Amino Acid Deletion Research Articles

NOTCH4ΔL12_16 sensitizes lung adenocarcinomas to EGFR-TKIs through transcriptional down-regulation of HES1

Resistance to epidermal growth factor tyrosine kinase inhibitors (EGFR-TKI) remains one of the major challenges in lung adenocarcinoma (LUAD) therapy. Here, we find an increased frequency of the L12_16 amino acid deletion mutation in the signal peptide region of NOTCH4 (NOTCH4ΔL12_16) in EGFR-TKI-sensitive patients. Functionally, exogenous induction of NOTCH4ΔL12_16 in EGFR-TKI -resistant LUAD cells sensitizes them to EGFR-TKIs. This process is mainly mediated by the reduction of the intracellular domain of NOTCH4 (NICD4) caused by the NOTCH4ΔL12_16 mutation, which results in a lower localization of NOTCH4 in the plasma membrane. Mechanistically, NICD4 transcriptionally upregulates the expression of HES1 by competitively binding to the gene promoter relative to p-STAT3. Because p-STAT3 can downregulate the expression of HES1 in EGFR-TKI-resistant LUAD cells, the reduction of NICD4 induced by NOTCH4ΔL12_16 mutation leads to a decrease in HES1. Moreover, inhibition of the NOTCH4-HES1 pathway using inhibitors and siRNAs abolishes the resistance of EGFR-TKI. Overall, we report that the NOTCH4ΔL12_16 mutation sensitizes LUAD patients to EGFR-TKIs through transcriptional down-regulation of HES1 and that targeted blockade of this signaling cohort could reverse EGFR-TKI -resistance in LUAD, providing a potential approach to overcome resistance to EGFR-TKI -therapy.

Mind the gap

Amino acid deletions are high-risk, high-reward mutations, yet structural consequences are poorly understood. In this issue of Structure, Woods etal. (2023) individually deleted 65 residues from a small α-helical protein, structurally assayed the 17 soluble variants, and developed a computational model of deletion solubility combining Rosetta and AlphaFold2.

KCNT2-Related Disorders: Phenotypes, Functional, and Pharmacological Properties.

Pathogenic variants in KCNT2 are rare causes of developmental epileptic encephalopathy (DEE). We herein describe the phenotypic and genetic features of patients with KCNT2-related DEE, and the in vitro functional and pharmacological properties of KCNT2 channels carrying 14 novel or previously untested variants. Twenty-five patients harboring KCNT2 variants were investigated: 12 were identified through an international collaborative network, 13 were retrieved from the literature. Clinical data were collected and included in a standardized phenotyping sheet. Novel variants were detected using exome sequencing and classified using ACMG criteria. Functional and pharmacological studies were performed by whole-cell electrophysiology in HEK-293 and SH-SY5Y cells. The phenotypic spectrum encompassed: (a) intellectual disability/developmental delay (21/22 individuals with available information), ranging from mild to severe/profound; (b) epilepsy (15/25); (c) neurological impairment, with altered muscle tone (14/22); (d) dysmorphisms (13/20). Nineteen pathogenic KCNT2 variants were found (9 new, 10 reported previously): 16 missense, 1 in-frame deletion of a single amino acid, 1 nonsense, and 1 frameshift. Among tested variants, 8 showed gain-of-function (GoF), and 6 loss-of-function (LoF) features when expressed heterologously in vitro. Quinidine and fluoxetine blocked all GoF variants, whereas loxapine and riluzole activated some LoF variants while blocking others. We expanded the phenotypic and genotypic spectrum of KCNT2-related disorders, highlighting novel genotype-phenotype associations. Pathogenic KCNT2 variants cause GoF or LoF in vitro phenotypes, and each shows a unique pharmacological profile, suggesting the need for in vitro functional and pharmacological investigation to enable targeted therapies based on the molecular phenotype. ANN NEUROL 2023;94:332-349.

Identification of the novel HLA-DPB1*1455:01N allele in a French living organ donor.

The novel HLA-DPB1*1455:01N allele differs from DPB1*05:01:01:01 by one amino acid deletion in exon 3.

Structural Dynamics Predominantly Determine the Adaptability of Proteins to Amino Acid Deletions.

The insertion or deletion (indel) of amino acids has a variety of effects on protein function, ranging from disease-forming changes to gaining new functions. Despite their importance, indels have not been systematically characterized towards protein engineering or modification goals. In the present work, we focus on deletions composed of multiple contiguous amino acids (mAA-dels) and their effects on the protein (mutant) folding ability. Our analysis reveals that the mutant retains the native fold when the mAA-del obeys well-defined structural dynamics properties: localization in intrinsically flexible regions, showing low resistance to mechanical stress, and separation from allosteric signaling paths. Motivated by the possibility of distinguishing the features that underlie the adaptability of proteins to mAA-dels, and by the rapid evaluation of these features using elastic network models, we developed a positive-unlabeled learning-based classifier that can be adopted for protein design purposes. Trained on a consolidated set of features, including those reflecting the intrinsic dynamics of the regions where the mAA-dels occur, the new classifier yields a high recall of 84.3% for identifying mAA-dels that are stably tolerated by the protein. The comparative examination of the relative contribution of different features to the prediction reveals the dominant role of structural dynamics in enabling the adaptation of the mutant to mAA-del without disrupting the native fold.

One amino acid drives the lipid droplet targeting sequence of a new noncoding RNA-encoded protein to mitochondrion.

Over the past decade, the majority of the mammalian genome considered to be noncoding has been revealed to be able to produce proteins. Many RNA molecules, mis-annotated as noncoding, actually are predicted to code for proteins. Some of those proteins have been identified and verified to play critical roles in multiple biological processes. The lipid droplet (LD) is a unique cellular organelle bound with a phospholipid monolayer membrane, and is closely associated with cellular lipid metabolism and metabolic disorders. However, it is still unclear how a protein targets to LDs. Here we identified a new protein on LDs, LDANP2, which is encoded by noncoding RNA, through a proteomics-based strategy. The key sequence for its localization on LDs, Truncation 3, is predicted to form an amphipathic helix. Surprisingly, the deletion of the first amino acid in Truncation 3 resulted in mitochondrial localization. How the types of amino acids would determine the LD or mitochondrial localizations of the protein was studied. The findings introduce a useful strategy to mine for new proteins and would provide clues to the understanding of how a protein would find its right organelle, with phospholipid monolayer or bilayer membrane.

Dysregulation of the WNK4-SPAK/OxSR1 pathway has a mild effect on NKCC2 activity

Background: The furosemide-sensitive Na + -K+ - 2Cl − cotransporter (NKCC2) reabsorbs 20% of filtered Na+ along the thick ascending limb (TAL), while and thiazide-sensitive NaCl cotransporter (NCC) reabsorbs 5-10% along the distal convoluted tubule (DCT). The WNK4-SPAK/OxSR1 pathway mediates NKCC2 phosphorylation at threonines 96 and 101 (pT96/pT101) and NCC phosphorylation at threonine 53 (pT53). This activates both cotransporters. A recent study reported that the commonly used pT96/pT101 pNKCC2 antibody cross-reacts with pT53 NCC in mice on the C57BL/6 background due to a 5 amino acid deletion, calling into question some previous findings regarding WNK4-SPAK/OxSR1 regulation of NKCC2. Although WNK4 is highly expressed along both TAL and DCT, dysregulation of WNK4-SPAK/OxSR1 pathway specifically causes Familial Hyperkalemic Hypertension (FHHt), which is exquisitely sensitive to thiazide diuretics, and thus a disease of NCC dysregulation. Using a new pNKCC2 antibody specific in C57BL/6 mice, we tested the hypothesis that the WNK4-SPAK/OxSR1 pathway more strongly activates NCC than it does NKCC2. Methods: A new pT96 NKCC2 antibody was validated in C57BL/6, 129Sv, and Slc12a3 −/− mice. The abundances of pT96 NKCC2 and pT53 NCC were evaluated in Wnk4 −/− , Oxsr1 −/− , Spak −/− , Oxsr1 −/− / Spak −/− mice, and several FHHt model, Cul3 +/−/Δ9 , Klhl3 −/− , and Klhl3 R528H/R528H mice. NKCC2 activity was confirmed by furosemide response test with thiazide pre-treatment in Klhl3 −/− mice. Both male and female were used and all genetically modified strains were on the C57BL/6 background. Results: The pT96 NKCC2 antibody detected pNKCC2 in C57BL/6 mice but not 129Sv mice, and did not cross-react with phosphorylated NCC. pT53 NCC was almost absent but pT96 NKCC2 was only slightly lower in Wnk4 −/− mice. pT53 NCC was almost absent with Spak deletion ( Spak −/− and Oxsr1 −/− / Spak −/− mice) but pT96 NKCC2 abundance did not differ from controls. Oxsr1 deletion ( Oxsr1 −/− and Oxsr1 −/− / Spak −/− mice) led to a modest lowering of pT96 NKCC2/total NKCC2 ratio but not pT96 NKCC2 abundance. Interestingly, immunofluorescence revealed that WNK4 expression was increased along not only DCT but also cortical TAL in Klhl3 −/− mice, but pT96 NKCC2 abundance was not changed. pT53 NCC abundance, but not pT96 NKCC2 abundance, was higher in two other FHHt mouse models with higher WNK4 ( Cul3 +/−/Δ9 and Klhl3 R528H/R528H mice). Consistent with no difference in pT96 NKCC2 abundance, urine Na + excretion after furosemide injection following thiazide treatment was similar between Klhl3 −/− and control mice. Conclusions: NCC is strongly phosphorylated by the WNK4-SPAK pathway, whereas NKCC2 is only mildly phosphorylated by the WNK4-OxSR1 pathway, suggesting another kinase can phosphorylate NKCC2. In FHHt models with Cul3 or Klhl3 mutations, NKCC2 phosphorylation is unchanged despite higher WNK4 abundance, explaining the thiazide-sensitivity of FHHt. Y.M. received a postdoctoral award from the Uehara Foundation; J.A.M. is funded by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK098141; M.C.B. is funded by CONACyT, Mexico, Grant 101720. 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.

CD4 T cell deficiency narrows virus-neutralizing IgG coverage for Polyomavirus and selects for antibody-escape variants

Abstract CD4 T cell deficiencies predispose patients to progressive multifocal leukoencephalopathy (PML), a brain disease caused by JC polyomavirus (JCPyV). PML is linked to immunomodulatory therapies that preferentially lower CD4 T cells in the CSF (natalizumab); but how CD4 T cells control JCPyV is undefined. “Blind spots” in the ability to neutralize JCPyV variants with mutations in the VP1 capsid protein were described in PML patients. We recently reported that a VP1 mAb selects for VP1-mutant escape variants both in vitroand in vivo; some of these variant viruses are neurovirulent. We hypothesized that alterations in the Ab response due to CD4 T cell insufficiency underlie emergence of Ab-escape mouse polyomavirus (MuPyV) variants. T-cell independent (TI) VP1-specific IgG (via anti-CD4 mAb depletion, CD40L blockade, MHCII KO mice, IL21R KO mice) neutralizes MuPyV but has reduced titer and avidity than anti-VP1 IgGs in CD4 T cell-sufficient mice. Serial passaging of MuPyV in vitrowith sera from TI mice selected an Ab-escape VP1 mutant containing amino acid deletions at residue 145–146 in an external loop of VP1 (MuPyV.145–146). In contrast to sera from healthy MuPyV-infected mice, TI sera failed to neutralize MuPyV.145–146. Depletion of CD4 T cells during persistent MuPyV infection, as a model of acquired CD4 T cell insufficiency (i.e. onset of immunomodulatory mAbs) via anti-CD4 mAb depletion, CD40L blockade, and anti-CD20 mAb B cell depletion also reduced anti-VP1 IgG titer and avidity. Ongoing studies seek to determine if a similar loss in recognition of the 145–146 VP1 epitope occurs. Our data shows that a narrowed antiviral IgG response due to CD4 T cell deficiency is a critical antecedent for outgrowth of Ab-escape PyV VP1 mutant viruses. Supported by grants from the NIH (5R01NS088367, 5R01NS092662, and R35NS127217) (AEL) Judy S. Finkelstein Memorial Award (KNA) T32 CA06395 (KNA)

The novel Nsp9-interacting host factor H2BE promotes PEDV replication by inhibiting endoplasmic reticulum stress-mediated apoptosis

Porcine epidemic diarrhoea (PED) caused by porcine epidemic diarrhoea virus (PEDV) has led to significant economic losses in the swine industry worldwide. Histone Cluster 2, H2BE (HIST2H2BE), the main protein component in chromatin, has been proposed to play a key role in apoptosis. However, the relationship between H2BE and PEDV remains unclear. In this study, H2BE was shown to bind and interact with PEDV nonstructural protein 9 (Nsp9) via immunoprecipitation-mass spectrometry (IP-MS). Next, we verified the interaction of Nsp9 with H2BE by immunoprecipitation and immunofluorescence. H2BE colocalized with Nsp9 in the cytoplasm and nuclei. PEDV Nsp9 upregulated the expression of H2BE by inhibiting the expression of IRX1. We demonstrated that overexpression of H2BE significantly promoted PEDV replication, whereas knockdown of H2BE by small interfering RNA (siRNA) inhibited PEDV replication. Overexpression of H2BE led to significantly inhibited GRP78 expression, phosphorylated PERK (p-PERK), phosphorylated eIF2 (p-eIF2), phosphorylated IRE1 (p-IRE1), and phosphorylated JNK (p-JNK); negatively regulated CHOP and Bax expression and caspase-9 and caspase-3 cleavage; and promoted Bcl-2 production. Knocking down H2BE exerted the opposite effects. Furthermore, we found that after deletion of amino acids 1–28, H2BE did not promote PEDV replication. In conclusion, these studies revealed the mechanism by which H2BE is associated with ER stress-mediated apoptosis to regulate PEDV replication. Nsp9 upregulates H2BE. H2BE plays a role in inhibiting apoptosis and thus facilitating viral replication, which depends on the N-terminal region of H2BE (amino acids 1–28). These findings provide a reference for host–PEDV interactions and offer the possibility for developing strategies for PEDV decontamination and prevention.

Assessing compatibility and viral fitness between poultry-adapted H9N2 and wild bird-derived neuraminidases

Exchange of viral segments between one or more influenza virus subtypes can contribute to a shift in virulence and adaptation to new hosts. Among several influenza subtypes, H9N2 is widely circulating in poultry populations worldwide and has the ability to infect humans. Here, we studied the reassortant compatibility between chicken H9N2 with N1–N9 gene segments of wild bird origin, either with an intact or truncated stalk. Naturally occurring amino acid deletions in the NA stalk of the influenza virus can lead to increased virulence in both mallard ducks and chickens. Our findings show extended genetic compatibility between chicken H9Nx gene segments and the wild-bird NA with and without 20 amino acid stalk deletion. Replication kinetics in avian, mammalian and human cell lines revealed that parental chH9N2 and rH9N6 viruses with intact NA-stalk replicated significantly better in avian DF1 cells compared to human A549 cells. After introducing a stalk deletion, an enhanced preference for replication in mammalian and human cell lines could be observed for rH9N2Δ(H6), rH9N6Δ and rH9N9Δ compared to the parental chH9N2 virus. This highlights the potential emergence of novel viruses with variable phenotypic traits, warranting the continuous monitoring of H9N2 and co-circulating subtypes in avian hosts.

Protein engineering

Protein engineering is the creation of new amino acid sequences to modify proteins. There are many potential applications where the addition, deletion or modification of amino acids can aid or hinder enzyme efficacy. As our knowledge of genetics has expanded rapidly, so has our knowledge of how this might be applied in reality. Proteins are coded for by the 20 amino acids found in nature – should we limit our efforts to those 20 amino acids? This issue also looks at research which is trying to see if we can move beyond this 20-code language of proteins, could engineering offer us new amino acids and new protein functions?Why do we need to do this? The world is under pressure to feed more people in more sustainable ways and treat people with medicine created more efficiently and sustainably. Protein engineering methods might help us reach this goal, producing more sustainable food possibly from unlikely sources, such as from pathogenic bacteria like Yersinia pestis, the causative agent of the plague which could help create lab-grown meat. Sustainable farming could be supported by protein engineering, reducing the carbon footprint of crops and supporting greater yields. I found it interesting that the human race has removed genetic diversity in our crops over time through farming techniques, so we now need to explore new ways of introducing diversity in order to make our crops work harder and be more resilient to pests and climate change.There are many questions and challenges in this area and the articles explore just some of these. They also give you an insight into how this can be applied in reality.As always, this issue also covers personal experiences in biochemistry for anyone interested in careers in the area check out the interviews, which includes interviews with two of the past 10 years' Colworth Medal winners, Professor Helen Walden and Dr Madan Babu, in celebration of the award's 60th anniversary, and write-ups of visits. I always love hearing about what people in the field do daily, as the roles and experiences people have in biochemistry can be so varied.

The clinical characteristics of pediatric patients infected by SARS-CoV-2 Omicron variant and whole viral genome sequencing analysis.

Pediatric population was generally less affected clinically by SARS-CoV-2 infection. Few pediatric cases of COVID-19 have been reported compared to those reported in infected adults. However, a rapid increase in the hospitalization rate of SARS-CoV-2 infected pediatric patients was observed during Omicron variant dominated COVID-19 outbreak. In this study, we analyzed the B.1.1.529 (Omicron) genome sequences collected from pediatric patients by whole viral genome amplicon sequencing using Illumina next generation sequencing platform, followed by phylogenetic analysis. The demographic, epidemiologic and clinical data of these pediatric patients are also reported in this study. Fever, cough, running nose, sore throat and vomiting were the more commonly reported symptoms in children infected by Omicron variant. A novel frameshift mutation was found in the ORF1b region (NSP12) of the genome of Omicron variant. Seven mutations were identified in the target regions of the WHO listed SARS-CoV-2 primers and probes. On protein level, eighty-three amino acid substitutions and fifteen amino acid deletions were identified. Our results indicate that asymptomatic infection and transmission among children infected by Omicron subvariants BA.2.2 and BA.2.10.1 are not common. Omicron may have different pathogenesis in pediatric population.

The bs5 allele of the susceptibility gene Bs5 of pepper (Capsicum annuum L.) encoding a natural deletion variant of a CYSTM protein conditions resistance to bacterial spot disease caused by Xanthomonas species

Key messageThe bs5 resistance gene against bacterial spot was identified by map-based cloning.The recessive bs5 gene of pepper (Capsicum annuum L.) conditions a non-hypersensitive resistance trait, characterized by a slightly swollen, pale green, photosynthetically active leaf tissue, following Xanthomonas euvesicatoria infection. The isolation of the bs5 gene by map-based cloning revealed that the bs5 protein was shorter by 2 amino acids as compared to the wild type Bs5 protein. The natural 2 amino acid deletion occurred in the cysteine-rich transmembrane domain of the tail-anchored (TA) protein, Ca_CYSTM1. The protein products of the wild type Bs5 and mutant bs5 genes were shown to be located in the cell membrane, indicating an unknown function in this membrane compartment. Successful infection of the Bs5 pepper lines was abolished by the 6 bp deletion in the TM encoding domain of the Ca_CYSTM1 gene in bs5 homozygotes, suggesting, that the resulting resistance might be explained by the lack of entry of the Xanthomonas specific effector molecules into the plant cells.

Evaluation of in silico pathogenicity prediction tools for the classification of small in-frame indels

BackgroundThe use of in silico pathogenicity predictions as evidence when interpreting genetic variants is widely accepted as part of standard variant classification guidelines. Although numerous algorithms have been developed and evaluated for classifying missense variants, in-frame insertions/deletions (indels) have been much less well studied.MethodsWe created a dataset of 3964 small (< 100 bp) indels predicted to result in in-frame amino acid insertions or deletions using data from gnomAD v3.1 (minor allele frequency of 1–5%), ClinVar and the Deciphering Developmental Disorders (DDD) study. We used this dataset to evaluate the performance of nine pathogenicity predictor tools: CADD, CAPICE, FATHMM-indel, MutPred-Indel, MutationTaster2021, PROVEAN, SIFT-indel, VEST-indel and VVP.ResultsOur dataset consisted of 2224 benign/likely benign and 1740 pathogenic/likely pathogenic variants from gnomAD (n = 809), ClinVar (n = 2882) and, DDD (n = 273). We were able to generate scores across all tools for 91% of the variants, with areas under the ROC curve (AUC) of 0.81–0.96 based on the published recommended thresholds. To avoid biases caused by inclusion of our dataset in the tools’ training data, we also evaluated just DDD variants not present in either gnomAD or ClinVar (70 pathogenic and 81 benign). Using this subset, the AUC of all tools decreased substantially to 0.64–0.87. Several of the tools performed similarly however, VEST-indel had the highest AUCs of 0.93 (full dataset) and 0.87 (DDD subset).ConclusionsAlgorithms designed for predicting the pathogenicity of in-frame indels perform well enough to aid clinical variant classification in a similar manner to missense prediction tools.

Identification and Analysis of Fungal-Specific Regions in the Aspergillus fumigatus Cu Exporter CrpA That Are Essential for Cu Resistance but Not for Virulence.

The opportunistic fungus Aspergillus fumigatus is the primary invasive mold pathogen in humans, and is responsible for an estimated 200,000 yearly deaths worldwide. Most fatalities occur in immunocompromised patients who lack the cellular and humoral defenses necessary to halt the pathogen's advance, primarily in the lungs. One of the cellular responses used by macrophages to counteract fungal infection is the accumulation of high phagolysosomal Cu levels to destroy ingested pathogens. A. fumigatus responds by activating high expression levels of crpA, which encodes a Cu+ P-type ATPase that actively transports excess Cu from the cytoplasm to the extracellular environment. In this study, we used a bioinformatics approach to identify two fungal-unique regions in CrpA that we studied by deletion/replacement, subcellular localization, Cu sensitivity in vitro, killing by mouse alveolar macrophages, and virulence in a mouse model of invasive pulmonary aspergillosis. Deletion of CrpA fungal-unique amino acids 1-211 containing two N-terminal Cu-binding sites, moderately increased Cu-sensitivity but did not affect expression or localization to the endoplasmic reticulum (ER) and cell surface. Replacement of CrpA fungal-unique amino acids 542-556 consisting of an intracellular loop between the second and third transmembrane helices resulted in ER retention of the protein and strongly increased Cu-sensitivity. Deleting CrpA N-terminal amino acids 1-211 or replacing amino acids 542-556 also increased sensitivity to killing by mouse alveolar macrophages. Surprisingly, the two mutations did not affect virulence in a mouse model of infection, suggesting that even weak Cu-efflux activity by mutated CrpA preserves fungal virulence.

Standing genetic variation affects phenotypic heterogeneity in an SCN5A-mutation founder population with excess sudden cardiac death

The Worm Study, ascertained from a multigeneration pedigree segregating a single amino acid deletion in SCN5A (c.4850_4852delTCT, p.(Phe1617del), rs749697698), is characterized by substantial phenotypic heterogeneity and overlap of sudden cardiac death, long-QT syndrome, cardiac conduction disease, Brugada syndrome, and isorhythmic atrioventricular dissociation. Linkage analysis for a synthetic trait derived from these phenotypes identified a single peak (logarithm of the odds [LOD] = 4.52) at the SCN5A/SCN10A/SCN11A locus on chromosome3. This study explored the role of additional genetic variation in the chromosome 3 locus as a source of phenotypic heterogeneity in the Worm Study population. Genotypes underlying the linkage peak (n = 70) were characterized using microarrays. Haplotypes were determined using family-aware phasing and a population-specific reference panel. Variants with minor allele frequencies >0.10 were tested for association with cardiac conduction disease and isorhythmic dissociation using LAMP and logistic regression. Only 1 haplotype carried the p.Phe1617del/rs749697698 deletion, suggesting relatively recent development (∼18 generations); this haplotype contained 5 other missense variants spanning SCN5A/SCN10A/SCN11A. Noncarrier haplotypes (n = 74) ranged in frequency from 0.5% to 5%. Although no variants were associated with cardiac conduction disease, a homozygous missense variant in SCN10A was associated with isorhythmic dissociation after correction for multiple comparisons (odds ratio 11.23; 95% confidence interval 2.76-23.39; P = 1.2 × 10-4). This variant (rs12632942) was previously associated with PR interval. Our data suggest that other variants, alongside a pathogenic mutation, are associated with phenotypic heterogeneity. Single-mutation screening may be insufficient to predict electrical heart disease in patients and family members. In the Worm Study population, segregating a pathogenic SCN5A mutation, compound variation in the SCN5A/SCN10A/SCN11A locus determines arrhythmic outcome.

A single amino acid deletion in the ER Ca2+ sensor STIM1 reverses the in vitro and in vivo effects of the Stormorken syndrome-causing R304W mutation.

Stormorken syndrome is a multiorgan hereditary disease caused by dysfunction of the endoplasmic reticulum (ER) Ca2+ sensor protein STIM1, which forms the Ca2+ release-activated Ca2+ (CRAC) channel together with the plasma membrane channel Orai1. ER Ca2+ store depletion activates STIM1 by releasing the intramolecular "clamp" formed between the coiled coil 1 (CC1) and CC3 domains of the protein, enabling the C terminus to extend and interact with Orai1. The most frequently occurring mutation in patients with Stormorken syndrome is R304W, which destabilizes and extends the STIM1 C terminus independently of ER Ca2+ store depletion, causing constitutive binding to Orai1 and CRAC channel activation. We found that in cis deletion of one amino acid residue, Glu296 (which we called E296del) reversed the pathological effects of R304W. Homozygous Stim1 E296del+R304W mice were viable and phenotypically indistinguishable from wild-type mice. NMR spectroscopy, molecular dynamics simulations, and cellular experiments revealed that although the R304W mutation prevented CC1 from interacting with CC3, the additional deletion of Glu296 opposed this effect by enabling CC1-CC3 binding and restoring the CC domain interactions within STIM1 that are critical for proper CRAC channel function. Our results provide insight into the activation mechanism of STIM1 by clarifying the molecular basis of mutation-elicited protein dysfunction and pathophysiology.

Different Variants of SARS-CoV-2: A Comprehensive Review on Mutation Patterns and Pathogenicity

Abstract: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), a single-stranded and positive- sense RNA virus, is exhibiting a great degree of mutation acclimated to variable environments, posing a great threat to human life, with numerous SARS-CoV-2 variants. This review aims to analyze emerging strains of SARS-CoV-2, their mutational pattern, their impact on viral pathogenicity, and the identification of the targets for the development of vaccines and therapeutics against the virus. SARSCoV- 2 consists of four structural protein codes viz, peplomer protein (S), an envelope protein (E), nucleoprotein capsid (N), and matrix protein (M), that are involved in the ingression of the viral genome into the host cell by interacting with the host cell receptor, i.e., human angiotensin-converting enzyme 2 (ACE2) (only S protein is shown to bind to ACE-2 receptor). The review includes consequences of mutations- D614G (pre-dominant mutation), E484K, N501Y, K417N, L452R, P681H, del69/70, del157/158, Ins214EPE, and so forth, in alpha, beta, gamma, delta, omicron including other strains of SARS-CoV-2. Mutations include substitution, deletion, and insertion of amino acids in the spike glycoprotein of the SARS-CoV-2, resulting in antibody neutralization, immune escape, etc. As a consequence of modifications in the nucleotide sequence of the viral genome code pushed on by exposure to various environments, these new strains influence the pathogenicity of the virus.

Structural and Biochemical Features of OXA-517: a Carbapenem and Expanded-Spectrum Cephalosporin Hydrolyzing OXA-48 Variant.

OXA-48-producing Enterobacterales have now widely disseminated throughout the world. Several variants have now been reported, differing by just a few amino-acid substitutions or deletions, mostly in the region of the loop β5-β6. As OXA-48 hydrolyzes carbapenems but lacks significant expanded-spectrum cephalosporin (ESC) hydrolytic activity, ESCs were suggested as a therapeutic option. Here, we have characterized OXA-517, a natural variant of OXA-48- with an Arg214Lys substitution and a deletion of Ile215 and Glu216 in the β5-β6 loop, capable of hydrolyzing at the same time ESC and carbapenems. MICs values of E. coli expressing blaOXA-517 gene revealed reduced susceptibility to carbapenems (similarly to OXA-48) and resistance to ESCs. Steady-state kinetic parameters revealed high catalytic efficiencies for ESCs and carbapenems. The blaOXA-517 gene was located on a ca. 31-kb plasmid identical to the prototypical IncL blaOXA-48-carrying plasmid except for an IS1R-mediated deletion of 30.7-kb in the tra operon. The crystal structure of OXA-517, determined to 1.86 Å resolution, revealed an expanded active site compared to that of OXA-48, which allows for accommodation of the bulky ceftazidime substrate. Our work illustrates the remarkable propensity of OXA-48-like carbapenemases to evolve through mutation/deletion in the β5-β6 loop to extend its hydrolysis profile to encompass most β-lactam substrates.

Genetic characterization of hepatitis B virus genotypes among patients with chronic infection in Sulaimaniyah city, Iraq.

Hepatitis B virus (HBV) genotypes are distributed unevenly throughout the world's regions. The researchers' goal in this study was to find out which HBV genotypes are now prevalent in the blood of chronic HBV patients in Iraq's Kurdistan Region's Sulaimaniyah governorate. Genotyping was carried out utilizing Polymerase Chain Reaction (PCR) type-specified primers. Thirty-three chronic HBV patients were included in the HBV genotyping assay. Phylogenic trees of Pre-S1/Pre S2/S genes' nucleotide sequences were constructed using 36 HBV isolates. All the patients had HBV genotype D. Additionally, two samples were further analyzed by sequencing and deposited in GenBank as HBV/Sul-1/2021 accession numbers MZ077051 and HBV/Sul-2/2021 accession numbers MZ077052. Phylogenic analysis indicated that the HBV isolates belong to sub-genotype D1/serotype ayw2. The HBV/Sul-2/2021 had two sequence deletion mutations from G61del-T87del, which accounted for 27 amino acid deletions, and ten other mutations were identified in the carboxylic terminus of the pre-S1 from Q104del-R113del. Accordingly, 37 amino acids were deleted in the S promoter region. Several other substitution mutations were recorded in both HBV isolates. Patients with chronic HBV were found to have the HBV sub-genotype D1/subtype ayw2 with no mixed genotypes. HBV/Sul-1/2022, a new strain with a 37-amino acid mutation, was found to be distinct from any previously known HBV isolates.