands of SNPs and samples have been hampered by the lack of a system that can perform genotyping with cost-effective throughput, accuracy, and reliability. To address this need, Orchid has developed an automated, ultra-high throughput system, SNPstream UHT, which uses multiplexed PCR in conjunction with our next generation SNP-ITtag array single base extension genotyping technology. The system employs oligonucleotide microarrays manufactured in a 384-well format on a novel glass-bottomed plate. Multiplexed PCR and genotyping are performed in homogeneous reactions, and assay results are read by direct two-color fluorescence on the SNPstream UHT Array Imager. The systems flexibility enables large projects involving thousands of SNPs and thousands of samples as well as small projects that have hundreds of SNPs and hundreds of samples to be done cost effectively. We have successfully demonstrated this system in greater than 1 000 000 genotyping assays with >96% of samples giving genotypes with >99% accuracy.

The main viral protease (3CL pro ) is indispensable for SARS-CoV-2 replication. We delineate the human protein substrate landscape of 3CL pro by TAILS substrate-targeted N-terminomics. We identify >100 substrates in human lung and kidney cells supported by analyses of SARS-CoV-2-infected cells. Enzyme kinetics and molecular docking simulations of 3CL pro 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 3CL pro 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 healthy lung cells. The 3CL pro substrate degradome establishes a foundational substrate atlas to accelerate exploration of SARS-CoV-2 pathology and drug design.

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.

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.

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.

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