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Pre-mRNA splicing must occur with extremely high fidelity. Spliceosomes assemble onto pre-mRNA guided by specific sequences (5' splice site, 3' splice site, and branchpoint). When splice sites are mutated, as in many hereditary diseases, the spliceosome can aberrantly select nearby pseudo- or "cryptic" splice sites, often resulting in nonfunctional protein. How the spliceosome distinguishes authentic splice sites from cryptic splice sites is poorly understood. We performed a Caenorhabditis elegans genetic screen to find cellular factors that affect the frequency with which the spliceosome uses cryptic splice sites and identified two alleles in core spliceosome component Prp8 that alter cryptic splicing frequency. Subsequent complementary genetic and structural analyses in yeast implicate these alleles in the stability of the spliceosome's catalytic core. However, despite a clear effect on cryptic splicing, high-throughput mRNA sequencing of these prp-8 mutant C. elegans reveals that overall alternative splicing patterns are relatively unchanged. Our data suggest the spliceosome evolved intrinsic mechanisms to reduce the occurrence of cryptic splicing and that these mechanisms are distinct from those that impact alternative splicing.

A class of translation inhibitors, exemplified by the natural product rocaglamide A (RocA), isolated from Aglaia genus plants, exhibits antitumor activity by clamping eukaryotic translation initiation factor 4A (eIF4A) onto polypurine sequences in mRNAs. This unusual inhibitory mechanism raises the question of how the drug imposes sequence selectivity onto a general translation factor. Here, we determined the crystal structure of the human eIF4A1⋅ATP analog⋅RocA⋅polypurine RNA complex. RocA targets the "bi-molecular cavity" formed characteristically by eIF4A1 and a sharply bent pair of consecutive purines in the RNA. Natural amino acid substitutions found in Aglaia eIF4As changed the cavity shape, leading to RocA resistance. This study provides an example of an RNA-sequence-selective interfacial inhibitor fitting into the space shaped cooperatively by protein and RNA with specific sequences.

Inherited retinal degenerations are a common cause of untreatable blindness worldwide, with retinitis pigmentosa and cone dystrophy affecting approximately 1 in 3500 and 1 in 10,000 individuals, respectively. A major limitation to the development of effective therapies is the lack of availability of animal models that fully replicate the human condition. Particularly for cone disorders, rodent, canine, and feline models with no true macula have substantive limitations. By contrast, the cone-rich macula of a nonhuman primate (NHP) closely mirrors that of the human retina. Consequently, well-defined NHP models of heritable retinal diseases, particularly cone disorders that are predictive of human conditions, are necessary to more efficiently advance new therapies for patients. We have identified 4 related NHPs at the California National Primate Research Center with visual impairment and findings from clinical ophthalmic examination, advanced retinal imaging, and electrophysiology consistent with achromatopsia. Genetic sequencing confirmed a homozygous R565Q missense mutation in the catalytic domain of PDE6C, a cone-specific phototransduction enzyme associated with achromatopsia in humans. Biochemical studies demonstrate that the mutant mRNA is translated into a stable protein that displays normal cellular localization but is unable to hydrolyze cyclic GMP (cGMP). This NHP model of a cone disorder will not only serve as a therapeutic testing ground for achromatopsia gene replacement, but also for optimization of gene editing in the macula and of cone cell replacement in general.

Retinal degeneration 3 (RD3) protein promotes accumulation of retinal membrane guanylyl cyclase (RetGC) in the photoreceptor outer segment and suppresses RetGC activation by guanylyl cyclase-activating proteins (GCAPs). Mutations truncating RD3 cause severe congenital blindness by preventing the inhibitory binding of RD3 to the cyclase. The high propensity of RD3 to aggregate in solution has prevented structural analysis. Here, we produced a highly soluble variant of human RD3 (residues 18-160) that is monomeric and can still bind and negatively regulate RetGC. The NMR solution structure of RD3 revealed an elongated backbone structure (70 Å long and 30 Å wide) consisting of a four-helix bundle with a long unstructured loop between helices 1 and 2. The structure reveals that RD3 residues previously implicated in the RetGC binding map to a localized and contiguous area on the structure, involving a loop between helices 2 and 3 and adjacent parts of helices 3 and 4. The NMR structure of RD3 was validated by mutagenesis. Introducing Trp85 or Phe29 to replace Cys or Leu, respectively, disrupts packing in the hydrophobic core and lowers RD3's apparent affinity for RetGC1. Introducing a positive charge at the interface (Glu32 to Lys) also lowered the affinity. Conversely, introducing Val in place of Cys93 stabilized the hydrophobic core and increased the RD3 affinity for the cyclase. The NMR structure of RD3 presented here provides a structural basis for elucidating RD3-RetGC interactions relevant for normal vision or blindness.

The atomic-level mechanisms that coordinate ligand release from protein pockets are only known for a handful of proteins. Here, we report results from accelerated molecular dynamics simulations for benzene dissociation from the buried cavity of the T4 lysozyme Leu99Ala mutant (L99A). In these simulations, benzene is released through a previously characterized, sparsely populated room-temperature excited state of the mutant, explaining the coincidence for experimentally measured benzene off rate and apo protein slow-timescale NMR relaxation rates between ground and excited states. The path observed for benzene egress is a multistep ligand migration from the buried cavity to ultimate release through an opening between the F/G-, H-, and I-helices and requires a number of cooperative multiresidue and secondary-structure rearrangements within the C-terminal domain of L99A. These rearrangements are identical to those observed along the ground state to excited state transitions characterized by molecular dynamic simulations run on the Anton supercomputer. Analyses of the molecular properties of the residues lining the egress path suggest that protein surface electrostatic potential may play a role in the release mechanism. Simulations of wild-type T4 lysozyme also reveal that benzene-egress-associated dynamics in the L99A mutant are potentially exaggerations of the substrate-processivity-related dynamics of the wild type.

Background: Mannose-binding lectin (MBL) is an innate immune protein with strong biologic plausibility for protecting against influenza virus-related sepsis and bacterial co-infection. In an autopsy cohort of 105 influenza-infected young people, carriage of the deleterious MBL gene MBL2_Gly54Asp("B") mutation was identified in 5 of 8 individuals that died from influenza-methicillin-resistant Staphylococcus aureus (MRSA) co-infection. We evaluated MBL2 variants known to influence MBL levels with pediatric influenza-related critical illness susceptibility and/or severity including with bacterial co-infections. Methods: We enrolled children and adolescents with laboratory-confirmed influenza infection across 38 pediatric intensive care units from November 2008 to June 2016. We sequenced MBL2 "low-producer" variants rs11003125("H/L"), rs7096206("Y/X"), rs1800450Gly54Asp("B"), rs1800451Gly57Glu("C"), rs5030737Arg52Cys("D") in patients and biologic parents. We measured serum levels and compared complement activity in low-producing homozygotes ("B/B," "C/C") to HYA/HYA controls. We used a population control of 1,142 healthy children and also analyzed family trios (PBAT/HBAT) to evaluate disease susceptibility, and nested case-control analyses to evaluate severity. Results: We genotyped 420 patients with confirmed influenza-related sepsis: 159 (38%) had acute lung injury (ALI), 165 (39%) septic shock, and 30 (7%) died. Although bacterial co-infection was diagnosed in 133 patients (32%), only MRSA co-infection (n = 33, 8% overall) was associated with death (p < 0.0001), present in 11 of 30 children that died (37%). MBL2 variants predicted serum levels and complement activation as expected. We found no association between influenza-related critical illness susceptibility and MBL2 variants using family trios (633 biologic parents) or compared to population controls. MBL2 variants were not associated with admission illness severity, septic shock, ALI, or bacterial co-infection diagnosis. Carriage of low-MBL producing MBL2 variants was not a risk factor for mortality, but children that died did have higher carriage of one or more B alleles (OR 2.3; p = 0.007), including 7 of 11 with influenza MRSA-related death (vs. 2 of 22 survivors: OR 14.5, p = 0.0002). Conclusions: MBL2 variants that decrease MBL levels were not associated with susceptibility to pediatric influenza-related critical illness or with multiple measures of critical illness severity. We confirmed a prior report of higher B allele carriage in a relatively small number of young individuals with influenza-MRSA associated death.

BACKGROUND:Osteomyelitis is a bone infection, most often caused by Staphylococcus aureus, in which neutrophils play a key role. Cathepsin G (CTSG) is a bactericidal serine protease stored in the neutrophil azurophilic granules. CTSG regulates inflammation, activating matrix metalloproteinases (MMPs), and coagulation. Lactoferrin (LF), a neutrophil glycoprotein, increases CTSG catalytic activity and induces inflammation. The aim of this study was to analyze a potential association between a CTSG gene polymorphism (Asn125Ser or N125S, rs45567233), that modifies CTSG activity, and could affect susceptibility to, or outcome of, bacterial osteomyelitis. METHODS:CTSG N125S polymorphism was genotyped in 329 osteomyelitis patients and 415 controls), Blood coagulation parameters, serum CTSG activity, LF, MMP-1, MMP-13, and soluble receptor activator for nuclear factor κ B ligand (sRANKL) levels were assessed in carriers of the different CTSG genotypes. RESULTS:CTSG N125S (AG) genotype was significantly more frequent among osteomyelitis patients than controls (15.5% vs. 9.4%, p = 0.014). CTSG N125S variant G allele (AG +GG) was also more frequent among osteomyelitis patients (8.1% vs. 4.7%, p = 0.01). Serum CTSG activity and LF levels were significantly higher in osteomyelitis patients carrying the G allele compared to those with the AA genotype, (p

Homozygosity for the p.C282Y substitution in the HFE protein encoded by the hemochromatosis gene on chromosome 6p (HFE) is a common genetic trait that increases susceptibility to iron overload. McLaren et al. used bivariate mixture modeling to analyze the joint population distribution of transferrin saturation (TS) and serum ferritin concentration (SF) measured for participants in the Hemochromatosis and Iron Overload Screening (HEIRS) Study. They identified four components (C1, C2, C3, and C4) with successively increasing means for TS and SF. They demonstrated that bivariate mixture modeling in TS and SF reflect the genetic locus of HFE and may isolate p.C282Y homozygotes from the general population. In the current study we used data from the another large cohort, the Australian HealthIron study of genetic and environmental modifiers of hereditary hemochromatosis, to validate the component analysis approach, to examine stability of component proportions over time and to determine if TS and SF values from an individual move between components at baseline and follow-up. Because sampling fractions from each p.C282Y / p.H63D genotype stratum are not equal, we used frequency weights based on the inverse of the probability of selection for invitation to participate. In the weighted female analytic cohorts, C4 captured most of C282Y homozygotes, and C2 was the largest component. We identified four components from the weighted male analytic cohort and C4 captured most of p.C282Y homozygotes. The bivariate mixture modeling approach suggested that the model is transferable from one white population to another, although estimated means within components may differ.

Gain-of-function variants in p110δ, the catalytic subunit of phosphatidylinositol 3-kinase (PI3K) expressed in lymphocytes, cause activated PI3-kinase δ syndrome (APDS), a primary immunodeficiency that is characterized by recurrent infections, viremia, lymphadenopathy, and autoimmunity. The mechanism of autoimmunity in APDS has not been well-understood. Here, we show the profound skewing of peripheral CD4+ T cells to a T follicular helper (TFH) phenotype in a patient with APDS bearing a novel p110δ variant, Y524S. We also saw a diminishment of transient Foxp3 expression in activated T cells. Mechanistic studies revealed that both the new variant and a previously described, pathogenic variant (E81K) enhanced an interaction between intracellular Osteopontin and p85α. This interaction had been shown in mice to promote TFH differentiation. Our results demonstrate a new influence of PI3K on human T cell differentiation that is unrelated to its lipid-kinase activity and suggest that TFH should be monitored in APDS patients.

During extracellular electron transfer, Geobacter sulfurreducens constitutively expresses the bc‐cytochrome CbcL, yet cells containing only this menaquinone oxidase fail to respire above −0.1 V vs. SHE. By identifying mutations within cbcL that permit growth at higher potentials, we provide evidence that this cytochrome is regulated by redox potential. Strains expressing only CbcLV205A, CbcLV205G, and CbcLF525Y were capable of growth with high potential electron acceptors including Fe(III) citrate, Mn(IV) oxides, and electrodes poised at +0.1 V vs. SHE. Electrochemical characterization of wild type CbcL revealed oxidative inactivation of electron transfer above −0.1 V, while CbcLV205A, CbcLV205G, and CbcLF525Y remained active. Growth yields of CbcLV205A, CbcLV205G, and CbcLF525Y were only 50 % of WT, consistent with CbcL‐dependent electron transfer conserving less energy. These data support the hypothesis that CbcL has evolved to rapidly shut off in response to redox potential, in order to divert electrons to higher yield oxidases that coexist in the Geobacter membrane. [ABSTRACT FROM AUTHOR]

The relative contribution of mutation and selection to the amino acid substitution rates observed in empirical matrices is unclear. Herein, we present a neutral continuous fitness-stability model, inspired by the Arrhenius law ( q i j = a i j e − Δ Δ G i j ). The model postulates that the rate of amino acid substitution ( i → j ) is determined by the product of a pre-exponential factor, which is influenced by the genetic code structure, and an exponential term reflecting the relative fitness of the amino acid substitutions. To assess the validity of our model, we computed changes in stability of 14,094 proteins, for which 137,073,638 in silico mutants were analyzed. These site-specific data were summarized into a 20 square matrix, whose entries, Δ Δ G i j , were obtained after averaging through all the sites in all the proteins. We found a significant positive correlation between these energy values and the disease-causing potential of each substitution, suggesting that the exponential term accurately summarizes the fitness effect. A remarkable observation was that amino acids that were highly destabilizing when acting as the source, tended to have little effect when acting as the destination, and vice versa (source → destination). The Arrhenius model accurately reproduced the pattern of substitution rates collected in the empirical matrices, suggesting a relevant role for the genetic code structure and a tuning role for purifying selection exerted via protein stability. [ABSTRACT FROM AUTHOR]

The expression of the Fasciola hepatica carboxylesterase type B (CestB) gene is known to be induced upon exposure to the anthelmintic triclabendazole (TCBZ), leading to a substantial rise in enzyme-specific activity. Furthermore, the nucleotide sequence of the CestB gene displays variations that can potentially result in radical amino acid substitutions at the ligand binding site. These substitutions hold the potential to impact both the ligand–protein interaction and the catalytic properties of the enzyme. Thus, the objective of our study was to identify novel CestB polymorphisms in TCBZ-resistant parasites and field isolates obtained from a highly endemic region in Central Mexico. Additionally, we aimed to assess these amino acid polymorphisms using 3D modeling against the metabolically oxidized form of the anthelmintic TCBZSOX. Our goal was to observe the formation of TCBZSOX-specific binding pockets that might provide insights into the role of CestB in the mechanism of anthelmintic resistance. We identified polymorphisms in TCBZ-resistant parasites that exhibited three radical amino acid substitutions at positions 147, 215, and 263. These substitutions resulted in the formation of a TCBZSOX-affinity pocket with the potential to bind the anthelmintic drug. Furthermore, our 3D modeling analysis revealed that these amino acid substitutions also influenced the configuration of the CestB catalytic site, leading to alterations in the enzyme’s interaction with chromogenic carboxylic ester substrates and potentially affecting its catalytic properties. However, it is important to note that the TCBZSOX-binding pocket, while significant for drug binding, was located separate from the enzyme’s catalytic site, rendering enzymatic hydrolysis of TCBZSOX impossible. Nonetheless, the observed increased affinity for the anthelmintic may provide an explanation for a drug sequestration type of anthelmintic resistance. These findings lay the groundwork for the future development of a molecular diagnostic tool to identify anthelmintic resistance in F. hepatica.

Mutations in CNGA3 and CNGB3, the genes encoding the subunits of the tetrameric cone photoreceptor cyclic nucleotide-gated ion channel, cause achromatopsia, a congenital retinal disorder characterized by loss of cone function. However, a small number of patients carrying the CNGB3/c.1208G>A;p.R403Q mutation present with a variable retinal phenotype ranging from complete and incomplete achromatopsia to moderate cone dysfunction or progressive cone dystrophy. By exploring a large patient cohort and published cases, we identified 16 unrelated individuals who were homozygous or (compound-)heterozygous for the CNGB3/c.1208G>A;p.R403Q mutation. In-depth genetic and clinical analysis revealed a co-occurrence of a mutant CNGA3 allele in a high proportion of these patients (10 of 16), likely contributing to the disease phenotype. To verify these findings, we generated a Cngb3R403Q/R403Q mouse model, which was crossbred with Cnga3-deficient (Cnga3-/-) mice to obtain triallelic Cnga3+/- Cngb3R403Q/R403Q mutants. As in human subjects, there was a striking genotype-phenotype correlation, since the presence of 1 Cnga3-null allele exacerbated the cone dystrophy phenotype in Cngb3R403Q/R403Q mice. These findings strongly suggest a digenic and triallelic inheritance pattern in a subset of patients with achromatopsia/severe cone dystrophy linked to the CNGB3/p.R403Q mutation, with important implications for diagnosis, prognosis, and genetic counseling.

Following the 47th American Society of Hematology Meeting in 2005, the late John Goldman and Tariq Mughal commenced a conference, the 1st Post-ASH Workshop, which brought together clinicians and scientists, to accelerate the adoption of new therapies for patients with myeloproliferative neoplasms (MPNs). The concept began with recognition of the CML success story following imatinib therapy, the discovery of JAK2V617F , and the demonstration that BCR-ABL1-negative MPNs are driven by abnormal JAK2 activation. This review is based on the presentations and deliberations at the XIIth Post-ASH Workshop on BCR-ABL1 positive and negative MPNs that took place on December 12 to 13, 2017, in Atlanta, Georgia, immediately following the 59th American Society of Hematology Meeting. We have selected some of the translational research and clinical topics, rather than an account of the proceedings. We discuss the role of immunotherapy in MPNs and the impact of the mutational landscape on TKI treatment in CML. We also consider how we might reduce TKI cardiovascular side effects, the potential role of nutrition as adjunctive nonpharmacologic intervention to reduce chronic inflammation in MPNs, and novel investigational therapies for MPNs.

BackgroundHepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide with limited treatment options. Mutation of β-catenin is one of the most frequent genetic events along hepatocarcinogenesis. β-catenin mutations can be in the form of point mutation or large N-terminal deletion. Studies suggested that different β-catenin mutations might have distinct oncogenic potential.MethodsWe tested the oncogenic activity of β-cateninS45Y, one of the most frequent point mutations of β-catenin, and ∆N90-β-catenin, a form of β-catenin with a large N-terminal deletion, in promoting HCC development in mice. Thus, we co-expressed β-cateninS45Y or ∆N90-β-catenin together with c-Met into the mouse liver using hydrodynamic injection.ResultsWe found that both β-catenin mutations were able to induce HCC formation in combination with c-Met at the same latency and efficiency. Tumors showed similar histological features and proliferation rates. However, immunohistochemistry showed predominantly nuclear staining of β-catenin in c-Met/∆N90-β-catenin HCC, but membrane immunoreactivity in c-Met/β-cateninS45Y HCC. qRT-PCR analysis demonstrated that both ∆N90-β-catenin and β-cateninS45Y induced the same effectors, although at somewhat different levels. In cultured cells, both ∆N90-β-catenin and β-cateninS45Y were capable of inducing TCF/LEF reporter expression, promoting proliferation, and inhibiting apoptosis.ConclusionsOur study suggests that β-cateninS45Y and ∆N90-β-catenin, in combination with the c-Met proto-oncogene, have similar oncogenic potential. Furthermore, nuclear staining of β-catenin does not always characterize β-catenin activity.

BackgroundWe report the successful treatment of the patient with osimertinib 80 mg/day following disease progression and a discordance in the detection of a mechanism of resistance epithelial growth factor receptor (EGFR) T790 M between liquid biopsy and tissue biopsy methods.Case presentationA 57-year-old Hispanic male patient initially diagnosed with an EGFR 19 deletion positive lung adenocarcinoma and clinically responded to initial erlotinib treatment. The patient subsequently progressed on erlotinib 150 mg/day and repeat biopsies both tissue and liquid were sent for next-generation sequencing (NGS). A T790 M EGFR mutation was detected in the blood sample using a liquid biopsy technique, but the tissue biopsy failed to show a T790 M mutation in a newly biopsied tissue sample. He was then successfully treated with osimertinib 80 mg/day, has clinically and radiologically responded, and remains on osimertinib treatment after 10 months.ConclusionsSecond-line osimertinib treatment, when administered at 80 mg/day, is both well tolerated and efficacious in a patient with previously erlotinib treated lung adenocarcinoma and a T790 M mutation detected by liquid biopsy.

The CTX-M β-lactamases have emerged as the most widespread extended-spectrum β-lactamases (ESBLs) in Gram-negative bacteria. These enzymes rapidly hydrolyze cefotaxime, but not the related cephalosporin, ceftazidime. ESBL variants have evolved, however, that provide enhanced ceftazidime resistance. We show here that a natural variant at a nonactive site, i.e. second-shell residue N106S, enhances enzyme stability but reduces catalytic efficiency for cefotaxime and ceftazidime and decreases resistance levels. However, when the N106S variant was combined with an active-site variant, D240G, that enhances enzyme catalytic efficiency, but decreases stability, the resultant double mutant exhibited higher resistance levels than predicted on the basis of the phenotypes of each variant. We found that this epistasis is due to compensatory effects, whereby increased stability provided by N106S overrides its cost of decreased catalytic activity. X-ray structures of the variant enzymes in complex with cefotaxime revealed conformational changes in the active-site loop spanning residues 103-106 that were caused by the N106S substitution and relieve steric strain to stabilize the enzyme, but also alter contacts with cefotaxime and thereby reduce catalytic activity. We noted that the 103-106 loop conformation in the N106S-containing variants is different from that of WT CTX-M but nearly identical to that of the non-ESBL, TEM-1 β-lactamase, having a serine at the 106 position. Therefore, residue 106 may serve as a "switch" that toggles the conformations of the 103-106 loop. When it is serine, the loop is in the non-ESBL, TEM-like conformation, and when it is asparagine, the loop is in a CTX-M-like, cefotaximase-favorable conformation.

B cells develop high affinity receptors during the course of affinity maturation, a cyclic process of mutation and selection. At the end of affinity maturation, a number of cells sharing the same ancestor (i.e. in the same "clonal family") are released from the germinal center; their amino acid frequency profile reflects the allowed and disallowed substitutions at each position. These clonal-family-specific frequency profiles, called "substitution profiles", are useful for studying the course of affinity maturation as well as for antibody engineering purposes. However, most often only a single sequence is recovered from each clonal family in a sequencing experiment, making it impossible to construct a clonal-family-specific substitution profile. Given the public release of many high-quality large B cell receptor datasets, one may ask whether it is possible to use such data in a prediction model for clonal-family-specific substitution profiles. In this paper, we present the method "Substitution Profiles Using Related Families" (SPURF), a penalized tensor regression framework that integrates information from a rich assemblage of datasets to predict the clonal-family-specific substitution profile for any single input sequence. Using this framework, we show that substitution profiles from similar clonal families can be leveraged together with simulated substitution profiles and germline gene sequence information to improve prediction. We fit this model on a large public dataset and validate the robustness of our approach on two external datasets. Furthermore, we provide a command-line tool in an open-source software package (https://github.com/krdav/SPURF) implementing these ideas and providing easy prediction using our pre-fit models.

DEAD-box proteins are an essential class of enzymes involved in all stages of RNA metabolism. The study of DEAD-box proteins is challenging in a native setting since they are structurally similar, often essential and display dosage sensitivity. Pharmacological inhibition would be an ideal tool to probe the function of these enzymes. In this work, we describe a chemical genetic strategy for the specific inactivation of individual DEAD-box proteins with small molecule inhibitors using covalent complementarity. We identify a residue of low conservation within the P-loop of the nucleotide-binding site of DEAD-box proteins and show that it can be mutated to cysteine without a substantial loss of enzyme function to generate electrophile-sensitive mutants. We then present a series of small molecules that rapidly and specifically bind and inhibit electrophile-sensitive DEAD-box proteins with high selectivity over the wild-type enzyme. Thus, this approach can be used to systematically generate small molecule-sensitive alleles of DEAD-box proteins, allowing for pharmacological inhibition and functional characterization of members of this enzyme family.

Optineurin (OPTN) mutations are linked to glaucoma pathology and E50K mutation shows massive cell death in photoreceptor cells and retinal ganglion cells. However, little is known about E50K-mediated mitochondrial dysfunction in photoreceptor cell degeneration. We here show that overexpression of E50K expression triggered BDNF deficiency, leading to Bax activation in RGC-5 cells. BDNF deficiency induced mitochondrial dysfunction by decreasing mitochondrial maximal respiration and reducing intracellular ATP level in RGC-5 cells. However, BDNF deficiency did not alter mitochondrial dynamics. Also, BDNF deficiency resulted in LC3-mediated mitophagosome formation in RGC-5 cells. These results strongly suggest that E50K-mediated BDNF deficiency plays a critical role in compromised mitochondrial function in glaucomatous photoreceptor cell degeneration.

Inhibition of soluble epoxide hydrolase (sEH, EPHX2) elicits potent cardiovascular protective effects in preclinical models of ischemic cardiovascular disease (CVD), and genetic polymorphisms in EPHX2 have been associated with developing ischemic CVD in humans. However, it remains unknown whether EPHX2 variants are associated with prognosis following an ischemic CVD event. We evaluated the association between EPHX2 p.Lys55Arg and p.Arg287Gln genotype with survival in 667 acute coronary syndrome (ACS) patients. No association with p.Arg287Gln genotype was observed (P = 0.598). Caucasian EPHX2 Arg55 carriers (Lys/Arg or Arg/Arg) had a significantly higher risk of 5-year mortality (adjusted hazard ratio [HR] 1.61, 95% confidence interval [CI] 1.01-2.55, P = 0.045). In an independent population of 2712 ACS patients, this association was not replicated (adjusted HR 0.92, 95% CI 0.70-1.21, P = 0.559). In a secondary analysis, Caucasian homozygous Arg55 allele carriers (Arg/Arg) appeared to exhibit a higher risk of cardiovascular mortality (adjusted HR 2.60, 95% CI 1.09-6.17). These results demonstrate that EPHX2 p.Lys55Arg and p.Arg287Gln polymorphisms do not significantly modify survival after an ACS event. Investigation of other sEH metabolism biomarkers in ischemic CVD appears warranted.

A 52 year-old man with Erdheim-Chester Disease (ECD) (a non-Langerhans polyostotic sclerosing histiocytosis) had next-generation sequencing (NGS) performed as part of his diagnostic workup. In addition to the tissue BRAF V600E mutation that is found in over 50% of ECD cases, he was also found to have a JAK2 V617F alteration in cell-free circulating tumor DNA (ctDNA) (liquid biopsy). The latter was thought to be an "incidental" finding, perhaps due to clonal hematopoiesis (though this usually occurs in older individuals), as his blood counts were normal and he had no splenomegaly. Approximately 13 months after the ctDNA test showing JAK2 V617F, he developed anemia, thrombocytopenia, and splenomegaly. Marrow biopsy then showed megakaryocytic atypia and markedly increased marrow fibrosis, consistent with WHO grade 2 of 3 myelofibrosis. Therefore, the patient was determined to have ECD with a typical BRAF V600E mutation, as well as primary myelofibrosis, with the latter diagnosis manifesting clinically over one year after the JAK2 V617F was first detected in ctDNA. He recently was started on the JAK2 inhibitor ruxolitinib. This case demonstrates that genomic alterations detected by liquid biopsy for evaluation of specific malignancies already present may serve as an early harbinger of hematological disease.

Dysregulation of YAP localization and activity is associated with pathological conditions such as cancer. Although activation of the Hippo phosphorylation cascade is known to cause cytoplasmic retention and inactivation of YAP, emerging evidence suggests that YAP can be regulated in a Hippo-independent manner. Here, we report that YAP is subject to non-proteolytic, K63-linked polyubiquitination by the SCFSKP2 E3 ligase complex (SKP2), which is reversed by the deubiquitinase OTUD1. The non-proteolytic ubiquitination of YAP enhances its interaction with its nuclear binding partner TEAD, thereby inducing YAP's nuclear localization, transcriptional activity, and growth-promoting function. Independently of Hippo signaling, mutation of YAP's K63-linkage specific ubiquitination sites K321 and K497, depletion of SKP2, or overexpression of OTUD1 retains YAP in the cytoplasm and inhibits its activity. Conversely, overexpression of SKP2 or loss of OTUD1 leads to nuclear localization and activation of YAP. Altogether, our study sheds light on the ubiquitination-mediated, Hippo-independent regulation of YAP.

CLC channels mediate passive Cl- conduction, while CLC transporters mediate active Cl- transport coupled to H+ transport in the opposite direction. The distinction between CLC-0/1/2 channels and CLC transporters seems undetectable by amino acid sequence. To understand why they are different functionally we determined the structure of the human CLC-1 channel. Its 'glutamate gate' residue, known to mediate proton transfer in CLC transporters, adopts a location in the structure that appears to preclude it from its transport function. Furthermore, smaller side chains produce a wider pore near the intracellular surface, potentially reducing a kinetic barrier for Cl- conduction. When the corresponding residues are mutated in a transporter, it is converted to a channel. Finally, Cl- at key sites in the pore appear to interact with reduced affinity compared to transporters. Thus, subtle differences in glutamate gate conformation, internal pore diameter and Cl- affinity distinguish CLC channels and transporters.

Congenital sodium diarrhea is a rare and life-threatening disorder characterized by a severe, secretory diarrhea containing high concentrations of sodium, leading to hyponatremia and metabolic acidosis. It may occur in isolation or in association with systemic features such as facial dysmorphism, choanal atresia, imperforate anus, and corneal erosions. Mutations in the serine protease inhibitor, Kunitz-Type 2 (SPINT2) gene have been associated with congenital sodium diarrhea and additional syndromic features. We present a child with congenital sodium diarrhea, cleft lip and palate, corneal erosions, optic nerve coloboma, and intermittent exotropia who was found to have biallelic mutations in SPINT2. One mutation, c.488A > G, predicting p.(Tyr163Cys), has been previously associated with a syndromic form of congenital sodium diarrhea. The other mutation, c.166_167dupTA, predicting p.(Asn57Thrfs*24) has not previously been reported and is likely a novel pathogenic variant for this disorder. We found only one other report of an optic nerve coloboma associated with SPINT2 mutations and this occurred in a patient with congenital tufting enteropathy. Our patient confirms an association of ocular coloboma with presumed loss of SPINT2 function.

Apoptosis is mediated through the extrinsic or intrinsic pathway. Key regulators of the intrinsic apoptotic pathway are the family of B cell lymphoma 2 (Bcl-2) proteins. The activity of the prototypical Bcl-2 protein is usually considered antiapoptotic. However, under some conditions, Bcl-2 associates with the orphan nuclear hormone receptors Nur77 and Nor-1, converting Bcl-2 into a proapoptotic molecule. Expression of Nur77 and Nor-1 is induced by a variety of signals, including those leading to apoptosis. Translocation of Nur77/Nor-1 to mitochondria results in their association with Bcl-2, exposing the Bcl-2 homology (BH) 3 domain and causing apoptosis. However, the molecular details of this interaction are incompletely understood. Here, through extensive Bcl-2 mutagenesis and functional assays, we identified residues within Bcl-2 that are essential for its interaction with Nur77/Nor-1. Although an initial report has suggested that an unstructured loop region between the Bcl-2 BH4 and BH3 domains is required for Bcl-2's interaction with Nur77/Nor-1, we found that it is dispensable for this interaction. Instead, we found important interacting residues at the BH4 domain and crucial interacting residues between the BH1 and BH2 domains. Bcl-2 alanine mutants at this region could no longer interact with Nur77/Nor-1 and could not initiate Nur77/Bcl-2-mediated cell death. However, they still retained their anti-apoptotic capability in two different death assays. These results establish crucial residues in Bcl-2 required for Nur77/Nor-1-mediated apoptosis and point to potential new strategies for manipulating Bcl-2 function.

Directed evolution has proven to be an invaluable tool for protein engineering; however, there is still a need for developing new approaches to continue to improve the efficiency and efficacy of these methods. Here, we demonstrate a new method for library design that applies a previously developed bioinformatic method, Statistical Coupling Analysis (SCA). SCA uses homologous enzymes to identify amino acid positions that are mutable and functionally important and engage in synergistic interactions between amino acids. We use SCA to guide a library of the protein luciferase and demonstrate that, in a single round of selection, we can identify luciferase mutants with several valuable properties. Specifically, we identify luciferase mutants that possess both red-shifted emission spectra and improved stability relative to those of the wild-type enzyme. We also identify luciferase mutants that possess a >50-fold change in specificity for modified luciferins. To understand the mutational origin of these improved mutants, we demonstrate the role of mutations at N229, S239, and G246 in altered function. These studies show that SCA can be used to guide library design and rapidly identify synergistic amino acid mutations from a small library.

The cardiac Na+-Ca2+ exchanger (NCX) plays a critical role in the heart by extruding Ca2+ after each contraction and thus regulates cardiac contractility. The activity of NCX is strongly inhibited by cytosolic protons, which suggests that intracellular acidification will have important effects on heart contractility. However, the mechanisms underlying this inhibition remain elusive. It has been suggested that pH regulation originates from the competitive binding of protons to two Ca2+-binding domains within the large cytoplasmic loop of NCX and requires inactivation by intracellular Na+ to fully develop. By combining mutagenesis and electrophysiology, we demonstrate that NCX pH modulation is an allosteric mechanism distinct from Na+ and Ca2+ regulation, and we show that cytoplasmic Na+ can affect the sensitivity of NCX to protons. We further identify two histidines (His 124 and His 165) that are important for NCX proton sensitivity and show that His 165 plays the dominant role. Our results reveal a complex interplay between the different allosteric mechanisms that regulate the activity of NCX. Because of the central role of NCX in cardiac function, these findings are important for our understanding of heart pathophysiology.

PurposeAs part of the Epilepsy Genetics Initiative, we re-evaluated clinically generated exome sequence data from 54 epilepsy patients and their unaffected parents to identify molecular diagnoses not provided in the initial diagnostic interpretation.MethodsWe compiled and analyzed exome sequence data from 54 genetically undiagnosed trios using a validated analysis pipeline. We evaluated the significance of the genetic findings by reanalyzing sequence data generated at Ambry Genetics, and from a number of additional case and control cohorts.ResultsIn 54 previously undiagnosed trios, we identified two de novo missense variants in SCN8A in the highly expressed alternative exon 5 A-an exon only recently added to the Consensus Coding Sequence database. One additional undiagnosed epilepsy patient harboring a de novo variant in exon 5 A was found in the Ambry Genetics cohort. Missense variants in SCN8A exon 5 A are extremely rare in the population, further supporting the pathogenicity of the de novo alterations identified.ConclusionThese results expand the range of SCN8A variants in epileptic encephalopathy patients and illustrate the necessity of ongoing reanalysis of negative exome sequences, as advances in the knowledge of disease genes and their annotations will permit new diagnoses to be made.

Amyloid-β (Aβ) and human islet amyloid polypeptide (hIAPP) aggregate to form amyloid fibrils that deposit in tissues and are associated with Alzheimer's disease (AD) and type II diabetes (T2D), respectively. Individuals with T2D have an increased risk of developing AD, and conversely, AD patients have an increased risk of developing T2D. Evidence suggests that this link between AD and T2D might originate from a structural similarity between aggregates of Aβ and hIAPP. Using the cryoEM method microelectron diffraction, we determined the atomic structures of 11-residue segments from both Aβ and hIAPP, termed Aβ(24-34) WT and hIAPP(19-29) S20G, with 64% sequence similarity. We observed a high degree of structural similarity between their backbone atoms (0.96-Å root mean square deviation). Moreover, fibrils of these segments induced amyloid formation through self- and cross-seeding. Furthermore, inhibitors designed for one segment showed cross-efficacy for full-length Aβ and hIAPP and reduced cytotoxicity of both proteins, although by apparently blocking different cytotoxic mechanisms. The similarity of the atomic structures of Aβ(24-34) WT and hIAPP(19-29) S20G offers a molecular model for cross-seeding between Aβ and hIAPP.

Angiopoietin-like 4 (ANGPTL4) is an endogenous inhibitor of lipoprotein lipase that modulates lipid levels, coronary atherosclerosis risk, and nutrient partitioning. We hypothesize that loss of ANGPTL4 function might improve glucose homeostasis and decrease risk of type 2 diabetes (T2D). We investigate protein-altering variants in ANGPTL4 among 58,124 participants in the DiscovEHR human genetics study, with follow-up studies in 82,766 T2D cases and 498,761 controls. Carriers of p.E40K, a variant that abolishes ANGPTL4 ability to inhibit lipoprotein lipase, have lower odds of T2D (odds ratio 0.89, 95% confidence interval 0.85-0.92, p = 6.3 × 10-10), lower fasting glucose, and greater insulin sensitivity. Predicted loss-of-function variants are associated with lower odds of T2D among 32,015 cases and 84,006 controls (odds ratio 0.71, 95% confidence interval 0.49-0.99, p = 0.041). Functional studies in Angptl4-deficient mice confirm improved insulin sensitivity and glucose homeostasis. In conclusion, genetic inactivation of ANGPTL4 is associated with improved glucose homeostasis and reduced risk of T2D.

The linker of nucleoskeleton and cytoskeleton (LINC) complex mediates intracellular cross talk between the nucleus and the cytoplasm. In striated muscle, the LINC complex provides structural support to the myocyte nucleus and plays an essential role in regulating gene expression and mechanotransduction. A wide range of cardiac and skeletal myopathies have been linked to mutations in LINC complex proteins. Studies utilizing tissue-specific knockout and mutant mouse models have revealed important insights into the roles of the LINC complex in striated muscle. In this chapter, we describe several feasible approaches for generating striated muscle-specific gene knockout and mutant mouse models to study LINC complex protein function in cardiac and skeletal muscle. The experimental procedures used for phenotyping and analysis of LINC complex knockout mice are also described.

Chinese sprangletop (Leptochloa chinensis (L.) Nees) is recognized as the most disturbing weed in rice fields in Africa, Australia, and Asia due to causing a significant reduction in yields. The habit of most lowland rice farmers in Indonesia is using high doses of herbicides more than once without any rotation, leading to increased weed resistance potential. Therefore, this study aimed to confirm the resistance level of L. chinensis to acetyl-CoA carboxylase (ACCase)-inhibiting herbicides using the whole-plant pot test method. We identified other herbicides that can control the resistant biotype and performed DNA sequencing on a sample to determine mutations present in the biotype′s ACCase gene through polymerase chain reaction. The herbicide dose–response experiment showed that the L. chinensis from Bantimurung Subdistrict, Maros Regency, South Sulawesi, exhibited resistance to ACCase inhibitors (metamifop and cyhalofop-butyl), which is the first case of resistance to ACCase herbicides reported in Indonesia. An acetolactate synthase inhibitor (bispyribac-sodium) and a 1-deoxy-d-xylulose-5-phosphate synthase (DOXP) inhibitor (clomazone) were effective at controlling the resistant biotypes of L. chinensis, so could considered for use in rotation or as an ingredient in mixed herbicides. Single-nucleotide substitution of guanine for thiamine at position 6081 (TGG; susceptible, TGT; Maros) that causes a Trp2027Cys mutation in the target gene contributes to the resistance of the Maros biotype to ACCase inhibitors. [ABSTRACT FROM AUTHOR]

The majority of seven-octapeptide repeat insertion (7-OPRI) carriers exhibit relatively early onset and a slowly progressive course. We have presented three cases of 7-OPRI, including two that are rapidly progressing, and compared the clinical and ancillary characteristics of the short-term and long-term disease course, as well as factors that influence disease course. The clinical and ancillary features of three new 7-OPRI patients in a Chinese pedigree were analyzed. Global data on 7-OPRI cases were then collected by reviewing the literature, and the cases were grouped according to clinical duration as per the WHO sCJD criteria, with a two-year cut-off. A Chinese pedigree has a glycine-to-glutamate substitution within the 7-OPRI insertion, which enhances the hydrophilicity of the prion protein. Two cases in this pedigree had a short disease course (consistent with the typical clinical and ancillary features of sCJD). In addition, the members of this pedigree had a later onset (p < 0.001) and shorter disease course (p < 0.001) compared to previously reported 7-OPRI cases with 129 cis-M and a similar age of onset and disease course to that of cases with 129 cis-V. The 7-OPRI cases with a shorter clinical course (n = 4) had a later onset (p = 0.021), higher rate of hyperintensity on MRI (p = 0.029) and higher frequency of 129 cis-V (p = 0.066) compared to those with a longer clinical course (n = 13). The clinical presentation of 7-OPRI is significantly heterogeneous. Codon 129 cis-V and amino acid substitution within repeat insertions are possible contributors to the short-term disease course of 7-OPRI. [ABSTRACT FROM AUTHOR]

Fasciola hepatica anthelmintic resistance may be associated with the catalytic activity of xenobiotic metabolizing enzymes. The gene expression of one of these enzymes, identified as carboxylesterase B (CestB), was previously described as inducible in adult parasites under anthelmintic treatment and exhibited a single nucleotide polymorphism at position 643 that translates into a radical amino acid substitution at position 215 from Glutamic acid to Lysine. Alphafold 3D models of both allelic sequences exhibited a significant affinity pocket rearrangement and different ligand-docking modeling results. Further bioinformatics analysis confirmed that the radical amino acid substitution is located at the ligand affinity site of the enzyme, affecting its affinity to serine hydrolase inhibitors and preferences for ester ligands. A field genotyping survey from parasite samples obtained from two developmental stages isolated from different host species from Argentina and Mexico exhibited a 37% allele distribution for 215E and a 29% allele distribution for 215K as well as a 34% E/K heterozygous distribution. No linkage to host species or geographic origin was found in any of the allele variants. [ABSTRACT FROM AUTHOR]

The resistance-nodulation-cell division (RND) superfamily of multidrug efflux systems are important players in mediating antibiotic resistance in gram-negative pathogens. Campylobacter jejuni , a major enteric pathogen, utilizes an RND-type transporter system, CmeABC, as the primary mechanism for extrusion of various antibiotics. Recently, a functionally potent variant of CmeABC (named RE-CmeABC) emerged in clinical Campylobacter isolates, conferring enhanced resistance to multiple antibiotic classes. Despite the clinical importance of RE-CmeABC, the molecular mechanisms for its functional gain and its evolutionary trajectory remain unknown. Here, we demonstrated that amino acid substitutions in RE-CmeB (inner membrane transporter), but not in RE-CmeA (periplasmic protein) and RE-CmeC (outer membrane protein), in conjunction with a nucleotide mutation in the promoter region of the efflux operon, are responsible for the functional gain of the multidrug efflux system. We also showed that RE- cmeABC is emerging globally and distributed in genetically diverse C. jejuni strains, suggesting its possible spread by horizontal gene transfer. Notably, many of RE- cmeABC harboring isolates were associated with the human host including strains from large disease outbreaks, indicating the clinical relevance and significance of RE-CmeABC. Evolutionary analysis indicated that RE- cmeB likely originated from Campylobacter coli , but its expansion mainly occurred in C. jejuni, possibly driven by antibiotic selection pressure. Additionally, RE- cmeB , but not RE- cmeA and RE- cmeC , experienced a selective sweep and was progressing to be fixed during evolution. Together, these results identify a mutation-based mechanism for functional gain in RE-CmeABC and reveal the key role of RE-CmeB in facilitating Campylobacter adaptation to antibiotic selection., Competing Interests: Competing interests statement:The authors declare no competing interest.

In 2024, several human infections with highly pathogenic clade 2.3.4.4b bovine influenza H5N1 viruses in the United States raised concerns about their capability for bovine-to-human or even human-to-human transmission. In this study, analysis of the hemagglutinin (HA) from the first-reported human-infecting bovine H5N1 virus (A/Texas/37/2024, Texas) revealed avian-type receptor binding preference. Notably, a Gln 226 Leu substitution switched Texas HA binding specificity to human-type receptors, which was enhanced when combined with an Asn 224 Lys mutation. Crystal structures of the Texas HA with avian receptor analog LSTa and its Gln 226 Leu mutant with human receptor analog LSTc elucidated the structural basis for this preferential receptor recognition. These findings highlight the need for continuous surveillance of emerging mutations in avian and bovine clade 2.3.4.4b H5N1 viruses.

Transmissible Spongiform Encephalopathies are fatal neurodegenerative diseases caused by the misfolding of the cellular prion protein (PrP C ) into its pathological isoform (PrP Sc ). Efficient transmission of PrP Sc occurs within the same species, but a species barrier limits interspecies transmission. While PrP structure is largely conserved among mammals, variations at the β2-α2 loop are observed, and even minor changes in the amino acid sequence of the β2-α2 loop can significantly affect transmission efficiency. The present study shows that the introduction of the elk/deer-specific amino acid substitutions at positions 169 (Ser to Asn) and 173 (Asn to Thr) into the mouse prion protein, which are associated with the structural rigidity of the β2-α2 loop, has a substantial impact on protein dynamics as well as on the misfolding pathways of the protein. Native state hydrogen-deuterium exchange studies coupled with mass spectrometry, show that the rigid loop substitutions stabilize not only the β2-α2 loop but also the C-terminal end of α3, suggesting that molecular interactions between these two segments are strengthened. Moreover, the energy difference between the native state and multiple misfolding-prone partially unfolded forms (PUFs) present at equilibrium, is increased. The decreased accessibility of the PUFs from the native state leads to a slowing down of the misfolding of the protein. The results of this study provide important insights into the early events of conformational conversion of prion protein into β-rich oligomers, and add to the evidence that the β2-α2 loop is a key determinant in prion protein aggregation.

Upon RNA virus infection, the signaling adaptor MAVS forms functional prion-like aggregates on the mitochondrial outer membrane, which serve as a central hub that links virus recognition to downstream antiviral innate immune responses. Multiple mechanisms regulating MAVS activation have been revealed; however, the checkpoint governing MAVS aggregation remains elusive. Here, we demonstrated that the palmitoylation of MAVS at cysteine 79 (C79), which is catalyzed mainly by the palmitoyl S-acyltransferase ZDHHC12, was essential for MAVS aggregation and antiviral innate immunity upon viral infection in macrophages. Notably, the systemic lupus erythematosus-associated mutation MAVS C79F was associated with defective palmitoylation, resulting in low type I interferon (IFN) production. Accordingly, Zdhhc12 deficiency apparently impaired RNA virus-induced type I IFN responses, and Zdhhc12-deficient mice were highly susceptible to lethal viral infection. These findings reveal a previously unknown mechanism by which the palmitoylation of MAVS is a checkpoint for its aggregation during viral infection to ensure timely activation of antiviral defense.

HLA-A*02:889 differs from HLA-A*02:01:01:01 by one nucleotide exchange at position 506(C>G) with an amino exchange., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Protein structure prediction and (re)design have gone through a revolution in the last 3 years. The tremendous progress in these fields has been almost exclusively driven by readily available machine learning algorithms applied to protein folding and sequence design problems. Despite these advancements, predicting site-specific mutational effects on protein stability and function remains an unsolved problem. This is a persistent challenge, mainly because the free energy of large systems is very difficult to compute with absolute accuracy and subtle changes to protein structures are hard to capture with computational models. Here, we describe the implementation and use of ESM-Scan, which uses the ESM zero-shot predictor to scan entire protein sequences for preferential amino acid changes, thus enabling in silico deep mutational scanning experiments. We benchmark ESM-Scan on its predictive capabilities for stability and functionality of sequence changes using three publicly available datasets and proceed by experimentally testing the tool's performance on a challenging test case of a blue-light-activated diguanylate cyclase from Methylotenera species (MsLadC), where it accurately predicted the importance of a highly conserved residue in a region involved in allosteric product inhibition. Our experimental results show that the ESM-zero shot model is capable of inferring the effects of a set of amino acid substitutions in their correlation between predicted fitness and experimental results. ESM-Scan is publicly available at https://huggingface.co/spaces/thaidaev/zsp., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Bacterial sortases are a family of cysteine transpeptidases in Gram-positive bacteria of which sortase A (SrtA) enzymes are responsible for ligating proteins to the peptidoglycan layer of the cell surface. Engineered versions of sortases are also used in sortase-mediated ligation (SML) strategies for a variety of protein engineering applications. Although a versatile tool, substrate recognition by Staphylococcus aureus SrtA (saSrtA), the most commonly utilized enzyme in SML, is stringent and relies on an LPXTG pentapeptide motif. Previous structural studies revealed that the requirement of a glycine in the binding motif may be due to potential steric hindrance of amino acids possessing a β-carbon by W194, a tryptophan located in the β7-β8 loop of the enzyme. Here, we measured the effect of seven single point mutants of W194 (A, D, F, G, N, S, Y) saSrtA using a FRET-based activity assay. We found that while the LPXTG motif remains a requirement for initial proteolytic cleavage, the nucleophile specificity of our variants is altered. In particular, W194A and W194S saSrtA recognize a D-Ala nucleophile and are able to perform ligation reactions. Notably, an LPXT(D-Ala) peptide was not cleaved by either mutant enzyme. We hypothesize that these variants may potentially be utilized to develop an irreversible sortase-mediated reaction. Taken together, this experiment reveals new insight into sortase specificity and possible future SML strategies., (© 2024 The Protein Society.)

A homozygous E40K mutation in the gene coding canine Cu/Zn-superoxide dismutase (cSOD1) causes degenerative myelopathy (DM) in dogs. A pathological hallmark of DM with the cSOD1 mutation is the aggregation of mutant cSOD1 proteins in neurons. The amino acid substitution E40K disrupts a salt bridge between Glu40 and Lys91 and is considered to destabilize the native state of cSOD1; however, the mechanism by which mutant cSOD1 aggregates remains unclear. Here, we show that mutant cSOD1 losing a copper and zinc ion forms oligomers crosslinked via disulfide bonds. The E40K substitution was found to result in the increased solvent exposure of the Cys7 side chain, which then attacked the disulfide bond (Cys57-Cys146) in cSOD1 to form disulfide-linked oligomers. We also successfully prevented the Cys7 exposure and thus the oligomerization of mutant cSOD1 by a fragment antibody that specifically recognizes the region around the mutation site. The fragment antibody covered the β-plug region, reinforcing the interactions compromised by the E40K substitution and thus contributing to the maintenance of the structural integrity of the β-barrel core of cSOD1. Taken together, we propose that the Cys7 exposure in cSOD1 upon the salt bridge disruption plays a central role in the aggregation mechanism of DM-associated mutant cSOD1., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Ancestral relationships among biological species are often represented and analyzed by means of phylogenetic trees. Substitution and distance matrices are two main types of matrices that are used in phylogeny analyses. Substitution matrices describe a frequency change of amino acids in nucleotide or protein sequence over time, while distance matrices estimate phylogeny using a matrix of pairwise distances based on a particular code or analytical concept. Recent investigation by Elena Fimmel and coworkers (Life 11:1338, 2021) showed that: 1. the robustness of a genetic code against point mutations can be described using the conductance measure, and 2. all possible point mutations of the genetic code can be represented as a weighted graph with weights that correspond to the probabilities of these mutations. In this article, we constructed and tested three novel distance matrices based on conductance measure, that take into account the point mutation robustness of the Standard Genetic Code (SGC). These distance matrices are based on maximum (CMAX), average (CAVG), and minimum (CMIN) conductance-optimized distances between codons coding for individual amino acids. The performance of those distance matrices was tested on a dataset of RecA proteins in Bacteria, Archaea (RadA homolog) and Eukarya (Rad51 homolog). RecA protein and its functional homologs were selected for this investigation since they are essential for the repair and maintenance of DNA, and consequently well conserved and present in all domains of life. PAM250 and BLOSUM62 matrices were usually used as a standard for distance matrix testing. PAM250 and BLOSUM62 substitution matrices specified accurately three biological domains of life according to Carl Woese and George Fox (Proc Natl Acad Sci U S A 74:5088, 1977). An identical result was obtained using three novel distance matrices (CMIN, CMAX, CAVG). This result supports the applicability of novel distance matrices based on the conductance method and suggests that further investigations based on this approach are justified., Competing Interests: Declaration of competing interest Authors declare no conflict of Interest for their Article., (Copyright © 2024 Elsevier B.V. All rights reserved.)

By comparing the high-coverage archaic genome sequences to those of modern humans, specific genetic differences have been identified. For example, a human-specific substitution has been found in neuro-oncological ventral antigen 1 (NOVA1) - an RNA-binding protein that regulates the alternative splicing of neuronal pre-mRNA. The amino acid substitution results in an isoleucine-to-valine change at position 197 in NOVA1 (archaic: I197, modern human: V197). Previous studies have utilised gene editing technology to compare the archaic and modern human forms of NOVA1 in cortical organoids, however, the structural and molecular details require further investigation. Using an in silico approach, the modern human (WT) and archaic (V197I) structures of NOVA1 were generated. Moreover, the structure of NOVA1 containing a glycine-to-valine substitution at position 68 (G68V), which occurs at the RNA-binding interface, was examined for comparison. Protein-RNA docking was subsequently performed to model the interaction of NOVA1 variants with RNA and the complexes were evaluated further using classical molecular dynamics (MD) simulations. Based on the MM-PBSA analysis, the binding free energies were similar between the WT (-956.8 ± 32.6 kcal/mol), V197I (-975.4 ± 65.6 kcal/mol), and G68V (-946.7 ± 34.3 kcal/mol) complexes. The findings highlight the binding and stability of protein-RNA complexes with only modest structural changes observed in the archaic and G68V variants compared to the WT NOVA1 protein. Further clarification is required to enhance our understanding of the impact of NOVA1 mutations on alternative splicing and disease development. In particular, delineating the effect of multiple mutations in the NOVA1 gene is of importance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The extensive use of β-lactam antibiotics has led to significant resistance, primarily due to hydrolysis by β-lactamases. OXA class D β-lactamases can hydrolyze a wide range of β-lactam antibiotics, rendering many treatments ineffective. We investigated the effects of single amino acid substitutions in OXA-10 on its substrate spectrum. Broad-spectrum variants with point mutations were searched and biochemically verified. Three key residues, G157D, A124T, and N73S, were confirmed in the variants, and their crystal structures were determined. Based on an enzyme kinetics study, the hydrolytic activity against broad-spectrum cephalosporins, particularly ceftazidime, was significantly enhanced by the G157D mutation in loop 2. The A124T or N73S mutation close to loop 2 also resulted in higher ceftazidime activity. All structures of variants with point mutations in loop 2 or nearby exhibited increased loop 2 flexibility, which facilitated the binding of ceftazidime. These results highlight the effect of a single amino acid substitution in OXA-10 on broad-spectrum drug resistance. Structure-activity relationship studies will help us understand the drug resistance spectrum of β-lactamases, enhance the effectiveness of existing β-lactam antibiotics, and develop new drugs.

The proportion of human isolates with reduced neuraminidase inhibitors (NAIs) susceptibility in highly pathogenic avian influenza (HPAI) H7N9 virus was high. These drug-resistant strains showed good replication capacity without serious loss of fitness. In the presence of oseltamivir, R229I substitution were found in HA1 region of the HPAI H7N9 virus before NA R292K appeared. HPAI H7N9 or H7N9/PR8 recombinant viruses were developed to study whether HA R229I could increase the fitness of the H7N9 virus bearing NA 292K. Replication efficiency was assessed in MDCK or A549 cells. Neuraminidase enzyme activity and receptor-binding ability were analyzed. Pathogenicity in C57 mice was evaluated. Antigenicity analysis was conducted through a two-way HI test, in which the antiserum was obtained from immunized ferrets. Transcriptomic analysis of MDCK infected with HPAI H7N9 24hpi was done. It turned out that HA R229I substitution from oseltamivir induction in HA1 region increased (1) replication ability in MDCK( P < 0.05) and A549( P < 0.05), (2) neuraminidase enzyme activity, (3) binding ability to both α2,3 and α2,6 receptor, (4) pathogenicity to mice(more weight loss; shorter mean survival day; viral titer in respiratory tract, P < 0.05; Pathological changes in pneumonia), (5) transcriptome response of MDCK, of the H7N9 virus bearing NA 292K. Besides, HA R229I substitution changed the antigenicity of H7N9/PR8 virus (>4-fold difference of HI titre). It indicated that through the fine-tuning of HA-NA balance, R229I increased the fitness and changed the antigenicity of H7N9 virus bearing NA 292K. Public health attention to this mechanism needs to be drawn.

Deciphering the specificity of T-cell receptor (TCR) repertoires is crucial for monitoring adaptive immune responses and developing targeted immunotherapies and vaccines. To elucidate the specificity of previously unseen TCRs, many methods employ the BLOSUM62 matrix to find TCRs with similar amino acid (AA) sequences. However, while BLOSUM62 reflects the AA substitutions within conserved regions of proteins with similar functions, the remarkable diversity of TCRs means that both TCRs with similar and dissimilar sequences can bind the same epitope. Therefore, reliance on BLOSUM62 may bias detection towards epitope-specific TCRs with similar biochemical properties, overlooking those with more diverse AA compositions. In this study, we introduce tcrBLOSUMa and tcrBLOSUMb, specialized AA substitution matrices for CDR3 alpha and CDR3 beta TCR chains, respectively. The matrices reflect AA frequencies and variations occurring within TCRs that bind the same epitope, revealing that both CDR3 alpha and CDR3 beta display tolerance to a wide range of AA substitutions and differ noticeably from the standard BLOSUM62. By accurately aligning distant TCRs employing tcrBLOSUMb, we were able to improve clustering performance and capture a large number of epitope-specific TCRs with diverse AA compositions and physicochemical profiles overlooked by BLOSUM62. Utilizing both the general BLOSUM62 and specialized tcrBLOSUM matrices in existing computational tools will broaden the range of TCRs that can be associated with their cognate epitopes, thereby enhancing TCR repertoire analysis., (© The Author(s) 2024. Published by Oxford University Press.)

In a recent publication in Nature, Motone et al. 1 report the development of a protein sequencing method using nanopores that enables the reading of long protein strands. This method allows for multi-pass re-reading and can detect single amino acid substitutions as well as post-translational modifications (PTMs)., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The Env protein of murine leukemia virus (MLV) is the prototype of a large clade of retroviral fusogens, collectively known as gamma-type Envs. Gamma-type Envs are found in retroviruses and endogenous retroviruses (ERVs) representing a broad range of vertebrate hosts. All gamma-type Envs contain a highly conserved stretch of 26-residues in the transmembrane subunit (TM) comprising two motifs, a putative immunosuppressive domain (ISD) and a CX 6 CC motif. Extraordinary conservation of the ISD and its invariant association with the CX 6 CC suggests a fundamental contribution to Env function. To investigate ISD function, we characterized several mutants with single amino acid substitutions at conserved positions in the MLV ISD. A majority abolished infectivity, although we did not observe a corresponding loss in intrinsic ability to mediate membrane fusion. Ratios of the surface subunit (SU) to capsid protein (CA) in virions were diminished for a majority of the ISD mutants, while TM:CA ratios were similar to wild type. Specific loss of SU reflected premature isomerization of the labile disulfide bond that links SU and TM prior to fusion. Indeed, all non-infectious mutants displayed significantly lower disulfide stability than wild-type Env. These results reveal a role for ISD positions 2, 3, 4, 7, and 10 in regulating a late step in entry after fusion peptide insertion but prior to creation of the fusion pore. This implies that the ISD is part of a larger domain, comprising the ISD and CX 6 CC motifs, that is critical for the formation and regulation of the metastable, intersubunit disulfide bond.IMPORTANCEThe gamma-type Env is a prevalent viral fusogen, found within retroviruses and endogenous retroviruses across vertebrate species and in filoviruses such as Ebolavirus. The fusion mechanism of gamma-type Envs is unique from other Class I fusogens such as those of influenza A virus and HIV-1. Gamma-type Envs contain a hallmark feature known as the immunosuppressive domain (ISD) that has been the subject of some controversy in the literature surrounding its putative immunosuppressive effects. Despite the distinctive conservation of the ISD, little has been done to investigate the role of this region for the function of this widespread fusogen. Our work demonstrates the importance of the ISD for the function of gamma-type Envs in infection, particularly in regulating the intermediate steps of membrane fusion. Understanding the fusion mechanism of gamma-type Envs has broad implications for understanding the entry of extant viruses and aspects of host biology connected to co-opted endogenous gamma-type Envs., Competing Interests: The authors declare no conflict of interest.