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The sodium-iodide symporter (NIS, SLC5A5) is expressed at the basolateral membrane of the thyroid follicular cell, and facilitates the thyroidal iodide uptake required for thyroid hormone biosynthesis. Biallelic loss-of-function mutations in NIS are a rare cause of dyshormonogenic congenital hypothyroidism. Affected individuals typically exhibit a normally sited, often goitrous thyroid gland, with absent uptake of radioiodine in the thyroid and other NIS-expressing tissues. We report a novel homozygous NIS mutation (c.1067 C>T, p.S356F) in four siblings from a consanguineous Indian kindred, presenting with significant hypothyroidism. Functional characterization of the mutant protein demonstrated impaired plasma membrane localization and cellular iodide transport.

Clusterin (CLU) is a secreted glycoprotein, heterodimeric in nature, and is expressed in a wide variety of tissues and body fluids such as serum and plasma. CLU has also been known to be a promising biomarker for cell death, malignancy, cancer progression, and resistance development. However, the lack of a CLU crystal structure obstructs understanding the possible role of reported mutations on the structure, and the subsequent effects on downstream signaling pathways and cancer progression. Considering the importance of crystal structure, a model structure of the pre-secretory isoform of CLU was built to predict the effect of mutations at the molecular level. Ab initio model was built using RaptorX, and loop refinement and energy minimization were carried out with ModLoop, ModRefiner, and GalaxyWeb servers. The cancer associated mutational spectra of CLU was retrieved from the cBioPortal server and 117 unique missense mutations were identified. Evolutionarily conserved regions and pathogenicity of mutations identified in CLU were analyzed using ConSurf and Rhapsody, respectively. Furthermore, sequence and structure-based mutational analysis were carried out with iSTABLE, DynaMut and PremPS servers. Molecular dynamics simulations were carried out with GROMACS for 50 ns to determine the stability of the wild type and mutant protein structures. A dynamically stable model structure of pre-secretory CLU (psCLU) which has high concurrence with the sequence based secondary structure predictions has been explored. Changes in the intra-atomic interactions and folding pattern between wild type and mutant structures were observed. To our conclusion, eleven mutations with the highest structural and functional significance have been predicted to have pathogenic and deleterious effects.Communicated by Ramaswamy H. Sarma.

This study aims to identify the underlying genetic cause of a Chinese patient with Leber congenital amaurosis (LCA). Detailed clinical data and family history were collected. A medical diagnostic panel sequencing covering 4450 genes was conducted. Two candidate disease-causing mutations detected in CEP290 were then validated with Sanger sequencing and bioinformatic analysis. Reverse transcription polymerase chain reaction (RT-PCR) and cDNA sequencing were performed to understand the effect of the novel CEP290 mutation on CEP290 mRNA splicing. A five-month-old LCA patient with both parents was enrolled. Medical diagnostic panel sequencing revealed that the patient is a compound heterozygote for two potentially pathogenic CEP290 mutations. Among them, c.1666dupA (p.I556NfsX20) was previously reported and has a significant association with LCA phenotype. A novel CEP290 mutation (c.3310–1_3313delGCTTA) was confirmed in both the patient and her father. RT-PCR and cDNA sequencing confirmed that the novel mutation affects the CEP290 mRNA splicing and results in a complete skipping of exon 29. The frameshift resulted in an early stop codon and truncation of the mutant protein by 1371 amino acid residues (p.L1104fsX6). Our report provided a new mutation to the spectrum of CEP290-related diseases. The data suggested that the c.3310–1_3313delGCTTA mutation affects the CEP290 mRNA splicing and the CEP290 protein function. This valuable information is important for future studies on the mRNA splicing of CEP290 and the pathogenesis of CEP290-related diseases.

We report the first pedigree of Lynch syndrome bearing a germ-line MSH2 missense mutation c.1808A>T (Asp603Val). Until now, this missense mutation, in exon 12 of MSH2, was identified as a variant of unknown significance in the International Society for Gastrointestinal Hereditary Tumours database. In vitro induction mutagenesis experiments indicated that the MSH2 mutant protein (Asp603Val) is easily degraded in embryonic stem cells, albeit there is no clinical information concerning this mutant. Our pedigree includes four patients with Lynch syndrome-associated malignancies and clinically matches the Amsterdam II criteria. The proband, a female, first had an endometrial cancer at the age of 49 and then mantle cell lymphoma, colonic and gastric adenocarcinomas and neuroendocrine carcinoma, successively. Her mother also had Lynch syndrome-associated malignancies, including colonic, uterine and gastric cancers, and her elder son had rectal cancer. In the germline of the proband and her son, an MSH2 missense mutation c.1808A>T was discovered. Immunohistochemical analyses indicated that the expression of the MSH2 protein was decreased in the tumors, such as gastric cancer and neuroendocrine carcinoma, due to the missense mutation c.1808A>T. This study showed that the MSH2 missense mutation c.1808A>T (Asp603Val) is a likely pathogenic mutation and is responsible for typical Lynch syndrome-associated malignancies, including neuroendocrine carcinoma.

Post-translational modification (PTM) is one of the major regulatory mechanism for protein activities. To understand the function of PTMs, mutants that prevent or mimic the modification are frequently utilized. The endogenous proteins are usually depleted while the point mutations are expressed. A common strategy to accomplish these tasks includes two-steps: First, a cell line stably expressing shRNA for protein depletion is generated, then an RNAi-resistance construct is introduced to express mutant. However, these steps are time- and labor-consuming. More importantly, shRNA and mutant protein are frequently expressed in different cells at different time, which significantly disturbs the conclusions. To overcome these technical problems, we developed a lentiviral based one-plasmid system that allowed concurrent expression of shRNA and mutant protein. The puromycin-resistant gene was inserted for the selection of stable-expression cells. Using this plasmid, we efficiently replaced the endogenous proteins with comparable levels of exogenous proteins for LDHB and PKM2, two glycolytic enzymes regulated by PTM in cancer cells. The system was also successfully exploited in evaluating the role of phosphorylation of LDHB serine 162 in multiple in vitro and in vivo assays. Thus, we have developed an efficient one-plasmid system to replace endogenous protein with point mutations for the functional study of PTM.

Macrocyclic peptides have the potential to address intracellular protein–protein interactions (PPIs) of high value therapeutic targets that have proven largely intractable to small molecules. Here, we report broadly applicable lessons for applying this modality to intracellular targets and specifically for advancing chemical matter to address KRAS, a protein that represents the most common oncogene in human lung, colorectal and pancreatic cancers yet is one of the most challenging targets in human disease. Specifically, we focused on KRpep-2d, an arginine-rich KRAS-binding peptide with a disulfide-mediated macrocyclic linkage and a protease-sensitive backbone. These latter redox and proteolytic labilities obviated cellular activity. Extensive structure–activity relationship studies involving macrocyclic linker replacement, stereochemical inversion, and backbone α-methylation, gave a peptide with on-target cellular activity. However, we uncovered an important generic insight – the arginine-dependent cell entry mechanism limited its therapeutic potential. In particular, we observed a strong correlation between net positive charge and histamine release in an ex vivo assay, thus making this series unsuitable for advancement due to the potentially fatal consequences of mast cell degranulation. This observation should signal to researchers that cationic-mediated cell entry – an approach that has yet to succeed in the clinic despite a long history of attempts – carries significant therapy-limiting safety liabilities. Nonetheless, the cell-active molecules identified here validate a unique inhibitory epitope on KRAS and thus provide valuable molecular templates for the development of therapeutics that are desperately needed to address KRAS-driven cancers – some of the most treatment-resistant human malignancies., Targeting undruggable intracellular proteins with peptides: novel on-target macrocyclic peptide inhibitors of KRAS with broad inhibition of proliferation of multiple KRAS-dependent cancer cell lines.

The bacterial protein Hfq is a member of a large family of Lsm proteins, which are characterized by a conserved tertiary structure and form a stable ring-shaped quaternary structure composed of several monomers. The bacterial proteins form hexamers, whereas their archaeal and eukaryotic homologues are heptamers. Until today, there is no explanation for the fact that highly homologous Lsm proteins with the same tertiary structure form multimers composed of a different number of monomers, and this issue generates a lot of discussion. The effect of the loop L4 of variable length on the quaternary structure of the Hfq protein was experimentally validated. The produced protein retains the hexameric quaternary structure, and the extended loop protrudes from the protein core in different directions. Therefore, attempts to change the quaternary structure of the protein failed, which confirms its high stability. The RNA-binding properties of the mutant protein remained virtually unchanged.

Although it has long been deemed "undruggable", with the development of drugs specifically binding the KRAS-G12C mutant protein, clinical trials that directly inhibit oncogenic RAS have recently made promising improvements. In particular, the covalent KRAS-G12C inhibitors sotorasib and adagrasib are used to treat patients with advanced non-small cell lung cancer (NSCLC) carrying KRAS-G12C mutations. Unfortunately, the vast majority of patients do not respond to KRAS-G12C inhibitor therapy, mainly due to intrinsic or acquired resistance caused by cellular, molecular, and genetic mechanisms. Improving the understanding of drug response in the tumor microenvironment may continue to promote the design, testing, and clinical application of KRAS-G12C inhibitors.

Few genetically dominant mutations involved in human disease have been fully explained at the molecular level. In cases where the mutant gene encodes a transcription factor, the dominant-negative mode of action of the mutant protein is particularly poorly understood. Here, we studied the genome-wide mechanism underlying a dominant-negative form of the SOX18 transcription factor (SOX18RaOp) responsible for both the classical mouse mutant Ragged Opossum and the human genetic disorder Hypotrichosis-lymphedema-telangiectasia-renal defect syndrome. Combining three single-molecule imaging assays in living cells together with genomics and proteomics analysis, we found that SOX18RaOp disrupts the system through an accumulation of molecular interferences which impair several functional properties of the wild-type SOX18 protein, including its target gene selection process. The dominant-negative effect is further amplified by poisoning the interactome of its wild-type counterpart, which perturbs regulatory nodes such as SOX7 and MEF2C. Our findings explain in unprecedented detail the multi-layered process that underpins the molecular aetiology of dominant-negative transcription factor function.

As the body of scientific research focusing on the severe acute respiratory syndrome coronavirus 2 or SARS-CoV-2 continues to grow, several mutations have been reported as very common across the globe. In this study, we analysed the SARS-CoV-2 nucleocapsid protein (N protein) with respect to the widely observed 28881-28883 GGG to AAC variant. One of the major functions of the SARS-CoV-2 nucleocapsid protein is virion packaging through its interactions with the membrane protein (M protein). Our goal was to investigate, using in silico studies, the interaction between the mutant nucleocapsid protein and the M protein and how it differed from that of wild type N-M protein interaction. The results showed significant differences in interactions between the two. The mutant protein was predicted to form 3 salt bridges with the M protein, while the wild type only formed 2. The mutant protein was also predicted to display less temperature sensitivity than its wild type counterpart. Supplementary Information The online version contains supplementary material available at 10.1186/s43141-021-00233-z.

Conformational fluctuations from ground-state to sparsely populated but functionally important excited states play a key role in enzyme catalysis. For Escherichia coli dihydrofolate reductase (DHFR), the release of the product tetrahydrofolate (THF) and oxidized cofactor NADP+ occurs through exchange between closed and occluded conformations of the Met20 loop. A "dynamic knockout" mutant of E. coli DHFR, where the E. coli sequence in the Met20 loop is replaced by the human sequence (N23PP/S148A), models human DHFR and is incapable of accessing the occluded conformation. 1H and 15N CPMG relaxation dispersion analysis for the ternary product complex of the mutant enzyme with NADP+ and the product analogue 5,10-dideazatetrahydrofolate (ddTHF) (E:ddTHF:NADP+) reveals the mechanism by which NADP+ is released when the Met20 loop cannot undergo the closed-to-occluded conformational transition. Two excited states were observed: one related to a faster, relatively high-amplitude conformational fluctuation in areas near the active site, associated with the shuttling of the nicotinamide ring of the cofactor out of the active site, and the other to a slower process where ddTHF undergoes small-amplitude motions within the binding site that are consistent with disorder observed in a room-temperature X-ray crystal structure of the N23PP/S148A mutant protein. These motions likely arise due to steric conflict of the pterin ring of ddTHF with the ribose-nicotinamide moiety of NADP+ in the closed active site. These studies demonstrate that site-specific kinetic information from relaxation dispersion experiments can provide intimate details of the changes in catalytic mechanism that result from small changes in local amino acid sequence.

Asthenozoospermia (ASZ) is a condition characterized by reduced forward motility of spermatozoa affecting approximately 19% of infertile men. A kinase anchor protein 4 (AKAP4) is an X-linked testis-specific gene and plays a major role in sperm motility and flagella formation. However, few studies have reported its association with ASZ. Here, we sequenced for exonic mutations of human AKAP4 gene by high-fidelity PCR/Sanger sequencing in peripheral blood samples from 150 ASZ patients and 150 fertile men. We reported the identification of three novel hemizygous mutations unique to four ASZ patients, including one patient carrying missense mutation c.454T>C (p.S152P), two patient carrying synonymous mutation c.1173T>C (p.H391H), and one patient carrying synonymous mutation c.2007 A>G (p.R669R). The p.S152P mutation was located in a precursor pro-polypeptide domain of AKAP4 protein, which was predicted to be damaging by SIFT and PolyPhen-2 and could cause the protein accumulation in the cytoplasm of COS-7 cells. The mature protein of AKAP4 was absent in spermatozoa of ASZ patient harboring AKAP4 p.S152P mutation. Further in vitro cellular assays showed that reactive oxygen species (ROS), malondialdehyde (MDA), myeloperoxidase (MPO) levels, and apoptotic cells were increased in GC2-spd cells by AKAP4 p.S152P mutant protein, whereas superoxide dismutase (SOD) level was decreased. AKAP4 p.H391H and p.R669R mutant proteins were coimmunoprecipitated with ribonuclease T2 (RNASET2) protein in GC2-spd cells, whereas no interaction between the AKAP4 p.S152P mutant protein and RNASET2 protein was observed. In addition, AKAP4 p.S152P mutant protein could decrease the activity of PKA/PI3K signaling. Overall, our study identifies a novel AKAP4 p.S152P mutation is associated with ASZ probably through affecting oxidative stress and cell apoptosis by regulating the interaction with RNASET2 and the activity of the PKA/PI3K signaling pathway.

FREM2 is a member of the FREM2–FRAS1–FREM1 protein complex which contributes to epithelial–mesenchymal coupling. We report a Thai woman with cryptophthalmos, dental anomalies, and oral vestibule defect. A compound heterozygous mutation (c.6499C>T; p.Arg2167Trp and c.641_642del; p.Glu214GlyfsTer135) in the FREM2 gene was identified. The frameshift variant p.Glu214GlyfsTer135 is de novo and novel. It is predicted to result in the loss of most of the functional domains. The p.Arg2167Trp mutation was predicted to disrupt both Ca2+ binding and conformational change. The Arg2167Trp mutant protein has been shown to cause partial loss of function, decrease its interaction with FREM1 and result in impaired function of the FRAS1–FREM2–FREM1 complex. Frem2 was shown to be expressed in the developing tooth and vestibular lamina. It is hypothesized that these mutations resulted in aberration of the FRAS1–FREM2–FREM1 protein complex, resulting in loss of nephronectin, basement membrane disruption, and abnormal epithelial–mesenchymal interactions leading to dental and oral vestibule malformations.

In this study, we have established a convenient and efficient approach named Modification of DNA Regions with Metagenomic DNA Fragments (MDRMDF) for protein engineering. Degenerate primers were designed corresponding to conserved regions of the gene of interest which were used for amplification of fragments with template of the metagenomic DNA. The resulting PCR products were used to replace the corresponding regions of the gene of interest to introduce modified gene for function-based screening. Therefore, this method can make full use of the metagenomic DNA sequences with unknown metagenomic gene information for efficient protein engineering. The β-xylosidase BH3683 was used to construct a MDRMDF library which was screened with a newly designed p-NPX-M9 medium-based strategy. As a result, a mutant protein Xyl-M56 showing high activity, improved pH stability and higher tolerance to organic solvents was obtained which may have potential for industrial application. The MDRMDF method may find wide application in enzyme engineering, metabolic engineering and other fields, especially offering a new methodological option for the directed evolution of proteins.

Architectural DNA-binding proteins (ADBPs) are abundant constituents of eukaryotic or bacterial chromosomes that bind DNA promiscuously and function in diverse DNA reactions. They generate large conformational changes in DNA upon binding yet can slide along DNA when searching for functional binding sites. Here we investigate the mechanism by which ADBPs diffuse on DNA by single-molecule analyses of mutant proteins rationally chosen to distinguish between rotation-coupled diffusion and DNA surface sliding after transient unbinding from the groove(s). The properties of yeast Nhp6A mutant proteins, combined with molecular dynamics simulations, suggest Nhp6A switches between two binding modes: a static state, in which the HMGB domain is bound within the minor groove with the DNA highly bent, and a mobile state, where the protein is traveling along the DNA surface by means of its flexible N-terminal basic arm. The behaviors of Fis mutants, a bacterial nucleoid-associated helix-turn-helix dimer, are best explained by mobile proteins unbinding from the major groove and diffusing along the DNA surface. Nhp6A, Fis, and bacterial HU are all near exclusively associated with the chromosome, as packaged within the bacterial nucleoid, and can be modeled by three diffusion modes where HU exhibits the fastest and Fis the slowest diffusion.

Disruption of axon-glia interactions in the peripheral nervous system has emerged as a major cause of arthrogryposis multiplex congenita (AMC), a condition characterized by multiple congenital postural abnormalities involving the major joints. Several genes crucially important to the biology of Schwann cells have now been implicated with AMC. One such gene is LGI4 which encodes a secreted glycoprotein. LGI4 is expressed and secreted by Schwann cells and binds its receptor ADAM22 on the axonal membrane to drive myelination. Homozygous mutations in LGI4 or ADAM22 results in severe congenital hypomyelination and joint contractures in mice. Recently bi-allelic LGI4 loss of function mutations has been described in three unrelated families with severe AMC. Two individuals in a fourth, non-consanguineous family were found to be compound heterozygous for two LGI4 missense mutations. It is not known how these missense mutations affect the biology of LGI4. Here we investigated whether these missense mutations affected the secretion of the protein, its ADAM22 binding capacity, or its myelination-promoting function. We demonstrate that the mutations largely affect the progression of the mutant protein through the endomembrane system resulting in severely reduced expression. Importantly, binding to ADAM22 and myelination-promoting activity appear largely unaffected, suggesting that treatment with chemical chaperones to improve secretion of the mutant proteins might prove beneficial.

Selective neuronal accumulation of misfolded proteins is a key step toward neurodegeneration in a wide range of neurodegenerative diseases, including Huntington's (HD) diseases. Our recent studies suggest that Hsp70-binding protein 1 (HspBP1), an Hsp70/CHIP inhibitor that reduces protein folding, is highly expressed in neuronal cells and accounts for the accumulation of the HD protein huntingtin (HTT) in neuronal cells. To further determine the role of HspBP1 in regulation of mutant protein accumulation, we investigated whether increasing expression of HspBP1 in glial cells can also induce the accumulation of endogenous mutant HTT in glial cells and yield non-cell-autonomous toxic effects. We performed stereotaxic injection of AAV to selectively express HspBP1 in astrocytes in the brains of HD140Q knock-in (KI) mice that express mutant HTT ubiquitously but do not display obvious neurodegeneration. However, HspBP1 expression in HD140Q astrocytes led to the increased accumulation of endogenous mutant HTT and robust neuronal loss in the striatum of HD140Q KI mice. In transgenic HD mice that selectively express mutant HTT in astrocytes, increased accumulation of mutant HTT in astrocytes via HspBP1 expression did not elicit neurodegeneration but could exacerbate neurological symptoms. Consistently, suppressing the expression of endogenous HspBp1 in the striatum of HD140Q KI mice via CRISPR/Cas9 led to a significant reduction of mutant HTT accumulation. Our findings suggest that although endogenous mutant HTT in astrocytes can exacerbate neurological symptoms, it mediates neurodegeneration only when mutant HTT is also accumulated in neuronal cells.

Acephalic spermatozoa syndrome (ASS) is a severe form of teratozoospermia, previous studies have shown that SUN5 mutations are the major cause of acephalic spermatozoa syndrome. This study is to identify the pathogenic mutations in SUN5 leading to ASS. PCR and Sanger sequence were performed to define the breakpoints and mutations in SUN5. Whole genome sequencing (WGS) was performed to detect heterozygous deletion. Western blotting and immunofluorescence analysis detected the expression level and localization of SUN5. Furthermore, the pathogenicity of the mutant SUN5 was predicted in silico and was verified by the experiments in vitro. We identified one novel homozygous missense mutation (c.775G>A; p.G259S) and one compound heterozygous including one reported missense mutation (c.1043A>T; p.N348I) and a large deletion that contains partial EFCAB8 ( NM_001143967 .1) and BPIFB2 ( NM_025227 ) and complete SUN5 ( NM_080675 ), and one recurrent homozygous splice-site mutation (c.340G>A; p.G114R) in SUN5 in three patients with ASS. Our results showed that SUN5 could not be detected in the patients' spermatozoa and the exogenous expression level of the mutant protein was decreased in transfected HEK-293T cells. This study expands the mutational spectrum of SUN5. We recommended a clinical diagnostic strategy for SUN5 genomic deletion to screen heterozygous deletions and indicated that the diagnostic value of screening for SUN5 mutations and deletions in infertile men with ASS.

In the present study, we report a human-inherited, impaired, adaptive immunity disorder, which predominantly manifested as a B cell differentiation defect, caused by a heterozygous IKZF3 missense variant, resulting in a glycine-to-arginine replacement within the DNA-binding domain of the encoded AIOLOS protein. Using mice that bear the corresponding variant and recapitulate the B and T cell phenotypes, we show that the mutant AIOLOS homodimers and AIOLOS–IKAROS heterodimers did not bind the canonical AIOLOS–IKAROS DNA sequence. In addition, homodimers and heterodimers containing one mutant AIOLOS bound to genomic regions lacking both canonical motifs. However, the removal of the dimerization capacity from mutant AIOLOS restored B cell development. Hence, the adaptive immunity defect is caused by the AIOLOS variant hijacking IKAROS function. Heterodimeric interference is a new mechanism of autosomal dominance that causes inborn errors of immunity by impairing protein function via the mutation of its heterodimeric partner. The zinc-finger transcription factor IKAROS is essential for B cell development. Taniuchi, Morio and colleagues identify a human kindred presenting with B cell immunodeficiency that was caused by a heterozygous missense mutation in IKZF3 encoding the related AIOLOS protein. AIOLOSG159R is a mutant protein that interferes with both wild-type AIOLOS and IKAROS by forming heterodimers that bind to aberrant DNA-binding sites and prevent normal expression of IKAROS-dependent genes.

To explore the causative mutation for autosomal recessive inheritance factor V (FV) deficiency in a Chinese family. Relative coagulation indexes and the FV antigen were tested by the one-stage clotting method and ELISA, respectively. At the same time, the calibrated automated thrombogram (CAT) was used to analyze the mutant protein function. All 25 exons, flanking sequences, 5' and 3' untranslated regions of the F5 were amplified by PCR and sequenced directly, while each suspected variant was verified by reverse sequencing. The possible impact of the mutant was analyzed by the corresponding bioinformatics software. The phenotypic tests showed that the proband's FV activity has decreased to 24%, whereas the FV antigen has also reduced to 28%. The genetic analysis revealed that she was a compound heterozygote for a frameshift variant from small deletion in the exon 13 (c.2390_2390delC, p.Pro798Leufs∗13) and a missense mutation in the exon 25 (c.6665A>G, p.Asp2222Gly). Meanwhile, the online bioinformatics software indicated that the frameshift variant was disease-causing. The pathogenic variant p.Pro798Leufs∗13 and the benign variant p.Asp2222Gly largely account for the decrease of the FV deficiency in this Chinese family, of which the pathogenic variant is firstly reported in the world.

Summary Our study reports a novel dominant COL1A1 mutation in OI. Using a zebrafish model, we confirmed that the glycine to serine substitution at position 608 of the COL1A1 protein has deleterious effects on bone development. Purpose Osteogenesis imperfecta (OI), also known as brittle bone disease, is a group of genetic disorders. Mutations in two genes, collagen type I alpha 1 chain (COL1A1) and collagen type I alpha 1 chain (COL1A2), which encode the pro-a1 (I) and pro-a2 (I) chains of type I collagen, respectively, the most abundant form of collagen in the human body, cause most cases of OI. Methods In this study, we used panel-based next-generation sequencing for prenatal diagnosis of a fetus whose ultrasound images suggested OI. A de novo mutation in COL1A1 gene was suspected to cause the phenotype. To validate the effect of this mutation in a zebrafish model, we constructed a plasmid containing the corresponding mutation in the zebrafish gene col1a1a (c.1744 G > A), and overexpressed the mutant protein in zebrafish larvae. Results We identified a novel COL1A1 mutation (c.1822 G > A; p.Gly608Ser) in the fetus but not in her parents by an skeletal dysplasias panel. Bioinformatic analysis showed that the affected residue (p.Gly608) is highly conserved from zebrafish to humans. In contrast to larvae expressing wild-type (WT) col1a1a and enhanced green fluorescent protein (EGFP), col1a1a mutation-expressing larvae showed significant spinal curvature and embryonic lethality, mimicking the phenotype of human OI. Conclusions Our study revealed the pathogenicity of a de novo mutation, c.1822 G > A, in human COL1A1, which expands the mutation spectrum of OI.

15 pags., 6 figs., 2 tabs., Chitin is the most widespread amino renewable carbohydrate polymer in nature and the second most abundant polysaccharide. Therefore, chitin and chitinolytic enzymes are becoming more importance for biotechnological applications in food, health and agricultural fields, the design of effective enzymes being a paramount issue. We report the crystal structure of the plant-type endo-chitinase Chit33 from Trichoderma harzianum and its D165A/E167A-Chit33-(NAG) complex, which showed an extended catalytic cleft with six binding subsites lined with many polar interactions. The major trait of Chit33 is the location of the non-conserved Asp117 and Arg274 acting as a clamp, fixing the distorted conformation of the sugar at subsite –1 and the bent shape of the substrate, which occupies the full catalytic groove. Relevant residues were selected for mutagenesis experiments, the variants being biochemically characterized through their hydrolytic activity against colloidal chitin and other polymeric substrates with different molecular weights and deacetylation percentages. The mutant S118Y stands out, showing a superior performance in all the substrates tested, as well as detectable transglycosylation capacity, with this variant providing a promising platform for generation of novel Chit33 variants with adjusted performance by further design of rational mutants’. The putative role of Tyr in binding was extrapolated from molecular dynamics simulation., This work was supported by grants from the Spanish Ministry of Economy and Competitiveness through grants PID2019-105838RB-C33/-C32, Fundacioón Ramón Areces (XIX Call of Research Grants in Life and Material Sciences, and EU EMFF-Blue Economy-2018 (Fish4Fish-863697). We are grateful to the staff of the Synchrotron Radiation Sources at Alba (Barcelona, Spain) for providing access and for technical assistance with BL13-XALOC beamline and to the Fundacioón Ramón Areces for an institutional grant to the Centre of Molecular Biology Severo Ochoa.

Monogenic autoinflammatory diseases are a group of rheumatologic disorders caused by dysregulation in the innate immune system. The molecular mechanisms of these disorders are linked to defects in inflammasome-mediated, NF-κB-mediated or interferon-mediated inflammatory signalling pathways, cytokine receptors, the actin cytoskeleton, proteasome complexes and various enzymes. As with other human disorders, disease-causing variants in a single gene can present with variable expressivity and incomplete penetrance. In some cases, pathogenic variants in the same gene can be inherited either in a recessive or dominant manner and can cause distinct and seemingly unrelated phenotypes, although they have a unifying biochemical mechanism. With an enhanced understanding of protein structure and functionality of protein domains, genotype-phenotype correlations are beginning to be unravelled. Many of the mutated proteins are primarily expressed in haematopoietic cells, and their malfunction leads to systemic inflammation. Disease presentation is also defined by a specific effect of the mutant protein in a particular cell type and, therefore, the resulting phenotype might be more deleterious in one tissue than in another. Many patients present with the expanded immunological disease continuum that includes autoinflammation, immunodeficiency, autoimmunity and atopy, which necessitate genetic testing.

Mutations in Myelin Protein Zero (MPZ) cause CMT1B, the second leading cause of CMT1. Many of the >200 mutations cause neuropathy through a toxic gain of function by the mutant protein such as ER retention, activation of the Unfolded Protein Response (UPR) or disruption of myelin compaction. While there is extensive literature on the loss of function consequences of MPZ in heterozygous Mpz +/- null mice, there is little known of the consequences of MPZ haploinsufficiency in humans. We identified six patients from different families with p.Tyr68Ter or p.Asp104fs heterozygous mutations of MPZ that are predicted to cause a premature termination and nonsense mediated decay of the mutant allele. Five patients were evaluated in Milan and one in Iowa City; all should be haploinsufficient for MPZ. Patients were evaluated clinically and by electrophysiology. Sensory ataxia dominated the clinical presentation with only mild weakness present in five of the six patients. Symptoms presented in adulthood in all patients and only one individual had a CMTNSv2 >5. Deep tendon reflexes were absent in all patients. Patients with likely MPZ loss of function due to mutations that cause haplodeficiency in MPZ have a mild, predominantly large fiber sensory neuropathy that serves as a human equivalent to the neuropathy observed in heterozygous Mpz null mice. Successful therapeutic approaches in treating Mpz deficient mice may be candidates for trials in these and similar patients.

Purpose: Familial exudative vitreoretinopathy (FEVR) is a blinding retinal vascular disease. Clinically, FEVR is characterized by incomplete vascularization of the peripheral retina and pathological neovascularization. Only about 50% of FEVR cases can be explained by known FEVR disease gene variations. This study aimed to identify novel genes associated with the FEVR phenotype and explore their pathogenic mechanisms. Materials and Methods: Exome sequencing analyses were conducted on one Chinese family with FEVR whose affected members did not exhibit pathogenic variants in the known FEVR genes (verified using Sanger sequencing analysis). Functions of the affected proteins were evaluated using reporter assays. Western blot analysis was used to detect mutant protein expression and the genes' pathogenic mechanisms. Results: A rare novel heterozygous variant in DLG1 (c.1792A>G; p.S598G) was identified. The amino acid residues surrounding the identified variant are highly conserved among vertebrates. A luciferase reporter assay revealed that the mutant DLG1 protein DLG1-S598G lost its ability to activate Wnt signaling. Moreover, a knockdown (KD) of DLG1 in human primary retinal endothelial cells impaired tube formation. Mechanistically, DLG1 KD led to a reduction in phosphorylated VEGFR2, an essential receptor for the angiogenic potency that signals the vascular endothelial growth factor molecule. Conclusions: The data reported here demonstrate that DLG1 is a novel candidate gene for FEVR.

Hereditary angioedema (HAE) is a rare condition characterized by episodic local edema involving various organs, which can be life-threatening in some cases. Among the three subtypes of the disease, HAE types I and II are known to be caused by heterozygous mutations in the SERPING1 gene encoding C1 inhibitor (C1INH). Although a number of mutations in the SERPING1 gene have been identified to date, the mechanisms how these mutations cause HAE are not completely understood. We herein performed detailed in vitro studies for a missense SERPING1 gene mutation p.S150F which we recently identified in a Japanese patient with HAE type I. We showed that the p.S150F-mutant C1INH was stably expressed within the cultured cells, while it was not secreted into the medium at all. Furthermore, we demonstrated that the mutant C1INH significantly prevented secretion of wild-type C1INH. Finally, the results suggested that the wild-type protein was not only retained but also degraded within the cytoplasm through interacting with the mutant protein. Our study clearly revealed a dominant-negative effect of the p.S150F-mutant C1INH against the wild-type C1INH.

Huntington's disease (HD) is a neurodegenerative, dominantly inherited genetic disease caused by expansion of the polyglutamine tract in the huntingtin gene. At the cellular level, HD is characterized by the accumulation of mutant huntingtin protein in brain cells, resulting in the development of the HD phenotype, which includes mental disorders, decreased cognitive abilities, and progressive motor impairments in the form of chorea. Despite numerous studies, no unambigous connection between the accumulation of mutant protein and selective death of striatal neurons has yet been established. Recent studies have shown impairments in the calcium homeostasis in striatal neurons in HD. These cells are extremely sensitive to changes in the cytoplasmic concentration of calcium and its excessive increase leads to their death. One of the possible ways to normalize the balance of calcium in striatal neurons is through the sigma 1 receptor (S1R), which act as a calcium sensor that also exhibits modulating chaperone activity upon the cell stress observed during the development of many neurodegenerative diseases. The fact that S1R is a ligand-operated protein makes it a new promising molecular target for the development of drug therapy of HD based on the agonists of this receptor.

The CADM family of proteins consists of four neuronal specific adhesion molecules (CADM1, CADM2, CADM3 and CADM4) that mediate the direct contact and interaction between axons and glia. In the peripheral nerve, axon-Schwann cell interaction is essential for the structural organization of myelinated fibres and is primarily mediated by the binding of CADM3, expressed in axons, to CADM4, expressed by myelinating Schwann cells. We have identified—by whole exome sequencing—three unrelated families, including one de novo patient, with axonal Charcot-Marie-Tooth disease (CMT2) sharing the same private variant in CADM3, Tyr172Cys. This variant is absent in 230 000 control chromosomes from gnomAD and predicted to be pathogenic. Most CADM3 patients share a similar phenotype consisting of autosomal dominant CMT2 with marked upper limb involvement. High resolution mass spectrometry analysis detected a newly created disulphide bond in the mutant CADM3 potentially modifying the native protein conformation. Our data support a retention of the mutant protein in the endoplasmic reticulum and reduced cell surface expression in vitro. Stochastic optical reconstruction microscopy imaging revealed decreased co-localization of the mutant with CADM4 at intercellular contact sites. Mice carrying the corresponding human mutation (Cadm3Y170C) showed reduced expression of the mutant protein in axons. Cadm3Y170C mice showed normal nerve conduction and myelin morphology, but exhibited abnormal axonal organization, including abnormal distribution of Kv1.2 channels and Caspr along myelinated axons. Our findings indicate the involvement of abnormal axon-glia interaction as a disease-causing mechanism in CMT patients with CADM3 mutations.

G555F mutant of Fibrinogen A alpha-chain (FGA) is reported to be associated with kidney amyloidosis. In the current study, we have modelled the G555F mutant and examined the mutation's effect on the structural and functional level. We have also docked Vitamin C and D3 on the mutant's amyloidogenic region to identify if these vitamins can bind amyloidogenic regions. Further, we analyzed if they could prevent or modulate amyloid formation by stopping critical interactions in amyloidogenic regions in FGA. We used the wild type FGA model protein as a control. Our docking and molecular dynamics simulation results indicate stronger Vitamin D3 binding than Vitamin C to the amyloidogenic region of the mutant protein. The RMSD, radius of gyration, and RMSF values were higher for the G555F mutant than the FGA wild type protein. Overall, the results support these vitamins' potential as a therapeutic and anti-amyloidogenic agent for FGA renal amyloidosis.

Mechanical stress induced by contractions constantly threatens the integrity of muscle Z-disc, a crucial force-bearing structure in striated muscle. The PDZ-LIM proteins have been proposed to function as adaptors in transducing mechanical signals to preserve the Z-disc structure, however the underlying mechanisms remain poorly understood. Here, we show that LDB3, a well-characterized striated muscle PDZ-LIM protein, modulates mechanical stress signaling through interactions with the mechanosensing domain in filamin C, its chaperone HSPA8, and PKCα in the Z-disc of skeletal muscle. Studies of Ldb3Ala165Val/+ mice indicate that the myopathy-associated LDB3 p.Ala165Val mutation triggers early aggregation of filamin C and its chaperones at muscle Z-disc before aggregation of the mutant protein. The mutation causes protein aggregation and eventually Z-disc myofibrillar disruption by impairing PKCα and TSC2-mTOR, two important signaling pathways regulating protein stability and disposal of damaged cytoskeletal components at a major mechanosensor hub in the Z-disc of skeletal muscle., Pathak et al. identify LDB3, a striated muscle PDZ-LIM protein, as a signaling adaptor in a major mechanosensor assembly through interactions with filamin C, HSPA8, and PKCα. When LDB3 is mutated, PKCα and TSC2-mTOR mediated homeostasis is impaired, leading to protein aggregation myopathy.

Background Factor VIII (FVIII) is activated by thrombin-catalyzed cleavage at three sites. Previous reports indicated that the A2 domain contained thrombin-interactive sites responsible for cleavage at Arg372 . We have also found that the A1 domain of FVIII bound to the anion-binding exosite I of thrombin. The present study focused, therefore, on thrombin interaction with A1 residues 337-372 containing clustered acidic and hirugen-like sequences. Aim To identify specific thrombin-interactive site(s) within the A1 acidic region of FVIII. Methods and results The synthetic peptide of residues 337-353 with sulfated Tyr346 (337-353S) significantly blocked thrombin-catalyzed FVIII activation and cleavage at Arg372 , while a corresponding peptide of residues 354-372 had no significant effect. Treatment with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide to cross-link thrombin and 340-350S suggested that the 344-349 clustered acidic region was involved in thrombin interaction. Alanine-substituted FVIII mutants, Y346A and D347A/D348A/D349A, depressed thrombin-catalyzed activation and cleavage at Arg372 , with peak activation at ~ 50% and cleavage rates of ~ 10% to 20% compared to wild type (WT). The peak level of thrombin-catalyzed activation and the cleavage rate at Arg372 using FVIII mutants with 337-346 residues substituted with hirugen-sequences (MKNNEEAEDY337-346GDFEEIPEEY) were ~ 1.5- and ~ 2.5-fold of WT, respectively. Surface plasmon resonance-based analysis demonstrated that the Kd for active-site modified thrombin interactions using Y346A and D347A/D348A/D349A mutants was ~ 3- to 6-fold higher than that of WT, and that the hirugen-hybrid mutant facilitated association kinetics ~ 1.8-fold of WT. Conclusion Residues 346-349 with sulfated Tyr provided a thrombin-interactive site responsible for activation and cleavage at Arg372 . A hirugen-hybrid A1 mutant showed more efficient thrombin-catalyzed cleavage at Arg372 .

Background: Factor VIII (FVIII) is activated by thrombin-catalyzed cleavage at three sites. Previous reports indicated that the A2 domain contained thrombin-interactive sites responsible for cleavage at Arg³⁷². We have also found that the A1 domain of FVIII bound to the anion-binding exosite I of thrombin. The present study focused, therefore, on thrombin interaction with A1 residues 337-372 containing clustered acidic and hirugen-like sequences. Aim: To identify specific thrombin-interactive site(s) within the A1 acidic region of FVIII. Methods and results: The synthetic peptide of residues 337-353 with sulfated Tyr³⁴⁶ (337-353S) significantly blocked thrombin-catalyzed FVIII activation and cleavage at Arg³⁷², while a corresponding peptide of residues 354-372 had no significant effect. Treatment with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide to cross-link thrombin and 340-350S suggested that the 344-349 clustered acidic region was involved in thrombin interaction. Alanine-substituted FVIII mutants, Y346A and D347A/D348A/D349A, depressed thrombin-catalyzed activation and cleavage at Arg³⁷², with peak activation at ~ 50% and cleavage rates of ~ 10% to 20% compared to wild type (WT). The peak level of thrombin-catalyzed activation and the cleavage rate at Arg³⁷² using FVIII mutants with 337-346 residues substituted with hirugen-sequences (MKNNEEAEDY337-346GDFEEIPEEY) were ~ 1.5- and ~ 2.5-fold of WT, respectively. Surface plasmon resonance-based analysis demonstrated that the Kd for active-site modified thrombin interactions using Y346A and D347A/D348A/D349A mutants was ~ 3- to 6-fold higher than that of WT, and that the hirugen-hybrid mutant facilitated association kinetics ~ 1.8-fold of WT. Conclusion: Residues 346-349 with sulfated Tyr provided a thrombin-interactive site responsible for activation and cleavage at Arg³⁷². A hirugen-hybrid A1 mutant showed more efficient thrombin-catalyzed cleavage at Arg³⁷²., 博士（医学）・甲第788号・令和3年3月15日, © 2020 International Society on Thrombosis and Haemostasis., This is the peer reviewed version of the following article: https://onlinelibrary.wiley.com/doi/10.1111/jth.15201, which has been published in final form at https://doi.org/10.1111/jth.15201. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.

While germline recessive loss-of-function mutations in SEC23B in humans cause a rare form of anaemia, heterozygous change-of-function mutations result in increased predisposition to cancer. SEC23B encodes SEC23 homologue B, a component of coat protein complex II (COPII), which canonically transports proteins from the endoplasmic reticulum (ER) to the Golgi. Despite the association of SEC23B with anaemia and cancer, the precise pathophysiology of these phenotypic outcomes remains unknown. Recently, we reported that mutant SEC23B has non-canonical COPII-independent function, particularly within the ER stress and ribosome biogenesis pathways, and that may contribute to the pathobiology of cancer predisposition. In this study, we hypothesized that wild-type SEC23B has a baseline function within such cellular stress response pathways, with the mutant protein reflecting exaggerated effects. Here, we show that the wild-type SEC23B protein localizes to the nucleus in addition to classical distribution at the ER/Golgi interface and identify multiple putative nuclear localization and export signals regulating nuclear–cytoplasmic transport. Unexpectedly, we show that, independently of COPII, wild-type SEC23B can also localize to cell nucleoli under proteasome inhibition conditions, with distinct distribution patterns compared to mutant cells. Unbiased proteomic analyses through mass spectrometry further revealed that wild-type SEC23B interacts with a subset of nuclear proteins, in addition to central proteins in the ER stress, protein ubiquitination, and EIF2 signalling pathways. We validate the genotype-specific differential SEC23B–UBA52 (ribosomal protein RPL40) interaction. Finally, utilizing patient-derived lymphoblastoid cell lines harbouring either wild-type or mutant SEC23B, we show that SEC23B levels increase in response to ER stress, further corroborating its role as a cellular stress response sensor and/or effector. Overall, these observations suggest that SEC23B, irrespective of mutation status, has unexplored roles in the cellular stress response pathway, with implications relevant to cancer and beyond that, CDAII and normal cell biology.

Genetic cardiomyopathy is caused by mutations in various genes. The accumulation of potentially proteotoxic mutant protein aggregates due to insufficient autophagy is a possible mechanism of disease development. The objective of this study was to investigate the distribution in the myocardium of such aggregates in relation to specific pathogenic genetic mutations in cardiomyopathy hearts. Hearts from 32 genetic cardiomyopathy patients, 4 non‐genetic cardiomyopathy patients and 5 controls were studied. Microscopic slices from an entire midventricular heart slice were stained for p62 (sequestosome‐1, marker for aggregated proteins destined for autophagy). The percentage of cardiomyocytes with p62 accumulation was higher in cardiomyopathy hearts (median 3.3%) than in healthy controls (0.3%; P