Mutation Of Motif Research Articles

Mutations in an unrecognized internal NPT2A PDZ motif disrupt phosphate transport and cause congenital hypophosphatemia.

The Na+-dependent phosphate cotransporter-2A (NPT2A, SLC34A1) is a primary regulator of extracellular phosphate homeostasis. Its most prominent structural element is a carboxy-terminal PDZ ligand that binds Na+/H+ Exchanger Regulatory Factor-1 (NHERF1, SLC9A3R1). NHERF1, a multidomain PDZ protein, establishes NPT2A membrane localization and is required for hormone-inhibitable phosphate transport. NPT2A also possesses an uncharacterized internal PDZ ligand. Two recent clinical reports describe congenital hypophosphatemia in children harboring Arg495His or Arg495Cys variants within the internal PDZ motif. The wild-type internal 494TRL496 PDZ ligand binds NHERF1 PDZ2, which we consider a regulatory domain. Ablating the internal PDZ ligand with a 494AAA496 substitution blocked hormone-inhibitable phosphate transport. Complementary approaches, including CRISPR/Cas9 technology, site-directed mutagenesis, confocal microscopy, and modeling, showed that NPT2A Arg495His or Arg495Cys variants do not support PTH or FGF23 action on phosphate transport. Coimmunoprecipitation experiments indicate that both variants bind NHERF1 similarly to WT NPT2A. However, in contrast with WT NPT2A, NPT2A Arg495His, or Arg495Cys variants remain at the apical membrane and are not internalized in response to PTH. We predict that Cys or His substitution of the charged Arg495 changes the electrostatics, preventing phosphorylation of the upstream Thr494, interfering with phosphate uptake in response to hormone action, and inhibiting NPT2A trafficking. We advance a model wherein the carboxy-terminal PDZ ligand defines apical localization NPT2A, while the internal PDZ ligand is essential for hormone-triggered phosphate transport.

Molecular Characterization of U6 Promoters from Orange-Spotted Grouper (Epinephelus coioides) and Its Application in DNA Vector-Based RNAi Technology.

The U6 promoter, a typical RNA polymerase III promoter, is widely used to transcribe small RNAs in vector-based siRNA systems. The RNAi efficiency is mainly dependent on the transcriptional activity of the U6 promoter. However, studies have found that U6 promoters isolated from some fishes do not work well in distantly related species. To isolate a U6 promoter with high transcriptional efficiency from fish, in this study, we cloned five U6 promoters in orange-spotted grouper, of which only the grouper U6-1 (GU6-1) promoter contains the OCT element in the distant region. Functional studies revealed that the GU6-1 promoter has high transcriptional ability, which could efficiently transcribe shRNA and result in target gene knockdown in vitro and in vivo. Subsequently, the deletion or mutation of the OCT motif resulted in a significant decrease in promoter transcriptional activity, demonstrating that the OCT element plays an important role in enhancing the grouper U6 promoter transcription. Moreover, the transcriptional activity of the GU6-1 promoter showed little species specificity. It not only works in the grouper but also possesses high transcriptional activity in the zebrafish. Knockdown of the mstn gene in zebrafish and grouper through shRNA driven by the GU6-1 promoter could promote fish growth, suggesting that the GU6-1 promoter can be used as a potential molecular tool in aquaculture practice.

Determinants of tumor immune evasion: the role of T cell exposed motif frequency and mutant amino acid exposure.

Few neoepitopes detected in tumor biopsies are immunogenic. Tumor-specific T cell responses require both the presentation of an epitope that differs from wildtype and the presence of T cells with neoepitope-cognate receptors. We show that mutations detected in tumor biopsies result in an increased frequency of rare amino acid combinations compared to the human proteome and gastrointestinal microorganisms. Mutations in a large data set of oncogene and tumor suppressor gene products were compared to wildtype, and to the count of corresponding amino acid motifs in the human proteome and gastrointestinal microbiome. Mutant amino acids in T cell exposed positions of potential neoepitopes consistently generated amino acid motifs that are less common in both proteome reference datasets. Approximately 10% of the mutant amino acid motifs are absent from the human proteome. Motif frequency does not change when mutants were positioned in the MHC anchor positions hidden from T cell receptors. Analysis of neoepitopes in GBM and LUSC cases showed less common T cell exposed motifs, and HLA binding preferentially placing mutant amino acids in an anchor position for both MHC I and MHC II. Cross-presentation of mutant exposed neoepitopes by MHC I and MHC II was particularly uncommon. Review of a tumor mutation dataset known to generate T cell responses showed immunogenic epitopes were those with mutant amino acids exposed to the T cell receptor and with exposed pentamer motifs present in the human and microbiome reference databases. The study illustrates a previously unrecognized mechanism of tumor immune evasion, as rare T cell exposed motifs produced by mutation are less likely to have cognate T cells in the T cell repertoire. The complex interactions of HLA genotype, binding positions, and mutation specific changes in T cell exposed motif underscore the necessity of evaluating potential neoepitopes in each individual patient.

Metabolic regulation of the two-pore domain potassium channel TASK-1

TASK-1 is a two-pore potassium ion channel and implicated in the maintenance of cell membrane potential. Little is known about the traffic regulation of TASK-1 under metabolic stress. The present study was designed to explore whether cell surface expression of TASK-1 is modulated by hypoxia. The trafficking of recombinant TASK-1 channels was studied in HEK293T cells under hypoxia induced by sodium cyanide (NaCN). Incubation of cells with NaCN significantly reduced TASK-1 fluorescence intensity at cell periphery, and this effect was inhibited by pretreatment with bisindolylmaleimide (BIM), a specific protein kinase C (PKC) inhibitor. Similar results were confirmed by quantifying extracellular HA-tagged TASK-1 on the cell surface using chemiluminescence assay. NaCN-induced reduction in TASK-1 at cell periphery was also prevented by co-expressing the dominant-negative dynamin 1 (K44E), or cholesterol depletion with MβCD, indicating a potential role of cholesterol and dynamin-dependent endocytosis in PKC-mediated reduction of cell surface TASK-1 by NaCN. Mutagenesis studies further showed that NaCN-induced reduction of TASK-1 at cell periphery was observed in cells expressing wild-type TASK-1 but not mutant TASK-1 with mutation of the dileucine motif (263-264 LL-AA) or the PKC phosphorylation consensus site (T400A). Moreover, Western blot analysis demonstrated that NaCN reduced the amount of wild-type TASK-1 but not the TASK-1 mutants (263-264 LL-AA and T400A) in the membrane fraction. In conclusion, our results demonstrate that cell surface TASK-1 channels are downregulated under metabolic inhibition, possibly through PKC-mediated endocytosis in a cholesterol and dynamin-dependent manner. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

CRISPR-Cas effector specificity and cleavage site determine phage escape outcomes.

CRISPR-mediated interference relies on complementarity between a guiding CRISPR RNA (crRNA) and target nucleic acids to provide defense against bacteriophage. Phages escape CRISPR-based immunity mainly through mutations in the protospacer adjacent motif (PAM) and seed regions. However, previous specificity studies of Cas effectors, including the class 2 endonuclease Cas12a, have revealed a high degree of tolerance of single mismatches. The effect of this mismatch tolerance has not been extensively studied in the context of phage defense. Here, we tested defense against lambda phage provided by Cas12a-crRNAs containing preexisting mismatches against the genomic targets in phage DNA. We find that most preexisting crRNA mismatches lead to phage escape, regardless of whether the mismatches ablate Cas12a cleavage in vitro. We used high-throughput sequencing to examine the target regions of phage genomes following CRISPR challenge. Mismatches at all locations in the target accelerated emergence of mutant phage, including mismatches that greatly slowed cleavage in vitro. Unexpectedly, our results reveal that a preexisting mismatch in the PAM-distal region results in selection of mutations in the PAM-distal region of the target. In vitro cleavage and phage competition assays show that dual PAM-distal mismatches are significantly more deleterious than combinations of seed and PAM-distal mismatches, resulting in this selection. However, similar experiments with Cas9 did not result in emergence of PAM-distal mismatches, suggesting that cut-site location and subsequent DNA repair may influence the location of escape mutations within target regions. Expression of multiple mismatched crRNAs prevented new mutations from arising in multiple targeted locations, allowing Cas12a mismatch tolerance to provide stronger and longer-term protection. These results demonstrate that Cas effector mismatch tolerance, existing target mismatches, and cleavage site strongly influence phage evolution.

Evolutionary aspects of mutation in functional motif and post-translational modifications in SARS-CoV-2 3CLpro (Mpro): an in-silico study.

The online version contains supplementary material available at 10.1007/s42485-023-00105-9.

Point mutations in the catalytic domain disrupt cellulose synthase (CESA6) vesicle trafficking and protein dynamics.

Cellulose, the main component of the plant cell wall, is synthesized by the multimeric cellulose synthase (CESA) complex (CSC). In plant cells, CSCs are assembled in the endoplasmic reticulum or Golgi and transported through the endomembrane system to the plasma membrane (PM). However, how CESA catalytic activity or conserved motifs around the catalytic core influence vesicle trafficking or protein dynamics is not well understood. Here, we used yellow fluorescent protein (YFP)-tagged AtCESA6 and created 18 mutants in key motifs of the catalytic domain to analyze how they affected seedling growth, cellulose biosynthesis, complex formation, and CSC dynamics and trafficking in Arabidopsis thaliana. Seedling growth and cellulose content were reduced by nearly all mutations. Moreover, mutations in most conserved motifs slowed CSC movement in the PM as well as delivery of CSCs to the PM. Interestingly, mutations in the DDG and QXXRW motifs affected YFP-CESA6 abundance in the Golgi. These mutations also perturbed post-Golgi trafficking of CSCs. The 18 mutations were divided into two groups based on their phenotypes; we propose that Group I mutations cause CSC trafficking defects whereas Group II mutations, especially in the QXXRW motif, affect protein folding and/or CSC rosette formation. Collectively, our results demonstrate that the CESA6 catalytic domain is essential for cellulose biosynthesis as well as CSC formation, protein folding and dynamics, and vesicle trafficking.

The alg-1 Gene Is Necessary for Orsay Virus Replication in Caenorhabditis elegans.

The establishment of the Orsay virus-Caenorhabditis elegans infection model has enabled the identification of host factors essential for virus infection. Argonautes are RNA interacting proteins evolutionary conserved in the three domains of life that are key components of small RNA pathways. C. elegans encodes 27 argonautes or argonaute-like proteins. Here, we determined that mutation of the argonaute-like gene 1, alg-1, results in a greater than 10,000-fold reduction in Orsay viral RNA levels, which could be rescued by ectopic expression of alg-1. Mutation in ain-1, a known interactor of ALG-1 and component of the RNA-induced silencing complex, also resulted in a significant reduction in Orsay virus levels. Viral RNA replication from an endogenous transgene replicon system was impaired by the lack of ALG-1, suggesting that ALG-1 plays a role during the replication stage of the virus life cycle. Orsay virus RNA levels were unaffected by mutations in the ALG-1 RNase H-like motif that ablate the slicer activity of ALG-1. These findings demonstrate a novel function of ALG-1 in promoting Orsay virus replication in C. elegans. IMPORTANCE All viruses are obligate intracellular parasites that recruit the cellular machinery of the host they infect to support their own proliferation. We used Caenorhabditis elegans and its only known infecting virus, Orsay virus, to identify host proteins relevant for virus infection. We determined that ALG-1, a protein previously known to be important in influencing worm life span and the expression levels of thousands of genes, is required for Orsay virus infection of C. elegans. This is a new function attributed to ALG-1 that was not recognized before. In humans, it has been shown that AGO2, a close relative protein to ALG-1, is essential for hepatitis C virus replication. This demonstrates that through evolution from worms to humans, some proteins have maintained similar functions, and consequently, this suggests that studying virus infection in a simple worm model has the potential to provide novel insights into strategies used by viruses to proliferate.

Tendril morphogenesis is regulated by a CsaTEN-CsaUFO module in cucumber.

Tendril is a morphological innovation during plant evolution, which provides the plants to obtain climbing ability. However, the tendril morphogenesis is poorly understood. A novel tendril morphogenesis defective mutant (tmd1) was identified in cucumber. The apical part of tendril was replaced by a leaf blade in tmd1 mutant, and it lost the climbing ability. Map-based cloning, qPCR detection, bioinformatic analysis, yeast one-hybrid assay, electrophoretic mobility shift assay, and luciferase assay were used to explore the molecular mechanism of CsaTMD1 in regulating tendril morphogenesis. CsaUFO was the candidate causal gene, and a fragment deletion within promoter impaired CsaUFO expression in tmd1 mutant. A conserved motif 1, which harbored two putative TCP transcription factor binding sites, was located within this deleted fragment. CsaTEN directly bound the motif 1 and positively regulated CsaUFO, and mutation in motif 1 removed this regulation. Our work shows a CsaTEN-CsaUFO module in regulating tendril morphogenesis, indicating that evolution of tendril in cucumber due to simply drive of CsaUFO by CsaTEN in tendril. Additionally, the conserved motif 1 provides a strategy for engineering tendril-less Cucurbitaceae crops.

Mechanistic insights into Schizosaccharomyces pombe GT-A family protein Pvg3 in the biosynthesis of pyruvylated β1,3-galactose of N-linked oligosaccharides

N-linked oligosaccharides in the fission yeast Schizosaccharomyces pombe contain large amounts of d-galactose (Gal), which mainly comprises α1,2- and α1,3-linked Gal except for pyruvylated β1,3-linked Gal (PvGalβ) at the non-reducing end. The PvGalβ unit of N-glycans is important for regulating nonsexual flocculation and invasive growth, but the mechanistic basis for β-galactosylation in fission yeast is poorly understood. To gain insight into this mechanism, we have characterized three genes previously identified to be involved in PvGalβ biosynthesis (pvg2, pvg3, and pvg5), with a focus on pvg3, which is predicted to contain a domain conserved in galactosyltransferase family 31 (GT31) proteins. Fluorescent microscopy revealed that Pvg3 is stably localized at the Golgi membrane, regardless of the presence of pvg2+ or pvg5+, suggesting that Pvg2 and Pvg5 are essential for the function of Pvg3 as a β1,3-galactosyltransferase, and not for its localization to the Golgi. Mutation of the GT31 family DXD motif and GT-A fold in Pvg3 resulted in loss of catalytic activity invivo, supporting the idea that Pvg3 is a GT-A type β1,3-galactosyltransferase. Docking simulations further indicated that Pvg3 can recognize donor and acceptor substrates suitable for β-(1→3) bond formation. Yeast two-hybrid assay showed that Pvg5 physically interacts with Pvg3 and the pyruvyltransferase Pvg1. Collectively, these results provide insight into β-galactosylation catalyzed by Pvg3 and the supporting role of Pvg5 in PvGalβ biosynthesis.

The L1 cell adhesion molecule constrains dendritic spine density in pyramidal neurons of the mouse cerebral cortex.

A novel function for the L1 cell adhesion molecule, which binds the actin adaptor protein Ankyrin was identified in constraining dendritic spine density on pyramidal neurons in the mouse neocortex. In an L1-null mouse mutant increased spine density was observed on apical but not basal dendrites of pyramidal neurons in diverse cortical areas (prefrontal cortex layer 2/3, motor cortex layer 5, visual cortex layer 4. The Ankyrin binding motif (FIGQY) in the L1 cytoplasmic domain was critical for spine regulation, as demonstrated by increased spine density and altered spine morphology in the prefrontal cortex of a mouse knock-in mutant (L1YH) harboring a tyrosine (Y) to histidine (H) mutation in the FIGQY motif, which disrupted L1-Ankyrin association. This mutation is a known variant in the human L1 syndrome of intellectual disability. L1 was localized by immunofluorescence staining to spine heads and dendrites of cortical pyramidal neurons. L1 coimmunoprecipitated with Ankyrin B (220 kDa isoform) from lysates of wild type but not L1YH forebrain. This study provides insight into the molecular mechanism of spine regulation and underscores the potential for this adhesion molecule to regulate cognitive and other L1-related functions that are abnormal in the L1 syndrome.

The force-dependent filamin A-G3BP1 interaction regulates phase-separated stress granule formation.

Filamin A (FLNA) is an actin crosslinking protein that mediates mechanotransduction. External and internal mechanical forces, through the actin cytoskeleton, can induce conformational changes of the FLNA molecule to expose cryptic binding sites for its binding partners. Here, we identified G3BP1 (Ras-GTPase activating protein SH3 domain-binding protein 1) as a new FLNA mechanobinding partner. Unlike other FLNA-binding partners to the mechanosensing domain repeat 21 (R21), G3BP1 requires an additional neighboring R22 to interact. We demonstrated that their interaction occurs in the cytosol of living cells in an actin polymerization-dependent manner. We also mapped the FLNA-binding site on G3BP1 and found that a F360A point mutation in the RNA recognition motif disrupts the interaction. RNA interferes with the FLNA-G3BP1 interaction and FLNA does not localize in RNA-rich stress granules (SGs). Disruption of the interaction is sufficient to promote phase-separated SG formation and arsenite treatment further stimulates formation of SGs. Taken together, these data identify G3BP1 as a new mechanobinding protein that interacts with the FLNA mechanosensing domain R21 and suggest that SG formation is partially regulated by mechanical force.

Methyltransferase K-D-K-E motif influences the intercellular transmission of Newcastle disease virus

ABSTRACT We previously demonstrated that two methyltransferase motifs, K-D-K-E and G-G-D, affect the pathogenicity of Newcastle disease virus (NDV) by regulating mRNA translation and virus transmission. Here, we compared the infectious centre area produced by the NDV strain, rSG10, and methyltransferase motifs mutant rSG10 strains in DF-1 cells. The results show that intercellular transmission was attenuated by methyltransferase motif mutations. We further determined the ability of mutant viruses to spread in cell-free and cell-to-cell situations. Cell-free transmission of rSG10-K1756A was not reduced, indicating that cell-to-cell transmission of rSG10-K1756A was decreased. Using a donor and target system, we demonstrated that NDV can spread from cell-to-cell directly. Furthermore, by comparing the protein distribution area of three strains when treated with 2% agar overlay, we found that rSG10-K1756A was defective in cell-to-cell transmission. Tunnelling nanotubes (TNTs) are an important mode for cell-to-cell transmission. Treatment of cells with cytochalasin D (CytoD) or nocodazole to inhibit the formation of TNTs, reduced protein levels in all strains, but rSG10-K1756A was the least affected. These results indicate that mutation of the K-D-K-E motif is likely to restricted the spread of NDV via TNTs. Finally, we observed that matrix protein (M) and fusion protein (F) promoted the formation of cellular extensions, which may be involved in the cell-to-cell spread of NDV. Our research reveals a novel mechanism by which methyltransferase motifs affect the cell-to-cell spread of NDV and provides insight into dissemination of paramyxoviruses.

Identification of Clinically Relevant HIV Vif Protein Motif Mutations through Machine Learning and Undersampling.

Human Immunodeficiency virus (HIV) and its clinical entity, the Acquired Immunodeficiency Syndrome (AIDS) continue to represent an important health burden worldwide. Although great advances have been made towards determining the way viral genetic diversity affects clinical outcome, genetic association studies have been hindered by the complexity of their interactions with the human host. This study provides an innovative approach for the identification and analysis of epidemiological associations between HIV Viral Infectivity Factor (Vif) protein mutations and four clinical endpoints (Viral load and CD4 T cell numbers at time of both clinical debut and on historical follow-up of patients. Furthermore, this study highlights an alternative approach to the analysis of imbalanced datasets, where patients without specific mutations outnumber those with mutations. Imbalanced datasets are still a challenge hindering the development of classification algorithms through machine learning. This research deals with Decision Trees, Naïve Bayes (NB), Support Vector Machines (SVMs), and Artificial Neural Networks (ANNs). This paper proposes a new methodology considering an undersampling approach to deal with imbalanced datasets and introduces two novel and differing approaches (MAREV-1 and MAREV-2). As theses approaches do not involve human pre-determined and hypothesis-driven combinations of motifs having functional or clinical relevance, they provide a unique opportunity to discover novel complex motif combinations of interest. Moreover, the motif combinations found can be analyzed through traditional statistical approaches avoiding statistical corrections for multiple tests.

PPARα Induces the Expression of CAR That Works as a Negative Regulator of PPARα Functions in Mouse Livers.

The nuclear receptor peroxisome proliferator-activated receptor α (PPARα) is a transcription factor that controls the transcription of genes responsible for fatty acid metabolism. We have recently reported a possible drug-drug interaction mechanism via the interaction of PPARα with the xenobiotic nuclear receptor constitutive androstane receptor (CAR). Drug-activated CAR competes with the transcriptional coactivator against PPARα and prevents PPARα-mediated lipid metabolism. In this study, to elucidate the crosstalk between CAR and PPARα, we focused on the influence of PPARα activation on CAR's gene expression and activation. Male C57BL/6N mice (8-12 weeks old, n = 4) were treated with PPARα and CAR activators (fenofibrate and phenobarbital, respectively), and hepatic mRNA levels were determined using quantitative reverse transcription PCR. Reporter assays using the mouse Car promoter were performed in HepG2 cells to determine the PPARα-dependent induction of CAR. CAR KO mice were treated with fenofibrate, and the hepatic mRNA levels of PPARα target genes were determined. Treatment of mice with a PPARα activator increased Car mRNA levels as well as genes related to fatty acid metabolism. In reporter assays, PPARα induced the promoter activity of the Car gene. Mutation of the putative PPARα-binding motif prevented PPARα-dependent induction of reporter activity. In electrophoresis mobility shift assay, PPARα bound to the DR1 motif of the Car promoter. Since CAR has been reported to attenuate PPARα-dependent transcription, CAR was considered a negative feedback protein for PPARα activation. Treatment with fenofibrate induced the mRNA levels of PPARα target genes in Car-null mice more than those in wild-type mice, suggesting that CAR functions as a negative feedback factor for PPARα.

Investigating the structural and dynamical basis for ion affinity in the ion-binding sites of signaling proteins.

Calcium binding in proteins is essential to numerous biological processes including signaling and homeostasis. EF-hands are one of the most widely-studied Ca2+ binding motifs. These highly-conserved motifs are found in signaling proteins, but despite numerous studies the relationship between sequence, conformational ensembles, and affinities is not well understood. To investigate EF-hand motifs, Lanthanides are commonly used as biophysically-tractable substitute for Ca2+ because the lanthanides provide a direct handle on ionic radius, thus enabling characterization of the protein response to changes in ion size. Here we seek to understand the sequence-structure relation in EF-hands to obtain a structural basis that will explain the sequence-affinity relation observed in nature and connect the response of EF-hands to global conformational changes in signaling proteins. Two-dimensional infrared (2D IR) spectroscopy and MD simulations are used to monitor the EF Hand motifs ability to bind to lanthanides outside the protein scaffold, as a minimal model system, and the changes in the secondary structure of each EF Hand motif in lanthanide metal solutions. The results show that the sequence of the EF Hand motifs plays a crucial role in the ability of the motif to bind to ions of specific radius. A single mutation of the EF Hand motif resulted in the following behavior: no binding, preferential binding to smaller ionic radii (<95 pm), and binding to a range of ionic radii (86-105 pm). The results are interpreted through simulations, which connect measured spectra to specific changes in the configurational ensembles.

The role of protein-protein interactions in Fluc dimerization stability and function.

Functional ion channel assembly often requires subunit oligomerization. Yet the molecular factors that define the stability of ion channel complexes in membranes remain to be delineated. Here we investigate this question by studying the dynamic dimerization reaction of the model system Fluc to map out the molecular and physical forces driving this reaction in membranes. Fluc is dual-topology homodimer, where dimerization confers the highly specific fluoride permeation pathway. Our previous analysis demonstrated that a sodium titratable mutant of Fluc participates in a two-step equilibrium dimerization reaction in membranes. First, membrane-dependent dimerization drives subunits to assemble and form a non-contact lipid-solvated stable dimer intermediate by burying a membrane defect imposed in the monomer. However, the fluoride conducting state requires the protein subunits to come closer and expel the lipids to form the fluoride permeation pathway. High resolution crystal structures of Fluc show numerous protein-protein interactions across the dimerization interface, suggesting these interactions might be involved in stabilizing the close-contact dimer state. Alternatively, these contacts may not play a major role in the overall dimer stability but may be important for conferring function. Here, we will investigate how disruption of these protein-protein interactions affects dimerization stability, channel activity or both. Site-directed mutagenesis was employed following four strategies of disruption: (1) reduction of van der Waals interactions by leucine-to-alanine substitutions; (2) removal of potential hydrogen bonds by threonine-to-valine substitutions; (3) decrease backbone flexibility by glycine-to-alanine mutations of the GxxxG dimerization motif; (4) and introduction of bulky tryptophan residues at the interface. Using single-molecule photobleaching analysis to investigate dimer stability in membranes combined with single channel bilayer recordings, these results will provide a quantitative characterization of the contribution of protein-protein interactions to Fluc dimer stability and activity.

Hotspot of de novo telomere addition stabilizes linear amplicons in yeast grown in sulfate-limiting conditions.

Evolution is driven by the accumulation of competing mutations that influence survival. A broad form of genetic variation is the amplification or deletion of DNA (≥50 bp) referred to as copy number variation (CNV). In humans, CNV may be inconsequential, contribute to minor phenotypic differences, or cause conditions such as birth defects, neurodevelopmental disorders, and cancers. To identify mechanisms that drive CNV, we monitored the experimental evolution of Saccharomyces cerevisiae populations grown under sulfate-limiting conditions. Cells with increased copy number of the gene SUL1, which encodes a primary sulfate transporter, exhibit a fitness advantage. Previously, we reported interstitial inverted triplications of SUL1 as the dominant rearrangement in a haploid population. Here, in a diploid population, we find instead that small linear fragments containing SUL1 form and are sustained over several generations. Many of the linear fragments are stabilized by de novo telomere addition within a telomere-like sequence near SUL1 (within the SNF5 gene). Using an assay that monitors telomerase action following an induced chromosome break, we show that this region acts as a hotspot of de novo telomere addition and that required sequences map to a region of <250 base pairs. Consistent with previous work showing that association of the telomere-binding protein Cdc13 with internal sequences stimulates telomerase recruitment, mutation of a four-nucleotide motif predicted to associate with Cdc13 abolishes de novo telomere addition. Our study suggests that internal telomere-like sequences that stimulate de novo telomere addition can contribute to adaptation by promoting genomic plasticity.

Haploinsufficiency as a Foreground Pathomechanism of Poirer-Bienvenu Syndrome and Novel Insights Underlying the Phenotypic Continuum of CSNK2B-Associated Disorders.

CSNK2B encodes for the regulatory subunit of the casein kinase II, a serine/threonine kinase that is highly expressed in the brain and implicated in development, neuritogenesis, synaptic transmission and plasticity. De novo variants in this gene have been identified as the cause of the Poirier-Bienvenu Neurodevelopmental Syndrome (POBINDS) characterized by seizures and variably impaired intellectual development. More than sixty mutations have been described so far. However, data clarifying their functional impact and the possible pathomechanism are still scarce. Recently, a subset of CSNK2B missense variants affecting the Asp32 in the KEN box-like domain were proposed as the cause of a new intellectual disability-craniodigital syndrome (IDCS). In this study, we combined predictive functional and structural analysis and in vitro experiments to investigate the effect of two CSNK2B mutations, p.Leu39Arg and p.Met132LeufsTer110, identified by WES in two children with POBINDS. Our data prove that loss of the CK2beta protein, due to the instability of mutant CSNK2B mRNA and protein, resulting in a reduced amount of CK2 complex and affecting its kinase activity, may underlie the POBINDS phenotype. In addition, the deep reverse phenotyping of the patient carrying p.Leu39Arg, with an analysis of the available literature for individuals with either POBINDS or IDCS and a mutation in the KEN box-like motif, might suggest the existence of a continuous spectrum of CSNK2B-associated phenotypes rather than a sharp distinction between them.

Spike and nsp6 are key determinants of SARS-CoV-2 Omicron BA.1 attenuation.

The SARS-CoV-2 Omicron variant is more immune evasive and less virulent than other major viral variants that have so far been recognized1-12. The Omicron spike (S) protein, which has an unusually large number of mutations, is considered to be the main driver of these phenotypes. Here we generated chimeric recombinant SARS-CoV-2 encoding the S gene of Omicron (BA.1 lineage) in the backbone of an ancestral SARS-CoV-2 isolate, and compared this virus with the naturally circulating Omicron variant. The Omicron S-bearing virus robustly escaped vaccine-induced humoral immunity, mainly owing to mutations in the receptor-binding motif; however, unlike naturally occurring Omicron, it efficiently replicated in cell lines and primary-like distal lung cells. Similarly, in K18-hACE2 mice, although virus bearing Omicron S caused less severe disease than the ancestral virus, its virulence was not attenuated to the level of Omicron. Further investigation showed that mutating non-structural protein 6 (nsp6) in addition to the S protein was sufficient to recapitulate the attenuated phenotype of Omicron. This indicates that although the vaccine escape of Omicron is driven by mutations in S, the pathogenicity of Omicron is determined by mutations both in and outside of the S protein.