A proteomics tool capable of identifying single proteins would be important for cell biology research and applications. Here, we demonstrate a nanopore-based single-molecule peptide reader sensitive ...to single–amino acid substitutions within individual peptides. A DNA-peptide conjugate was pulled through the biological nanopore MspA by the DNA helicase Hel308. Reading the ion current signal through the nanopore enabled discrimination of single–amino acid substitutions in single reads. Molecular dynamics simulations showed these signals to result from size exclusion and pore binding. We also demonstrate the capability to “rewind” peptide reads, obtaining numerous independent reads of the same molecule, yielding an error rate of <10
in single amino acid variant identification. These proof-of-concept experiments constitute a promising basis for the development of a single-molecule protein fingerprinting and analysis technology.
The distribution of fitness effects (DFE) encompasses the fraction of deleterious, neutral, and beneficial mutations. It conditions the evolutionary trajectory of populations, as well as the rate of ...adaptive molecular evolution (
). Inferring DFE and
from patterns of polymorphism, as given through the site frequency spectrum (SFS) and divergence data, has been a longstanding goal of evolutionary genetics. A widespread assumption shared by previous inference methods is that beneficial mutations only contribute negligibly to the polymorphism data. Hence, a DFE comprising only deleterious mutations tends to be estimated from SFS data, and
is then predicted by contrasting the SFS with divergence data from an outgroup. We develop a hierarchical probabilistic framework that extends previous methods to infer DFE and
from polymorphism data alone. We use extensive simulations to examine the performance of our method. While an outgroup is still needed to obtain an unfolded SFS, we show that both a DFE, comprising both deleterious and beneficial mutations, and
can be inferred without using divergence data. We also show that not accounting for the contribution of beneficial mutations to polymorphism data leads to substantially biased estimates of the DFE and
We compare our framework with one of the most widely used inference methods available and apply it on a recently published chimpanzee exome data set.
SARS-CoV-2 variants with spike (S)-protein D614G mutations now predominate globally. We therefore compare the properties of the mutated S protein (S
) with the original (S
). We report here ...pseudoviruses carrying S
enter ACE2-expressing cells more efficiently than those with S
. This increased entry correlates with less S1-domain shedding and higher S-protein incorporation into the virion. Similar results are obtained with virus-like particles produced with SARS-CoV-2 M, N, E, and S proteins. However, D614G does not alter S-protein binding to ACE2 or neutralization sensitivity of pseudoviruses. Thus, D614G may increase infectivity by assembling more functional S protein into the virion.
Identifying pathogenic variants and underlying functional alterations is challenging. To this end, we introduce MutPred2, a tool that improves the prioritization of pathogenic amino acid ...substitutions over existing methods, generates molecular mechanisms potentially causative of disease, and returns interpretable pathogenicity score distributions on individual genomes. Whilst its prioritization performance is state-of-the-art, a distinguishing feature of MutPred2 is the probabilistic modeling of variant impact on specific aspects of protein structure and function that can serve to guide experimental studies of phenotype-altering variants. We demonstrate the utility of MutPred2 in the identification of the structural and functional mutational signatures relevant to Mendelian disorders and the prioritization of de novo mutations associated with complex neurodevelopmental disorders. We then experimentally validate the functional impact of several variants identified in patients with such disorders. We argue that mechanism-driven studies of human inherited disease have the potential to significantly accelerate the discovery of clinically actionable variants.
Interleukin-33 (IL-33) is a member of the IL-1 family and is involved in polarization of T cells toward a T helper 2 (Th2) cell phenotype. IL-33 is thought to be activated via caspase-1-dependent ...proteolysis, similar to the proinflammatory cytokines IL-1β and IL-18, but this remains unproven. Here we showed that IL-33 was processed by caspases activated during apoptosis (caspase-3 and -7) but was not a physiological substrate for caspases associated with inflammation (caspase-1, -4, and -5). Furthermore, caspase-dependent processing of IL-33 was not required for ST2 receptor binding or ST2-dependent activation of the NF-κB transcription factor. Indeed, caspase-dependent proteolysis of IL-33 dramatically attenuated IL-33 bioactivity in vitro and in vivo. These data suggest that IL-33 does not require proteolysis for activation, but rather, that IL-33 bioactivity is diminished through caspase-dependent proteolysis within apoptotic cells. Thus, caspase-mediated proteolysis acts as a switch to dampen the proinflammatory properties of IL-33.
Proteins have distinct structural and functional constraints at different sites that lead to site-specific preferences for particular amino acid residues as the sequences evolve. Heterogeneity in the ...amino acid substitution process between sites is not modeled by commonly used empirical amino acid exchange matrices. Such model misspecification can lead to artefacts in phylogenetic estimation such as long-branch attraction. Although sophisticated site-heterogeneous mixture models have been developed to address this problem in both Bayesian and maximum likelihood (ML) frameworks, their formidable computational time and memory usage severely limits their use in large phylogenomic analyses. Here we propose a posterior mean site frequency (PMSF) method as a rapid and efficient approximation to full empirical profile mixture models for ML analysis. The PMSF approach assigns a conditional mean amino acid frequency profile to each site calculated based on a mixture model fitted to the data using a preliminary guide tree. These PMSF profiles can then be used for in-depth tree-searching in place of the full mixture model. Compared with widely used empirical mixture models with k classes, our implementation of PMSF in IQ-TREE (http://www.iqtree.org) speeds up the computation by approximately k/1.5-fold and requires a small fraction of the RAM. Furthermore, this speedup allows, for the first time, full nonparametric bootstrap analyses to be conducted under complex site-heterogeneous models on large concatenated data matrices. Our simulations and empirical data analyses demonstrate that PMSF can effectively ameliorate long-branch attraction artefacts. In some empirical and simulation settings PMSF provided more accurate estimates of phylogenies than the mixture models from which they derive.
Background:
There is conflicting literature regarding the association of the BRAF V600E mutation and aggressive clinicopathological features of papillary thyroid cancer (PTC). Nevertheless, some ...propose that BRAF status be incorporated into the management of patients with PTC, specifically recommendations regarding lymph node dissection. We therefore performed a meta-analysis to examine the relationship between BRAF and clinicopathological features of PTC.
Methods:
A literature search was performed within PubMed and EMBASE databases using the following Medical Subject Headings (MeSH) and keywords: “braf,” “mutation,” “thyroid,” “neoplasm(s),” “tumor,” “cancer,” and “carcinoma.” Individual study-specific odds ratios and confidence intervals were calculated, as were Mantel-Haenszel pooled odds ratios for the combined studies.
Results:
Thirty-two studies including 6372 patients were reviewed. BRAF mutation was associated with lymph node metastases (LNM), advanced stage, extrathyroidal extension, tumor size, male gender, multifocality, absence of capsule, classic PTC, and tall-cell variant PTC. There was no association with age or vascular invasion. Only two studies were prospective; nine included consecutive patients, whereas one included randomly selected patients; and only two included patients who had undergone routine central lymph node dissection and were thus evaluable for the presence of LNM.
Conclusion:
Meta-analysis found that BRAF mutation is associated with LNM, stage, extrathyroidal extension, tumor size, male gender, multifocality, absence of capsule, classic PTC, and tall-cell variant PTC in PTC. However, almost all studies were retrospective and only two of 32 included patients who had undergone routine central lymph node dissection, emphasizing the need for well-designed studies to appropriately examine this association before making important clinical decisions.
Epidermal growth factor receptor (EGFR) mutations are the second most common oncogenic driver event in non-small cell lung cancer (NSCLC). Classical activating mutations (exon 19 deletions and the ...L858R point mutation) comprise the vast majority of EGFR mutations and are well defined as strong predictors for good clinical response to EGFR tyrosine kinase inhibitors (EGFRi). However, low frequency mutations including point mutations, deletions, insertions and duplications occur within exons 18–25 of the EGFR gene in NSCLC and are associated with poorer responses to EGFRi. Despite an increased uptake of more sensitive detection methods to identify rare EGFR mutations in patients, our understanding of the biology of these rare EGFR mutations is poor compared to classical mutations. In particular, clinical data focused on these mutations is lacking due to their rarity and challenges in trial recruitment, resulting in an absence of effective treatment strategies for many low frequency EGFR mutations. In this review, we describe the structural and mechanistic features of rare EGFR mutations in NSCLC and discuss the preclinical and clinical evidence for EGFRi response for individual rare EGFR mutations. We also discuss EGFRi sensitivity for complex EGFR mutations, and conclude by offering a perspective on the outstanding questions and future steps required to make advances in the treatment of NSCLC patients that harbour rare EGFR mutations.
Monoclonal antibodies (mAbs) offer a treatment option for individuals with severe COVID-19 and are especially important in high-risk individuals where vaccination is not an option. Given the ...importance of understanding the evolution of resistance to mAbs by SARS-CoV-2, we reviewed the available in vitro neutralization data for mAbs against live variants and viral constructs containing spike mutations of interest. Unfortunately, evasion of mAb-induced protection is being reported with new SARS-CoV-2 variants. The magnitude of neutralization reduction varied greatly among mAb-variant pairs. For example, sotrovimab retained its neutralization capacity against Omicron BA.1 but showed reduced efficacy against BA.2, BA.4 and BA.5, and BA.2.12.1. At present, only bebtelovimab has been reported to retain its efficacy against all SARS-CoV-2 variants considered here. Resistance to mAb neutralization was dominated by the action of epitope single amino acid substitutions in the spike protein. Although not all observed epitope mutations result in increased mAb evasion, amino acid substitutions at non-epitope positions and combinations of mutations also contribute to evasion of neutralization. This Review highlights the implications for the rational design of viral genomic surveillance and factors to consider for the development of novel mAb therapies.
The Sorting Intolerant from Tolerant (SIFT) algorithm predicts the effect of coding variants on protein function. It was first introduced in 2001, with a corresponding website that provides users ...with predictions on their variants. Since its release, SIFT has become one of the standard tools for characterizing missense variation. We have updated SIFT's genome-wide prediction tool since our last publication in 2009, and added new features to the insertion/deletion (indel) tool. We also show accuracy metrics on independent data sets. The original developers have hosted the SIFT web server at FHCRC, JCVI and the web server is currently located at BII. The URL is http://sift-dna.org (24 May 2012, date last accessed).
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