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Bird feathers are the product of interactions between natural and artificial selection. Feather-related traits are important for chicken selection and breeding. Frizzle feather is characterized by the abnormally development of feathers in chickens. In the current study, frizzle feather characteristics were observed in a local breed called Xiushui Yellow Chicken in Jiangxi, China. To determine the molecular mechanisms that underlie frizzle feather in Xiushui Yellow Chicken, four populations of three breeds (Xiushui Yellow Chicken with frizzle feathers, Xiushui Yellow Chicken with normal feathers, Guangfeng White-Ear Yellow Chicken, and Ningdu Yellow Chicken) were selected for whole-genome resequencing. Using a comparative genome strategy and genome-wide association study, a missense mutation (g.5281494A>G) and a 15-bp deletion (g.5285437-5285451delGATGCCGGCAGGACG) in KRT75L4 were identified as candidate mutations associated with frizzle feather in Xiushui Yellow Chicken. Based on genotyping performed in a large Xiushui Yellow Chicken population, the g.5285437-5285451delGATGCCGGCAGGACG mutation in KRT75L4 was confirmed as the putative causative mutation of frizzle feather. These results deepen the understanding of the molecular mechanisms responsible for frizzle feather, as well as facilitating the molecular detection and selection of the feather phenotype in Xiushui Yellow Chickens., (© 2021 Stichting International Foundation for Animal Genetics.)

Otogelin-like protein (encoded by Otogl) was highly structural similar to the gelforming mucin proteins. Although human OTOG mutations have been linked to deafness, the biological function of OTOGL in male germ cell development remains enigmatic. In screening 336 patients with non-obstructive azoospermia (NOA), OTOGL displays the high mutant ratio (13.99 %). Then, we examined the expression of OTOGL in developing mouse testes. Otogl mRNA and protein are continually expressed in postnatal developing testes from postnatal day 0 (P0) testes to P21 testes exhibiting a decreased trend with the age growth. We thus generated a global Otogl knockout mouse (KO) model using the CRISPR/Cas9 technology; however, Otogl KO mice displayed normal development and fertility. Further histological analysis of Otogl knockout mouse testes revealed that all types of spermatogenic cells are present in Otogl KO seminiferous tubules. Together, our study suggested that OTOGL is nonessential for male germ cell development and spermatogenesis.

Hyperekplexia is a rare sensorimotor syndrome characterized by pathological startle reflex in response to unexpected trivial stimuli for which there is no specific treatment. Neonates suffer from hypertonia and are at high risk of sudden death due to apnea episodes. Mutations in the human SLC6A5 gene encoding the neuronal glycine transporter GlyT2 may disrupt the inhibitory glycinergic neurotransmission and cause a presynaptic form of the disease. The phenotype of missense mutations giving rise to protein misfolding but maintaining residual activity could be rescued by facilitating folding or intracellular trafficking. In this report, we characterized the trafficking properties of two mutants associated with hyperekplexia (A277T and Y707C, rat numbering). Transporter molecules were partially retained in the endoplasmic reticulum showing increased interaction with the endoplasmic reticulum chaperone calnexin. One transporter variant had export difficulties and increased ubiquitination levels, suggestive of enhanced endoplasmic reticulum-associated degradation. However, the two mutant transporters were amenable to correction by calnexin overexpression. Within the search for compounds capable of rescuing mutant phenotypes, we found that the arachidonic acid derivative N-arachidonoyl glycine can rescue the trafficking defects of the two variants in heterologous cells and rat brain cortical neurons. N-arachidonoyl glycine improves the endoplasmic reticulum output by reducing the interaction transporter/calnexin, increasing membrane expression and improving transport activity in a comparable way as the well-established chemical chaperone 4-phenyl-butyrate. This work identifies N-arachidonoyl glycine as a promising compound with potential for hyperekplexia therapy., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Missense variants are present amongst the healthy population, but some of them are causative of human diseases. A classification of variants associated with "healthy" or "diseased" states is therefore not always straightforward. A deeper understanding of the nature of missense variants in health and disease, the cellular processes they may affect, and the general molecular principles which underlie these differences is essential to offer mechanistic explanations of the true impact of pathogenic variants. Here, we have formalised a statistical framework which enables robust probabilistic quantification of variant enrichment across full-length proteins, their domains, and 3D structure-defined regions. Using this framework, we validate and extend previously reported trends of variant enrichment in different protein structural regions (surface/core/interface). By examining the association of variant enrichment with available functional pathways and transcriptomic and proteomic (protein half-life, thermal stability, abundance) data, we have mined a rich set of molecular features which distinguish between pathogenic and population variants: Pathogenic variants mainly affect proteins involved in cell proliferation and nucleotide processing and are enriched in more abundant proteins. Additionally, rare population variants display features closer to common than pathogenic variants. We validate the association between these molecular features and variant pathogenicity by comparing against existing in silico variant impact annotations. This study provides molecular details into how different proteins exhibit resilience and/or sensitivity towards missense variants and provides the rationale to prioritise variant-enriched proteins and protein domains for therapeutic targeting and development. The ZoomVar database, which we created for this study, is available at fraternalilab.kcl.ac.uk/ZoomVar. It allows users to programmatically annotate missense variants with protein structural information and to calculate variant enrichment in different protein structural regions., Competing Interests: The authors have declared that no competing interests exist.

The nuclear envelope (NE) is a critical barrier between the cytosol and nucleus that is key for compartmentalization within the cell and serves an essential role in organizing and protecting genomic DNA. Rupturing of the NE through loss of constitutive NE proteins and/or mechanical force applied to the nucleus results in the unregulated mixing of cytosolic and nuclear compartments, leading to DNA damage and genomic instability. Nuclear rupture has recently gained interest as a mechanism that may participate in various NE-associated diseases as well as cancer. Remarkably, these rupturing events are often transient, with cells being capable of rapidly repairing nuclear ruptures. Recently, we identified Barrier-to-Autointegration Factor (BAF), a DNA-binding protein involved in post-mitotic NE reformation and cytosolic viral regulation, as an essential protein for nuclear rupture repair. During interphase, the highly mobile cytosolic BAF is primed to monitor for a compromised NE by rapidly binding to newly exposed nuclear DNA and subsequently recruiting the factors necessary for NE repair. This review highlights the recent findings of BAF's roles in rupture repair, and offers perspectives on how regulatory factors that control BAF activity may potentially alter the cellular response to nuclear ruptures and how BAF may participate in human disease.

Fragile X syndrome (FXS) is the most frequent form of inherited intellectual disability and the best-described monogenic cause of autism. CGG-repeat expansion in the FMR1 gene leads to FMR1 silencing, loss-of-expression of the Fragile X Mental Retardation Protein (FMRP), and is a common cause of FXS. Missense mutations in the FMR1 gene were also identified in FXS patients, including the recurrent FMRP-R138Q mutation. To investigate the mechanisms underlying FXS caused by this mutation, we generated a knock-in mouse model (Fmr1 R138Q ) expressing the FMRP-R138Q protein. We demonstrate that, in the hippocampus of the Fmr1 R138Q mice, neurons show an increased spine density associated with synaptic ultrastructural defects and increased AMPA receptor-surface expression. Combining biochemical assays, high-resolution imaging, electrophysiological recordings, and behavioural testing, we also show that the R138Q mutation results in impaired hippocampal long-term potentiation and socio-cognitive deficits in mice. These findings reveal the functional impact of the FMRP-R138Q mutation in a mouse model of FXS.

Temperature is a major factor regulating plant growth. To reproduce at extreme temperatures, plants must develop normal reproductive organs when exposed to temperature changes. However, little is known about the underlying molecular mechanisms. Here, we identified the maize ( Zea mays ) mutant thermosensitive vanishing tassel1 - R ( tvt1 - R ), which lacks tassels at high (restrictive) temperatures due to shoot apical meristem (SAM) arrest, but forms normal tassels at moderate (permissive) temperatures. The critical stage for phenotypic conversion in tvt1 - R mutants is V2 to V6 (Vn, where "n" is the number of leaves with collars visible). Positional cloning and allelism and complementation tests revealed that a G-to-A mutation causing a Arg 277 -to-His 277 substitution in ZmRNRL1, a ribonucleotide reductase (RNR) large subunit (RNRL), confers the tvt1 - R mutant phenotype. RNR regulates the rate of deoxyribonucleoside triphosphate (dNTP) production for DNA replication and damage repair. By expression, yeast two-hybrid, RNA sequencing, and flow cytometric analyses, we found that ZmRNRL1- tvt1 - R failed to interact with all three RNR small subunits at 34°C due to the Arg 277 -to-His 277 substitution, which could impede RNR holoenzyme (α 2 β 2 ) formation, thereby decreasing the dNTP supply for DNA replication. Decreased dNTP supply may be especially severe for the SAM that requires a continuous, sufficient dNTP supply for rapid division, as demonstrated by the SAM arrest and tassel absence in tvt1 - R mutants at restrictive temperatures. Our study reveals a novel mechanism of temperature-gated tassel formation in maize and provides insight into the role of RNRL in SAM maintenance., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Interpretation of the colossal number of genetic variants identified from sequencing applications is one of the major bottlenecks in clinical genetics, with the inference of the effect of amino acid-substituting missense variations on protein structure and function being especially challenging. Here we characterize the three-dimensional (3D) amino acid positions affected in pathogenic and population variants from 1,330 disease-associated genes using over 14,000 experimentally solved human protein structures. By measuring the statistical burden of variations (i.e., point mutations) from all genes on 40 3D protein features, accounting for the structural, chemical, and functional context of the variations' positions, we identify features that are generally associated with pathogenic and population missense variants. We then perform the same amino acid-level analysis individually for 24 protein functional classes, which reveals unique characteristics of the positions of the altered amino acids: We observe up to 46% divergence of the class-specific features from the general characteristics obtained by the analysis on all genes, which is consistent with the structural diversity of essential regions across different protein classes. We demonstrate that the function-specific 3D features of the variants match the readouts of mutagenesis experiments for BRCA1 and PTEN, and positively correlate with an independent set of clinically interpreted pathogenic and benign missense variants. Finally, we make our results available through a web server to foster accessibility and downstream research. Our findings represent a crucial step toward translational genetics, from highlighting the impact of mutations on protein structure to rationalizing the variants' pathogenicity in terms of the perturbed molecular mechanisms., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

A highly infectious coronavirus, SARS-CoV-2, has spread in many countries. This virus recognizes its receptor, angiotensin-converting enzyme 2 (ACE2), using the receptor binding domain of its spike protein subunit S1. Many missense mutations are reported in various human populations for the ACE2 gene. In the current study, we predict the affinity of many ACE2 variants for binding to S1 protein using different computational approaches. The dissociation process of S1 from some variants of ACE2 is studied in the current work by molecular dynamics approaches. We study the relation between structural dynamics of ACE2 in closed and open states and its affinity for S1 protein of SARS-CoV-2.

The epilepsy-linked gene SV2A , has a number of potential roles in the synaptic vesicle (SV) life cycle. However, how loss of SV2A function translates into presynaptic dysfunction and ultimately seizure activity is still undetermined. In this study, we examined whether the first SV2A mutation identified in human disease (R383Q) could provide information regarding which SV2A-dependent events are critical in the translation to epilepsy. We utilized a molecular replacement strategy in which exogenous SV2A was expressed in mouse neuronal cultures of either sex, which had been depleted of endogenous SV2A to mimic the homozygous human condition. We found that the R383Q mutation resulted in a mislocalization of SV2A from SVs to the plasma membrane, but had no effect on its activity-dependent trafficking. This SV2A mutant displayed reduced mobility when stranded on the plasma membrane and reduced binding to its interaction partner synaptotagmin-1 (Syt1). Furthermore, the R383Q mutant failed to rescue reduced expression and dysfunctional activity-dependent trafficking of Syt1 in the absence of endogenous SV2A. This suggests that the inability to control Syt1 expression and trafficking at the presynapse may be key in the transition from loss of SV2A function to seizure activity. SIGNIFICANCE STATEMENT SV2A is a synaptic vesicle (SV) protein, the absence or dysfunction of which is linked to epilepsy. However, the series of molecular events that result in this neurological disorder is still undetermined. We demonstrate here that the first human mutation in SV2A identified in an individual with epilepsy displays reduced binding to synaptotagmin-1 (Syt1), an SV protein essential for synchronous neurotransmitter release. Furthermore, this mutant cannot correct alterations in both Syt1 expression and trafficking when expressed in the absence of endogenous SV2A (to mimic the homozygous human condition). This suggests that the inability to control Syt1 expression and trafficking may be key in the transition from loss of SV2A function to seizure activity., (Copyright © 2020 the authors.)

The distribution of fitness effects of mutation plays a central role in constraining protein evolution. The underlying mechanisms by which mutations lead to fitness effects are typically attributed to changes in protein specific activity or abundance. Here, we reveal the importance of a mutation's collateral fitness effects, which we define as effects that do not derive from changes in the protein's ability to perform its physiological function. We comprehensively measured the collateral fitness effects of missense mutations in the Escherichia coli TEM-1 β-lactamase antibiotic resistance gene using growth competition experiments in the absence of antibiotic. At least 42% of missense mutations in TEM-1 were deleterious, indicating that for some proteins collateral fitness effects occur as frequently as effects on protein activity and abundance. Deleterious mutations caused improper posttranslational processing, incorrect disulfide-bond formation, protein aggregation, changes in gene expression, and pleiotropic effects on cell phenotype. Deleterious collateral fitness effects occurred more frequently in TEM-1 than deleterious effects on antibiotic resistance in environments with low concentrations of the antibiotic. The surprising prevalence of deleterious collateral fitness effects suggests they may play a role in constraining protein evolution, particularly for highly expressed proteins, for proteins under intermittent selection for their physiological function, and for proteins whose contribution to fitness is buffered against deleterious effects on protein activity and protein abundance., Competing Interests: The authors declare no competing interest.

The human cytochrome P450 enzyme CYP4Z1 remains an understudied enzyme despite its association with poor prognosis and overexpression in breast cancer. Hence, CYP4Z1 has previously been suggested as an anti-breast cancer target. In the present study we employed extended mutation analysis to increase our understanding of the substrate binding mode of this enzyme. In a combined in vitro and in silico approach we show for the first time that residue Arg487 plays an important role in substrate recognition and binding of CYP4Z1. Using a large array of recombinant CYP4Z1 mutants we show that, apart from Asn381, all other postulated binding residues only play an auxiliary role in substrate recognition and binding. Different substrate interaction motifs were identified via dynamic pharmacophores (dynophores) and their impact on catalytically competent substrate binding was classified. These new insights on the substrate recognition and binding mode represent an important step towards the rational design of CYP4Z1 prodrugs and guide further investigations into the so far poorly understood physiological role of CYP4Z1., Competing Interests: Conflict of interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Voltage-gated calcium-channels (VGCCs) are heteromers consisting of several subunits. Mutations in the genes coding for VGCC subunits have been reported to be associated with autism spectrum disorder (ASD). In a previous study, we identified electrophysiologically relevant missense mutations of Ca V β 2 subunits of VGCCs. From this, we derived the hypothesis that several Ca V β 2 -mutations associated with ASD show common features sensitizing LTCCs and/or enhancing currents. Using a Ca V β 2d backbone, we performed extensive whole-cell and single-channel patch-clamp analyses of Ba 2+ currents carried by Ca v 1.2 pore subunits co-transfected with the previously described Ca V β 2 mutations (G167S, S197F) as well as a recently identified point mutation (V2D). Furthermore, the interaction of the mutated Ca V β 2d subunits with the RGK protein Gem was analyzed by co-immunoprecipitation assays and electrophysiological studies. Patch-clamp analyses revealed that all mutations increase Ba 2+ currents, e.g. by decreasing inactivation or increasing fraction of active sweeps. All Ca V β 2 mutations interact with Gem, but differ in the extent and characteristics of modulation by this RGK protein (e.g. decrease of fraction of active sweeps: Ca V β 2d_G167S > Ca V β 2d_V2D > Ca V β 2d_S197F ). In conclusion, patch-clamp recordings of ASD-associated Ca V β 2d mutations revealed differential modulation of Ba 2+ currents carried by Ca V 1.2 suggesting kind of an "electrophysiological fingerprint" each. The increase in current finally observed with all Ca V β 2d mutations analyzed might contribute to the complex pathophysiology of ASD and by this indicate a possible underlying molecular mechanism., Competing Interests: Declaration of Competing Interest None., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: In recent years, Ecuador and other South American countries have experienced an increase in arboviral diseases. A rise in dengue infections was followed by introductions of chikungunya and Zika, two viruses never before seen in many of these areas. Furthermore, the latest socioeconomic and political instability in Venezuela and the mass migration of its population into the neighboring countries has given rise to concerns of infectious disease spillover and escalation of arboviral spread in the region., Results: We performed phylogeographic analyses of dengue (DENV) and chikungunya (CHIKV) virus genomes sampled from a surveillance site in Ecuador in 2014-2015, along with genomes from the surrounding countries. Our results revealed at least two introductions of DENV, in 2011 and late 2013, that initially originated from Venezuela and/or Colombia. The introductions were subsequent to increases in the influx of Venezuelan and Colombian citizens into Ecuador, which in 2013 were 343% and 214% higher than in 2009, respectively. However, we show that Venezuela has historically been an important source of DENV dispersal in this region, even before the massive exodus of its population, suggesting already established paths of viral distribution. Like DENV, CHIKV was introduced into Ecuador at multiple time points in 2013-2014, but unlike DENV, these introductions were associated with the Caribbean. Our findings indicated no direct CHIKV connection between Ecuador, Colombia, and Venezuela as of 2015, suggesting that CHIKV was, at this point, not following the paths of DENV spread., Conclusion: Our results reveal that Ecuador is vulnerable to arbovirus import from many geographic locations, emphasizing the need of continued surveillance and more diversified prevention strategies. Importantly, increase in human movement along established paths of viral dissemination, combined with regional outbreaks and epidemics, may facilitate viral spread and lead to novel virus introductions. Thus, strengthening infectious disease surveillance and control along migration routes and improving access to healthcare for the vulnerable populations is of utmost importance.

Aromatase (CYP19A1) converts androgens into estrogens and is required for female sexual development and growth and development in both sexes. CYP19A1 is a member of cytochrome P450 family of heme-thiolate monooxygenases located in the endoplasmic reticulum and depends on reducing equivalents from the reduced nicotinamide adenine dinucleotide phosphate via the cytochrome P450 oxidoreductase coded by POR. Both the CYP19A1 and POR genes are highly polymorphic, and mutations in both these genes are linked to disorders of steroid biosynthesis. We have previously shown that R264C and R264H mutations in CYP19A1, as well as mutations in POR, reduce CYP19A1 activity. The R264C is a common polymorphic variant of CYP19A1, with high frequency in Asian and African populations. Polymorphic alleles of POR are found in all populations studied so far and, therefore, may influence activities of CYP19A1 allelic variants. So far, the effects of variations in POR on enzymatic activities of allelic variants of CYP19A1 or any other steroid metabolizing cytochrome P450 proteins have not been studied. Here we are reporting the effects of three POR variants on the aromatase activities of two CYP19A1 variants, R264C, and R264H. We used bacterially expressed and purified preparations of WT and variant forms of CYP19A1 and POR and constructed liposomes with embedded CYP19A1 and POR proteins and assayed the CYP19A1 activities using radiolabeled androstenedione as a substrate. With the WT-POR as a redox partner, the R264C-CYP19A1 showed only 15% of aromatase activity, but the R264H had 87% of aromatase activity compared to WT-CYP19A1. With P284L-POR as a redox partner, R264C-CYP19A1 lost all activity but retained 6.7% of activity when P284T-POR was used as a redox partner. The R264H-CYP19A1 showed low activities with both the POR-P284 L as well as the POR-P284 T. When the POR-Y607C was used as a redox partner, the R264C-CYP19A1 retained approximately 5% of CYP19A1 activity. Remarkably, The R264H-CYP19A1 had more than three-fold higher activity compared to WT-CYP19A1 when the POR-Y607C was used as the redox partner, pointing toward a beneficial effect. The slight increase in activity of R264C-CYP19A1 with the P284T-POR and the three-fold increase in activity of the R264H-CYP19A1 with the Y607C-POR point toward a conformational effect and role of protein-protein interaction governed by the R264C and R264H substitutions in the CYP19A1 as well as P284 L, P284 T and Y607C variants of POR. These studies demonstrate that the allelic variants of P450 when present with a variant form of POR may show different activities, and combined effects of variations in the P450 enzymes as well as in the POR should be considered when genetic data are available. Recent trends in the whole-exome and whole-genome sequencing as diagnostic tools will permit combined evaluation of variations in multiple genes that are interdependent and may guide treatment options by adjusting therapeutic interventions based on laboratory analysis., (Copyright © 2019. Published by Elsevier Ltd.)

17β-Hydroxysteroid dehydrogenases (17β-HSDs) catalyze the reduction of 17-ketosteroids and the oxidation of 17β-hydroxysteroids to regulate the production of androgens and estrogens. Among them, 17β-HSD type 3 (HSD17B3) is expressed almost exclusively in testicular Leydig cells and contributes to development of male sexual characteristics by converting androstenedione (A4) to testosterone (T). Mutations of HSD17B3 genes cause a 46,XY disorder of sexual development (46,XY DSD) as a result of low T production. Therefore, the evaluation of 17β-HSD3 enzymatic activity is important for understanding and diagnosing this disorder. We adapted a method that easily evaluates enzymatic activity of 17β-HSD3 by quantifying the conversion from A4 to T using androgen receptor (AR)-mediated transactivation. HEK293 cells were transduced to express human HSD17B3, and incubated medium containing A4. Depending on the incubation time with HSD17B3-expressing cells, the culture media progressively increased luciferase activities in CV-1 cells, transfected with the AR expression vector and androgen-responsive reporter. Culture medium from HSD17B1 and HSD17B5-expressing cells also increased the luciferase activities. This system is also applicable to detect the conversion of 11-ketoandrostenedione to 11-ketotestosterone by HSD17B3. Establishment of HEK293 cells expressing various missense mutations in the HSD17B3 gene associated with 46,XY DSD revealed that this system is effective to evaluate the enzymatic activities of mutant proteins., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Hairlessness is usually a rare trait in pigs; however, in this study, we found hairless (HR) pigs at a relatively high frequency in 1 pig herd. We observed that, the lower hair shaft density of HR pigs could be mainly attributed to the lower hair follicle density, and during the embryonic period, d 39-45 were a critical stage for the formation of the hair follicle. In this regard, d 41 during gestation was a particularly important point. Hair follicle morphogenesis occurring at an early stage of embryo development is similar to humans and mice. Further analyses of association studies based on single-nucleotide polymorphism chip as well as sequence data, mRNA sequencing, immunohistochemistry, and comparative genomics demonstrated that microtubule-associated protein 2 (MAP2) is a key gene responsible for hair follicle density and 1 missense mutation of A-to-G at rs328005415 in MAP2, causing a valine-to-methionine substitution leads to the HR phenotype. Considering the high homology between pigs and humans, our research has some significance for the study of the mechanisms of skin development, hair morphogenesis, and hair loss in humans by showing that the pig may be a more appropriate model in which to study these processes.-Jiang, Y., Jiang, Y., Zhang, H., Mei, M., Song, H., Ma, X., Jiang, L., Yu, Z., Zhang, Q., Ding, X. A mutation in MAP2 is associated with prenatal hair follicle density.

Seizure-related apnea is common and can be lethal. Its mechanisms however remain unclear and preventive strategies are lacking. We postulate that brainstem spreading depolarization (SD), previously associated with lethal seizures in animal models, initiates apnea upon invasion of brainstem respiratory centers. To study this, we assessed effects of brainstem seizures on brainstem function and respiration in male and female mice carrying a homozygous S218L missense mutation that leads to gain-of-function of voltage-gated Ca V 2.1 Ca 2+ channels and high risk for fatal seizures. Recordings of brainstem DC potential and neuronal activity, cardiorespiratory activity and local tissue oxygen were performed in freely behaving animals. Brainstem SD occurred during all spontaneous fatal seizures and, unexpectedly, during a subset of nonfatal seizures. Seizure-related SDs in the ventrolateral medulla correlated with respiratory suppression. Seizures induced by stimulation of the inferior colliculus could evoke SD that spread in a rostrocaudal direction, preceding local tissue hypoxia and apnea, indicating that invasion of SD into medullary respiratory centers initiated apnea and hypoxia rather than vice versa Fatal outcome was prevented by timely resuscitation. Moreover, NMDA receptor antagonists MK-801 and memantine prevented seizure-related SD and apnea, which supports brainstem SD as a prerequisite for brainstem seizure-related apnea in this animal model and has translational value for developing strategies that prevent fatal ictal apnea. SIGNIFICANCE STATEMENT Apnea during and following seizures is common, but also likely implicated in sudden unexpected death in epilepsy (SUDEP). This underlines the need to understand mechanisms for potentially lethal seizure-related apnea. In the present work we show, in freely behaving SUDEP-prone transgenic mice, that apnea is induced when spontaneous brainstem seizure-related spreading depolarization (SD) reaches respiratory nuclei in the ventrolateral medulla. We show that brainstem seizure-related medullary SD is followed by local hypoxia and recovers during nonfatal seizures, but not during fatal events. NMDA receptor antagonists prevented medullary SD and apnea, which may be of translational value., (Copyright © 2019 the authors.)

Protease inhibitors have the highest potency among antiviral therapies against HIV-1 infections, yet the virus can evolve resistance. Darunavir (DRV), currently the most potent Food and Drug Administration-approved protease inhibitor, retains potency against single-site mutations. However, complex combinations of mutations can confer resistance to DRV. While the interdependence between mutations within HIV-1 protease is key for inhibitor potency, the molecular mechanisms that underlie this control remain largely unknown. In this study, we investigated the interdependence between the L89V and L90M mutations and their effects on DRV binding. These two mutations have been reported to be positively correlated with one another in HIV-1 patient-derived protease isolates, with the presence of one mutation making the probability of the occurrence of the second mutation more likely. The focus of our investigation is a patient-derived isolate, with 24 mutations that we call "KY"; this variant includes the L89V and L90M mutations. Three additional KY variants with back-mutations, KY(V89L), KY(M90L), and the KY(V89L/M90L) double mutation, were used to experimentally assess the individual and combined effects of these mutations on DRV inhibition and substrate processing. The enzymatic assays revealed that the KY(V89L) variant, with methionine at residue 90, is highly resistant, but its catalytic function is compromised. When a leucine to valine mutation at residue 89 is present simultaneously with the L90M mutation, a rescue of catalytic efficiency is observed. Molecular dynamics simulations of these DRV-bound protease variants reveal how the L90M mutation induces structural changes throughout the enzyme that undermine the binding interactions.

The structurally dynamic amyloidogenic protein α-synuclein (αS) is universally recognized as a key player in Parkinson's disease (PD). Copper, which acts as a neuronal signaling agent, is also an effector of αS structure, aggregation, and localization in vivo. In humans, αS is known to carry an acetyl group on the starting methionine residue, capping the N-terminal free amine which was a known high-affinity Cu II binding site. We now report the first detailed characterization data using electron paramagnetic resonance (EPR) spectroscopy to describe the Cu II coordination modes of N-terminally acetylated αS ( NAc αS). Through use of EPR hyperfine structure analyses and the Peisach-Blumberg correlation, an N3O1 binding mode was established that involves the single histidine residue at position 50 and a lower population of a second Cu II -binding mode that may involve a C-terminal contribution. We additionally generated an N-terminally acetylated disease-relevant variant, NAc H50Q, that promotes a shift in the Cu II binding site to the C-terminus of the protein. Moreover, fibrillar NAc H50Q-Cu II exhibits enhanced parallel β-sheet character and increased hydrophobic surface area compared to NAc αS-Cu II and to both protein variants that lack a coordinated cupric ion. The results presented herein demonstrate the differential impact of distinct Cu II binding sites within NAc αS, revealing that C-terminal Cu II binding exacerbates the structural consequences of the H50Q missense mutation. Likewise, the global structural modifications that result from N-terminal capping augment the properties of Cu II coordination. Hence, consideration of the effect of Cu II on NAc αS and NAc H50Q misfolding may shed light on the extrinsic or environmental factors that influence PD pathology.

Mutations in cardiac myosin binding protein C (cMyBPC) are a major cause of hypertrophic cardiomyopathy (HCM). In particular, a single amino acid substitution of tyrosine to serine at residue 237 in humans (residue 235 in mice) has been linked to HCM with strong disease association. Although cMyBPC truncations, deletions and insertions, and frame shift mutations have been studied, relatively little is known about the functional consequences of missense mutations in cMyBPC. In this study, we characterized the functional and structural effects of the HCM-causing Y235S mutation by performing mechanical experiments and molecular dynamics simulations (MDS). cMyBPC null mouse myocardium was virally transfected with wild-type (WT) or Y235S cMyBPC (KO Y235S ). We found that Y235S cMyBPC was properly expressed and incorporated into the cardiac sarcomere, suggesting that the mechanism of disease of the Y235S mutation is not haploinsufficiency or poison peptides. Mechanical experiments in detergent-skinned myocardium isolated from KO Y235S hearts revealed hypercontractile behavior compared to KO WT hearts, evidenced by accelerated cross-bridge kinetics and increased Ca 2+ sensitivity of force generation. In addition, MDS revealed that the Y235S mutation causes alterations in important intramolecular interactions, surface conformations, and electrostatic potential of the C1 domain of cMyBPC. Our combined in vitro and in silico data suggest that the Y235S mutation directly disrupts internal and surface properties of the C1 domain of cMyBPC, which potentially alters its ligand-binding interactions. These molecular changes may underlie the mechanism for hypercontractile cross-bridge behavior, which ultimately results in the development of cardiac hypertrophy and in vivo cardiac dysfunction., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Most pathogenic missense mutations cause specific molecular phenotypes through protein destabilization. However, how protein destabilization is manifested as a given molecular phenotype is not well understood. We develop here a structural and energetic approach to describe mutational effects on specific traits such as function, regulation, stability, subcellular targeting or aggregation propensity. This approach is tested using large-scale experimental and structural perturbation analyses in over thirty mutations in three different proteins (cancer-associated NQO1, transthyretin related with amyloidosis and AGT linked to primary hyperoxaluria type I) and comprising five very common pathogenic mechanisms (loss-of-function and gain-of-toxic function aggregation, enzyme inactivation, protein mistargeting and accelerated degradation). Our results revealed that the magnitude of destabilizing effects and, particularly, their propagation through the structure to promote disease-associated conformational states largely determine the severity and molecular mechanisms of disease-associated missense mutations. Modulation of the structural perturbation at a mutated site is also shown to cause switches between different molecular phenotypes. When very common disease-associated missense mutations were investigated, we also found that they were not among the most deleterious possible missense mutations at those sites, and required additional contributions from codon bias and effects of CpG sites to explain their high frequency in patients. Our work sheds light on the molecular basis of pathogenic mechanisms and genotype-phenotype relationships, with implications for discriminating between pathogenic and neutral changes within human genome variability from whole genome sequencing studies.

The most common inherited cardiac disorder, hypertrophic cardiomyopathy (HCM), is characterized by thickening of heart muscle, for which genetic mutations in cardiac myosin-binding protein C3 ( c-MYBPC3 ) gene, is the leading cause. Notably, patients with HCM display a heterogeneous clinical presentation, onset and prognosis. Thus, delineating the molecular mechanisms that explain how disparate c-MYBPC3 variants lead to HCM is essential for correlating the impact of specific genotypes on clinical severity. Herein, five c-MYBPC3 missense variants clinically associated with HCM were investigated; namely V1 (R177H), V2 (A216T), V3 (E258K), V4 (E441K) and double mutation V5 (V3 + V4), all located within the C1 and C2 domains of MyBP-C, a region known to interact with sarcomeric protein, actin. Injection of the variant complementary RNAs in zebrafish embryos was observed to recapitulate phenotypic aspects of HCM in patients. Interestingly, V3- and V5-cRNA injection produced the most severe zebrafish cardiac phenotype, exhibiting increased diastolic/systolic myocardial thickness and significantly reduced heart rate compared with control zebrafish. Molecular analysis of recombinant C0-C2 protein fragments revealed that c-MYBPC3 variants alter the C0-C2 domain secondary structure, thermodynamic stability and importantly, result in a reduced binding affinity to cardiac actin. V5 (double mutant), displayed the greatest protein instability with concomitant loss of actin-binding function. Our study provides specific mechanistic insight into how c-MYBPC3 pathogenic variants alter both functional and structural characteristics of C0-C2 domains leading to impaired actin interaction and reduced contractility, which may provide a basis for elucidating the disease mechanism in HCM patients with c- MYBPC3 mutations., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Aims: Familial partial lipodystrophic syndrome 3 (FPLD3) is associated with mutations in the transcription factor PPARγ. One of these mutations, the P467L, confers a dominant negative effect. We and others have previously investigated the pathophysiology associated with this mutation using a humanized mouse model that recapitulates most of the clinical symptoms observed in patients who have been phenotyped under different experimental conditions. One of the key clinical manifestations observed, both in humans and mouse models, is the ectopic accumulation of fat in the liver. With this study we aim to dissect the molecular mechanisms that contribute to the excessive accumulation of lipids in the liver and characterize the negative effect of this PPARγ mutation on the activity of PPARα in vivo when activated by fibrates., Material and Methods: P465L-PPAR mutant and wild-type mice were divided into 8 experimental groups, 4 different conditions per genotype. Briefly, mice were fed a chow diet or a high-fat diet (HFD 45% Kcal from fat) for a period of 28 days and treated with WY14643 or vehicle for five days before culling. At the end of the experiment, tissues and plasma were collected. We performed extensive gene expression, fatty acid composition and histological analysis in the livers. The serum collected was used to measure several metabolites and to perform basic lipoprotein profile., Results: P465L mice showed increased levels of insulin and free fatty acids (FFA) as well as increased liver steatosis. They also exhibit decreased levels of very low density lipoproteins (VLDL) when fed an HFD. We also provide evidence of impaired expression of a number of well-established PPARα target genes in the P465L mutant livers., Conclusion: Our data demonstrate that P465L confers partial resistance to the hypolipidemic action of fibrates. These results show that the fatty liver phenotype observed in P465L mutant mice is not only the consequence of dysfunctional adipose tissue, but also involves defective liver metabolism. All in all, the deleterious effects of P465L-PPARγ mutation may be magnified by their collateral negative effect on PPARα function., (© 2018 John Wiley & Sons Ltd.)

Melanopsin is a blue light-sensitive opsin photopigment involved in a range of non-image forming behaviours, including circadian photoentrainment and the pupil light response. Many naturally occurring genetic variants exist within the human melanopsin gene (OPN4), yet it remains unclear how these variants affect melanopsin protein function and downstream physiological responses to light. Here, we have used bioinformatic analysis and in vitro expression systems to determine the functional phenotypes of missense human OPN4 variants. From 1242 human OPN4 variants collated in the NCBI Short Genetic Variation database (dbSNP), we identified 96 that lead to non-synonymous amino acid substitutions. These 96 missense mutations were screened using sequence alignment and comparative approaches to select 16 potentially deleterious variants for functional characterisation using calcium imaging of melanopsin-driven light responses in HEK293T cells. We identify several previously uncharacterised OPN4 mutations with altered functional properties, including attenuated or abolished light responses, as well as variants demonstrating abnormal response kinetics. These data provide valuable insight into the structure-function relationships of human melanopsin, including several key functional residues of the melanopsin protein. The identification of melanopsin variants with significantly altered function may serve to detect individuals with disrupted melanopsin-based light perception, and potentially highlight those at increased risk of sleep disturbance, circadian dysfunction, and visual abnormalities.

Protamines (PRMs), important proteins of chromatin condensation in spermiogenesis, are promising candidate genes to explore markers of sperm motility. The coding and in-silico predicted promoter regions of these genes were investigated in 102 crossbred and 32 purebred cattle. Also, mRNA quantification was done to explore its possibility as diagnostic tool of infertility. The PCR-SSCP analysis indicated there were two band patterns only in fragment I of the PRM1 and fragment II of the PRM2 gene. The sequence analysis revealed A152G and G179A transitions in the PRM1 gene. Similarly, G35A, A49G and A64G transitions were identified in the PRM2 gene which resulted in altered amino acid sequences from arginine (R) to glutamine (Q), from arginine (R) to glycine (G) and from arginine (R) to glycine (G), respectively. This caused the reduction in molecular weight of PRM2 from 2157.66 to 1931.33 Da due to reduction in the number of basic amino acids. These altered properties of the PRM2 protein led to the reduction in Mass Motility (MM: P < 0.01), Initial Progressive Motility (IPM; P < 0.05) and Post Thaw Motility (PTM; P < 0.05) in crossbred bulls. The least squares analysis of variance indicated there was an effect of PRM2 haplotypes on MM (P = 0.0069), IPM (P = 0.0306) and PTM (P = 0.0500) in crossbred cattle and on PTM (P = 0.0408) in the overall cattle population. Based on the RT-qPCR analysis, however, there was not any significant variation of PRM1 and PRM2 gene expression among sperm of Vrindavani bulls with relatively lesser and greater sperm motility., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Objective: To study the functional properties of six novel missense mutations of the NR5A1 gene encoding the steroidogenic factor 1 (SF-1) identified in six patients with 46,XY disorders of sex development (DSD) and to describe their relative phenotype-genotype relationship., Design: Genetic and functional studies., Setting: University department., Patient(s): Six 46,XY DSD patients., Intervention(s): None., Main Outcome Measure(s): Sanger sequencing and multiplex ligation-dependent probe amplification analysis to identify the mutations or deletions/duplications of the NR5A1 gene. Functional studies by transactivation assays to predict the impact of mutations on molecular function., Result(s): NR5A1 exons sequencing identified in six 46,XY DSD patients six novel mutations: p.T40R, p.T47C, p.G328W, p.A351E, p.R427W, and p.Q460R. Five missense variants were heterozygous, and one was homozygous (p.R427W). Functional analysis revealed a significant loss of DNA-binding and transactivation ability for all variants, except for p.Q460R, which showed a modest reduced activity compared with that of the wild-type protein. Phenotypes associated with these mutations varied from males with spontaneous puberty, substantial T production, and possible fertility, to females with and without müllerian structures and primary amenorrhea., Conclusion(s): We describe six novel mutations in NR5A1 gene and showed that they might affect protein structure, therefore compromising seriously the SF-1 role in regulating gonadal development. Clinically, we suggest that NR5A1 analysis should be performed whenever atypical sex organs are evidenced or there is an abnormal sexual development, to have proper diagnosis and better management of patients., (Copyright © 2018 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Empathy is crucial for our emotional experience and social interactions, and its abnormalities manifest in various psychiatric disorders. Observational fear is a useful behavioral paradigm for assessing affective empathy in rodents. However, specific genes that regulate observational fear remain unknown. Here we showed that 129S1/SvImJ mice carrying a unique missense variant in neurexin 3 (Nrxn3) exhibited a profound and selective enhancement in observational fear. Using the CRISPR/Cas9 system, the arginine-to-tryptophan (R498W) change in Nrxn3 was confirmed to be the causative variant. Selective deletion of Nrxn3 in somatostatin-expressing (SST+) interneurons in the anterior cingulate cortex (ACC) markedly increased observational fear and impaired inhibitory synaptic transmission from SST+ neurons. Concordantly, optogenetic manipulation revealed that SST+ neurons in the ACC bidirectionally controlled the degree of socially transmitted fear. Together, these results provide insights into the genetic basis of behavioral variability and the neurophysiological mechanism controlling empathy in mammalian brains., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The production of cytokines in response to DNA-damage events may be an important host defense response to help prevent the escape of pre-cancerous cells. The innate immune pathways involved in these events are known to be regulated by cellular molecules such as stimulator of interferon genes (STING), which controls type I interferon and pro-inflammatory cytokine production in response to the presence of microbial DNA or cytosolic DNA that has escaped from the nucleus. STING signaling has been shown to be defective in a variety of cancers, such as colon cancer and melanoma, actions that may enable damaged cells to escape the immunosurveillance system. Here, we report through examination of databases that STING signaling may be commonly suppressed in a greater variety of tumors due to loss-of-function mutation or epigenetic silencing of the STING/cGAS promoter regions. In comparison, RNA activated innate immune pathways controlled by RIG-I/MDA5 were significantly less affected. Examination of reported missense STING variants confirmed that many exhibited a loss-of-function phenotype and could not activate cytokine production following exposure to cytosolic DNA or DNA-damage events. Our data imply that the STING signaling pathway may be recurrently suppressed by a number of mechanisms in a considerable variety of malignant disease and be a requirement for cellular transformation.

N-glycosylation is a common posttranslational modification of secreted and membrane proteins, catalyzed by the two enzymatic isoforms of the oligosaccharyltransferase, STT3A and STT3B. Missense mutations are the most common mutations in inherited diseases; however, missense mutations that generate extra, non-native N-glycosylation sites have not been well characterized. Coagulation factor VIII (FVIII) contains five consensus N-glycosylation sites outside its functionally dispensable B domain. We developed a computer program that identified hemophilia A mutations in FVIII that can potentially create ectopic glycosylation sites. We determined that 18 of these ectopic sites indeed become N-glycosylated. These sites span the domains of FVIII and are primarily associated with a severe disease phenotype. Using STT3A and STT3B knockout cells, we determined that ectopic glycosylation exhibited different degrees of dependence on STT3A and STT3B. By separating the effects of ectopic N-glycosylation from those due to underlying amino acid changes, we showed that ectopic glycans promote the secretion of some mutants, but impair the secretion of others. However, ectopic glycans that enhanced secretion could not functionally replace a native N -glycan in the same domain. Secretion-deficient mutants, but not mutants with elevated secretion levels, show increased association with the endoplasmic reticulum chaperones BiP (immunoglobulin heavy chain-binding protein) and calreticulin. Though secreted to different extents, all studied mutants exhibited lower relative activity than wild-type FVIII. Our results reveal differential impacts of ectopic N-glycosylation on FVIII folding, trafficking and activity, which highlight complex disease-causing mechanisms of FVIII missense mutations. Our findings are relevant to other secreted and membrane proteins with mutations that generate ectopic N -glycans., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Splicing factor mutations are characteristic of myelodysplastic syndromes (MDS) and related myeloid neoplasms and implicated in their pathogenesis, but their roles in the development of MDS have not been fully elucidated. In the present study, we investigated the consequence of mutant Srsf2 expression using newly generated Vav1-Cre -mediated conditional knockin mice. Mice carrying a heterozygous Srsf2 P95H mutation showed significantly reduced numbers of hematopoietic stem and progenitor cells (HSPCs) and differentiation defects both in the steady-state condition and transplantation settings. Srsf2 -mutated hematopoietic stem cells (HSCs) showed impaired long-term reconstitution compared with control mice in competitive repopulation assays. Although the Srsf2 mutant mice did not develop MDS under the steady-state condition, when their stem cells were transplanted into lethally irradiated mice, the recipients developed anemia, leukopenia, and erythroid dysplasia, which suggests the role of replicative stress in the development of an MDS-like phenotype in Srsf2 -mutated mice. RNA sequencing of the Srsf2 -mutated HSPCs revealed a number of abnormal splicing events and differentially expressed genes, including several potential targets implicated in the pathogenesis of hematopoietic malignancies, such as Csf3r , Fyn , Gnas , Nsd1 , Hnrnpa2b1 , and Trp53bp1 Among the mutant Srsf2 -associated splicing events, most commonly observed were the enhanced inclusion and/or exclusion of cassette exons, which were caused by the altered consensus motifs for the recognition of exonic splicing enhancers. Our findings suggest that the mutant Srsf2 leads to a compromised HSC function by causing abnormal RNA splicing and expression, contributing to the deregulated hematopoiesis that recapitulates the MDS phenotypes, possibly as a result of additional genetic and/or environmental insults., (© 2018 by The American Society of Hematology.)

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that have common molecular and pathogenic characteristics, such as aberrant accumulation and ubiquitylation of TDP-43; however, the mechanisms that drive this process remain poorly understood. We have recently identified CCNF mutations in familial and sporadic ALS and FTD patients. CCNF encodes cyclin F, a component of an E3 ubiquitin-protein ligase (SCF cyclin F ) complex that is responsible for ubiquitylating proteins for degradation by the ubiquitin-proteasome system. In this study, we examined the ALS/FTD-causing p.Ser621Gly (p.S621G) mutation in cyclin F and its effect upon downstream Lys48-specific ubiquitylation in transfected Neuro-2A and SH-SY5Y cells. Expression of mutant cyclin F S621G caused increased Lys48-specific ubiquitylation of proteins in neuronal cells compared to cyclin F WT . Proteomic analysis of immunoprecipitated Lys48-ubiquitylated proteins from mutant cyclin F S621G -expressing cells identified proteins that clustered within the autophagy pathway, including sequestosome-1 (p62/SQSTM1), heat shock proteins, and chaperonin complex components. Examination of autophagy markers p62, LC3, and lysosome-associated membrane protein 2 (Lamp2) in cells expressing mutant cyclin F S621G revealed defects in the autophagy pathway specifically resulting in impairment in autophagosomal-lysosome fusion. This finding highlights a potential mechanism by which cyclin F interacts with p62, the receptor responsible for transporting ubiquitylated substrates for autophagic degradation. These findings demonstrate that ALS/FTD-causing mutant cyclin F S621G disrupts Lys48-specific ubiquitylation, leading to accumulation of substrates and defects in the autophagic machinery. This study also demonstrates that a single missense mutation in cyclin F causes hyper-ubiquitylation of proteins that can indirectly impair the autophagy degradation pathway, which is implicated in ALS pathogenesis.

The middle disordered hydrophobic region of the prion protein plays a critical role in conformational conversion of the protein, with pathogenic as well as protective mutations being localized to this region. In particular, it has been shown that the G127V mutation in this region of the human prion protein (huPrP) is protective against the spread of prion disease, but the mechanism of protection remains unknown. In this study, quantitative analyses of the kinetics of fibril formation by wild-type mouse prion protein (moPrP) and G126V moPrP (equivalent to G127V huPrP) reveal important differences: the critical concentration is higher, the lag phase is longer, and the initial effective rate constant of fibril growth is slower for the mutant variant. The study offers a simple biophysical explanation for why the G127V mutation in huPrP would be protective in humans: the ∼5-fold increase in critical concentration caused by the mutation likely results in the critical concentration (below which fibril formation cannot occur) being higher that the concentration of the protein present in and on cells in vivo.

Ribosomal protein synthesis results in the genetically programmed incorporation of amino acids into a growing polypeptide chain. Faithful amino acid incorporation that accurately reflects the genetic code is critical to the structure and function of proteins as well as overall proteome integrity. Errors in protein synthesis are generally detrimental to cellular processes yet emerging evidence suggest that proteome diversity generated through mistranslation may be beneficial under certain conditions. Cumulative translational error rates have been determined at the organismal level, however codon specific error rates and the spectrum of misincorporation errors from system to system remain largely unexplored. In particular, until recently technical challenges have limited the ability to detect and quantify comparatively rare amino acid misincorporation events, which occur orders of magnitude less frequently than canonical amino acid incorporation events. We now describe a technique for the quantitative analysis of amino acid incorporation that provides the sensitivity necessary to detect mistranslation events during translation of a single codon at frequencies as low as 1 in 10,000 for all 20 proteinogenic amino acids, as well as non-proteinogenic and modified amino acids. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Despite having physiological functions completely different from superoxide dismutase 1 (SOD1), profilin 1 (PFN1) also carries mutations causing amyotrophic lateral sclerosis (ALS) with a striking similarity to that triggered by SOD1 mutants. Very recently, the C71G-PFN1 has been demonstrated to cause ALS by a gain of toxicity and the acceleration of motor neuron degeneration preceded the accumulation of its aggregates. Here by atomic-resolution NMR determination of conformations and dynamics of WT-PFN1 and C71G-PFN1 in aqueous buffers and in membrane mimetics DMPC/DHPC bicelle and DPC micelle, we deciphered that: 1) the thermodynamic destabilization by C71G transforms PFN1 into coexistence with the unfolded state, which is lacking of any stable tertiary/secondary structures as well as restricted ps-ns backbone motions, thus fundamentally indistinguishable from ALS-causing SOD1 mutants. 2) Most strikingly, while WT-PFN1 only weakly interacts with DMPC/DHPC bicelle without altering the native structure, C71G-PFN1 acquires abnormal capacity in strongly interacting with DMPC/DHPC bicelle and DPC micelle, energetically driven by transforming the highly disordered unfolded state into a non-native helical structure, similar to what has been previously observed on ALS-causing SOD1 mutants. Our results imply that one potential mechanism for C71G-PFN1 to initiate ALS might be the abnormal interaction with membranes as recently established for SOD1 mutants., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Regulator of G protein signaling 2 (RGS2) plays a significant role in alleviating vascular contraction and promoting vascular relaxation due to its GTPase accelerating protein activity toward G α q. Mice lacking RGS2 display a hypertensive phenotype, and several RGS2 missense mutations have been found predominantly in hypertensive human subjects. However, the mechanisms whereby these mutations could impact blood pressure is unknown. Here, we selected 16 rare, missense mutations in RGS2 identified in various human exome sequencing projects and evaluated their ability to inhibit intracellular calcium release mediated by angiotensin II receptor type 1 (AT1R). Four of them had reduced function and were further investigated to elucidate underlying mechanisms. Low protein expression, protein mislocalization, and reduced G protein binding were identified as likely mechanisms of the malfunctioning mutants. The Q2L mutant had 50% lower RGS2 than wild-type (WT) protein detected by Western blot. Confocal microscopy demonstrated that R44H and D40Y had impaired plasma membrane targeting; only 46% and 35% of those proteins translocated to the plasma membrane when coexpressed with G α q Q209L compared with 67% for WT RGS2. The R188H mutant had a significant reduction in G α q binding affinity (10-fold increase in K i compared with WT RGS2 in a flow cytometry competition binding assay). This study provides functional data for 16 human RGS2 missense variants on their effects on AT1R-mediated calcium mobilization and provides molecular understanding of those variants with functional loss in vitro. These molecular behaviors can provide insight to inform antihypertensive therapeutics in individuals with variants having reduced function., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

The phosphatase and tensin homolog (PTEN) gene plays a crucial role in signal transduction by negatively regulating the PI3K signaling pathway. It is the most frequent mutated gene in many human-related cancers. Considering its critical role, a functional analysis of missense mutations of PTEN gene was undertaken in this study. Thirty five nonsynonymous single nucleotide polymorphisms (nsSNPs) within the coding region of the PTEN gene were selected for our in silico investigation, and five nsSNPs (G129E, C124R, D252G, H61D, and R130G) were found to be deleterious based on combinatorial predictions of different computational tools. Moreover, molecular dynamics (MD) simulation was performed to investigate the conformational variation between native and all the five mutant PTEN proteins having predicted deleterious nsSNPs. The results of MD simulation of all mutant models illustrated variation in structural attributes such as root-mean-square deviation, root-mean-square fluctuation, radius of gyration, and total energy; which depicts the structural stability of PTEN protein. Furthermore, mutant PTEN protein structures also showed a significant variation in the solvent accessible surface area and hydrogen bond frequencies from the native PTEN structure. In conclusion, results of this study have established the deleterious effect of the all the five predicted nsSNPs on the PTEN protein structure. Thus, results of the current study can pave a new platform to sort out nsSNPs that can be undertaken for the confirmation of their phenotype and their correlation with diseased status in case of control studies., (© 2016 International Union of Biochemistry and Molecular Biology, Inc.)

Functional impairments or trafficking defects of inhibitory glycine receptors (GlyRs) have been linked to human hyperekplexia/startle disease and autism spectrum disorders. We found that a lack of synaptic integration of GlyRs, together with disrupted receptor function, is responsible for a lethal startle phenotype in a novel spontaneous mouse mutant shaky , caused by a missense mutation, Q177K, located in the extracellular β8-β9 loop of the GlyR α1 subunit. Recently, structural data provided evidence that the flexibility of the β8-β9 loop is crucial for conformational transitions during opening and closing of the ion channel and represents a novel allosteric binding site in Cys-loop receptors. We identified the underlying neuropathological mechanisms in male and female shaky mice through a combination of protein biochemistry, immunocytochemistry, and both in vivo and in vitro electrophysiology. Increased expression of the mutant GlyR α1 Q177K subunit in vivo was not sufficient to compensate for a decrease in synaptic integration of α1 Q177K β GlyRs. The remaining synaptic heteromeric α1 Q177K β GlyRs had decreased current amplitudes with significantly faster decay times. This functional disruption reveals an important role for the GlyR α1 subunit β8-β9 loop in initiating rearrangements within the extracellular-transmembrane GlyR interface and that this structural element is vital for inhibitory GlyR function, signaling, and synaptic clustering. SIGNIFICANCE STATEMENT GlyR dysfunction underlies neuromotor deficits in startle disease and autism spectrum disorders. We describe an extracellular GlyR α1 subunit mutation (Q177K) in a novel mouse startle disease mutant shaky Structural data suggest that during signal transduction, large transitions of the β8-β9 loop occur in response to neurotransmitter binding. Disruption of the β8-β9 loop by the Q177K mutation results in a disruption of hydrogen bonds between Q177 and the ligand-binding residue R65. Functionally, the Q177K change resulted in decreased current amplitudes, altered desensitization decay time constants, and reduced GlyR clustering and synaptic strength. The GlyR β8-β9 loop is therefore an essential regulator of conformational rearrangements during ion channel opening and closing., (Copyright © 2017 Schaefer, Berger et al.)

Progressive encephalopathy with oedema, hypsarrhythmia, and optic atrophy (PEHO) syndrome is an early childhood onset, severe autosomal recessive encephalopathy characterized by extreme cerebellar atrophy due to almost total granule neuron loss. By combining homozygosity mapping in Finnish families with Sanger sequencing of positional candidate genes and with exome sequencing a homozygous missense substitution of leucine for serine at codon 31 in ZNHIT3 was identified as the primary cause of PEHO syndrome. ZNHIT3 encodes a nuclear zinc finger protein previously implicated in transcriptional regulation and in small nucleolar ribonucleoprotein particle assembly and thus possibly to pre-ribosomal RNA processing. The identified mutation affects a highly conserved amino acid residue in the zinc finger domain of ZNHIT3. Both knockdown and genome editing of znhit3 in zebrafish embryos recapitulate the patients' cerebellar defects, microcephaly and oedema. These phenotypes are rescued by wild-type, but not mutant human ZNHIT3 mRNA, suggesting that the patient missense substitution causes disease through a loss-of-function mechanism. Transfection of cell lines with ZNHIT3 expression vectors showed that the PEHO syndrome mutant protein is unstable. Immunohistochemical analysis of mouse cerebellar tissue demonstrated ZNHIT3 to be expressed in proliferating granule cell precursors, in proliferating and post-mitotic granule cells, and in Purkinje cells. Knockdown of Znhit3 in cultured mouse granule neurons and ex vivo cerebellar slices indicate that ZNHIT3 is indispensable for granule neuron survival and migration, consistent with the zebrafish findings and patient neuropathology. These results suggest that loss-of-function of a nuclear regulator protein underlies PEHO syndrome and imply that establishment of its spatiotemporal interaction targets will be the basis for developing therapeutic approaches and for improved understanding of cerebellar development., (© The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Human cytochrome P450 11B2 (CYP11B2) is an essential enzyme in the steroid hormone biosynthesis, which catalyzes the last three reaction steps of the aldosterone synthesis. These reactions comprise a hydroxylation at position C11 of the steroid intermediate deoxycorticosterone yielding corticosterone, followed by a hydroxylation at position C18 yielding 18-hydroxy-corticosterone and a subsequent oxidation of the hydroxyl group at C18, which results in the formation of aldosterone. Alterations in the amino acid sequence of CYP11B2 often cause severe disease patterns. We previously described a procedure for expression and purification of human CYP11B2 employing recombinant E. coli, which allows the rapid characterization of CYP11B2 mutants on a molecular level. This system was now utilized for the examination of the influence of the polymorphism at position 173 in combination with the mutation V386A on the activity of CYP11B2. Our in vitro findings show that the combination of the V386A mutation with the variant CYP11B2 173 Arg only slightly reduces the 18-hydroxylase and 18-oxidase activity, whereas the V386A mutation with the CYP11B2 173 Lys variant almost abolishes the 18-hydroxylation and 18-oxidation. In both cases the 11-hydroxylase activity is not affected. These findings highlight the importance of the genetic background of an enzyme when regarding the effect of clinical mutations.

Hemoglobin (Hb) is a potent oxidant outside the erythrocyte. The tyrosines α140 and β145 play an important role in the structure and function of Hb by forming switch and hinge contacts. These carboxy-terminal residues of the alpha and beta chains, respectively, were replaced to phenylalanine and several different methods were used to characterize the obtained mutants including a comet and plasmid DNA cleavage assay. It was observed that the genotoxic effect was 40% higher for αY140F compared with the wildtype, the βY145F and the double (αY140/β145F) mutants as determined by the comet assay. Cleavage of purified plasmid DNA after Hb application also revealed that the αY140F mutant showed 2-fold higher activity, while the βY145F and αY140/β145F mutants reduced the activity compared to wildtype Hb. This study clearly indicates that the penultimate tyrosines are involved in the genotoxicity of Hb.

Interleukin-10 receptor [IL10R] mutations are associated with severe childhood inflammatory bowel disease [IBD]. Two unrelated patients who died of very early-onset severe IBD and sepsis were identified as harbouring the same compound heterozygous mutations in IL10RA [p.R101W; p.T179T]. A third patient was found to be homozygous for p.T179T. The missense change of p.R101W has been reported. The synonymous change of p.T179T, with a minor allele frequency of 0.035% in the population, was novel. The p.T179T mutation was located before the 5' splice donor site, leading to exon skipping and out-of-frame fusion of exons 3 and 5, causing altered STAT3 phosphorylation in IL10-induced peripheral blood mononuclear cells. The patient developed colitis at 6 years of age, the oldest reported age of onset among patients with IL10RA mutations, and did not suffer from perianal disease. We report three paediatric patients with a rare, synonymous p.T179T variant causing a splicing error in IL10RA., (Copyright © 2016 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Inherited retinal disorders (IRDs) result in severe visual impairments in children and adults. A challenge in the field of retinal degenerations is identifying mechanisms of photoreceptor cell death related to specific genetic mutations. Mutations in the gene TULP1 have been associated with two forms of IRDs, early-onset retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). TULP1 is a cytoplasmic, membrane-associated protein shown to be involved in transportation of newly synthesized proteins destined for the outer segment compartment of photoreceptor cells; however, how mutant TULP1 causes cell death is not understood. In this study, we provide evidence that common missense mutations in TULP1 express as misfolded protein products that accumulate within the endoplasmic reticulum (ER) causing prolonged ER stress. In an effort to maintain protein homeostasis, photoreceptor cells then activate the unfolded protein response (UPR) complex. Our results indicate that the two major apoptotic arms of the UPR pathway, PERK and IRE1, are activated. Additionally, we show that retinas expressing mutant TULP1 significantly upregulate the expression of CHOP, a UPR signaling protein promoting apoptosis, and undergo photoreceptor cell death. Our study demonstrates that the ER-UPR, a known mechanism of apoptosis secondary to an overwhelming accumulation of misfolded protein, is involved in photoreceptor degeneration caused by missense mutations in TULP1. These observations suggest that modulating the UPR pathways might be a strategy for therapeutic intervention.

Background: Missense mutation of VHL gene is frequently detected in type 2 VHL diseases and linked to a wide range of pVHL functions and stability. Certain mutant pVHLs retain ability to regulate HIFs but lose their function by instability. In this case, regulating of degradation of mutant pVHLs, can be postulated as therapeutic method., Method: The stability and cellular function of missense mutant pVHLs were determine in HEK293T transient expressing cell and 786-O stable cell line. Ubiquitination assay of mutant VHL proteins was performed in vitro system. Anticancer effect of adenovirus mediated shUCP expressing was evaluated using ex vivo mouse xenograft assay., Results: Three VHL missense mutants (V155A, L158Q, and Q164R) are directly ubiquitinated by E2-EPF UCP (UCP) in vitro. Mutant pVHLs are more unstable than wild type in cell. Missense mutant pVHLs interact with UCP directly in both in vitro and cellular systems. Lacking all of lysine residues of pVHL result in resistance to ubiquitination thereby increase its stability. Missense mutant pVHLs maintained the function of E3 ligase to ubiquitinate HIF-1α in vitro. In cells expressing mutant pVHLs, Glut-1 and VEGF were relatively upregulated compared to their levels in cells expressing wild-type. Depletion of UCP restored missense mutant pVHLs levels and inhibited cell growth. Adenovirus-mediated shUCP RNA delivery inhibited tumor growth in ex vivo mouse xenograft model., Conclusion: These data suggest that targeting of UCP can be one of therapeutic method in type 2 VHL disease caused by unstable but functional missense mutant pVHL.

Objectives: To elucidate the involvement of MYO6 mutations, known to be responsible for DFNA22/DFNB37, in Japanese hearing loss patients through the use of genetic analysis., Methods: Genomic variations responsible for hearing loss were identified by massively parallel DNA sequencing (MPS) of 63 target candidate genes in 1120 Japanese hearing loss patients, and the detailed clinical features for the patients with MYO6 mutations were collected and analyzed., Results: Four mutations were successfully found in 7 families exhibiting autosomal dominant inheritance. All of the patients showed progressive hearing loss, but hearing type and onset age varied. Further, none of the affected patients showed any associated symptoms, such as hypertrophic cardiomyopathy or retinitis pigmentosa., Conclusions: MPS is powerful tool for the identification of rare causative deafness gene mutations, such as MYO6. The clinical characteristics noted in the present study not only confirmed the findings of previous reports but provided important new clinical information., (© The Author(s) 2015.)

Gain-of-toxic-function mutations in Seipin (Asparagine 88 to Serine (N88S) and Serine 90 to Leucine (S90L) mutations, both of which disrupt the N-glycosylation) cause autosomal dominant motor neuron diseases. However, the mechanism of how these missense mutations lead to motor neuropathy is unclear. Here, we analyze the impact of disruption of N-glycosylation of Seipin on synaptic transmission by over-expressing mutant Seipin in cultured cortical neurons via lentiviral infection. Immunostaining shows that over-expressed Seipin is partly colocalized with synaptic vesicle marker synaptophysin. Electrophysiological recordings reveal that the Seipin mutation significantly decreases the frequency, but not the amplitudes of miniature excitatory post-synaptic currents and miniature inhibitory post-synaptic currents. The amplitude of both evoked excitatory post-synaptic currents and inhibitory post-synaptic current is also compromised by mutant Seipin over-expression. The readily releasable pool and vesicular release probability of synaptic vesicles are both altered in neurons over-expressing Seipin-N88S, whereas neither γ-amino butyric acid (GABA) nor α-Amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA) induced whole cell currents are affected. Moreover, electron microscopy analysis reveals decreased number of morphologically docked synaptic vesicles in Seipin-N88S-expressing neurons. These data demonstrate that Seipin-N88S mutation impairs synaptic neurotransmission, possibly by regulating the priming and docking of synaptic vesicles at the synapse., (© 2013 International Society for Neurochemistry.)

The protein kinase C γ (PKCγ) undergoes multistep activation and participates in various cellular processes in Purkinje cells. Perturbations in its phosphorylation state, conformation or localization can disrupt kinase signalling, such as in spinocerebellar ataxia type 14 (SCA14) that is caused by missense mutations in PRKCG encoding for PKCγ. We previously showed that SCA14 mutations enhance PKCγ membrane translocation upon stimulation owing to an altered protein conformation. As the faster translocation did not result in an increased function, we examined how SCA14 mutations induce this altered conformation of PKCγ and what the consequences of this conformational change are on PKCγ life cycle. Here, we show that SCA14-related PKCγ-V138E exhibits an exposed C-terminus as shown by fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy in living cells, indicative of its partial unfolding. This conformational change was associated with faster phorbol 12-myristate 13-acetate-induced translocation and accumulation of fully phosphorylated PKCγ in the insoluble fraction, which could be rescued by coexpressing PDK1 kinase that normally triggers PKCγ autophosphorylation. We propose that the SCA14 mutation V138E causes unfolding of the C1B domain and exposure of the C-terminus of the PKCγ-V138E molecule, resulting in a decrease of functional kinase in the soluble fraction. Here, we show that the mutation V138E of the protein kinase C γ (PKCγ) C1B domain (PKCγ-V138E), which is implicated in spinocerebellar ataxia type 14, exhibits a partially unfolded C-terminus. This leads to unusually fast phorbol 12-myristate 13-acetate-induced membrane translocation and accumulation of phosphorylated PKCγ-V138E in the insoluble fraction, causing loss of the functional kinase. In contrast to general chaperones, coexpression of PKCγ's 'natural chaperone', PDK1 kinase, could rescue the PKCγ-V138E phenotype., (© 2013 International Society for Neurochemistry.)