The completion of the human genome project at the beginning of the 21st century, along with the rapid advancement of sequencing technologies thereafter, has resulted in exponential growth of ...biological data. In genetics, this has given rise to numerous variation databases, created to store and annotate the ever‐expanding dataset of known mutations. Usually, these databases focus on variation at the sequence level. Few databases focus on the analysis of variation at the 3D level, that is, mapping, visualizing, and determining the effects of variation in protein structures. Additionally, these Web servers seldom incorporate tools to help analyze these data. Here, we present the Human Mutation Analysis (HUMA) Web server and database. HUMA integrates sequence, structure, variation, and disease data into a single, connected database. A user‐friendly interface provides click‐based data access and visualization, whereas a RESTful Web API provides programmatic access to the data. Tools have been integrated into HUMA to allow initial analyses to be carried out on the server. Furthermore, users can upload their private variation datasets, which are automatically mapped to public data and can be analyzed using the integrated tools. HUMA is freely accessible at https://huma.rubi.ru.ac.za.
The Human Mutation Analysis (HUMA) web server has been developed as a platform for analyzing variation in humans. HUMA integrates sequence, structure, variation and disease data into a single, connected database. Access to this data is provided via a user‐friendly interface and a RESTful web API. Furthermore, users can upload private variation datasets, which are automatically mapped to public data, and tools have been integrated into the platform to allow analyses of this data to be carried out.
Embryonic stem (ES) cells are distinguished by their ability to undergo unlimited self-renewal although retaining pluripotency, the capacity to specify cells of all germ layers. Alternative splicing ...contributes to these biological processes by vastly increasing the protein coding repertoire, enabling genes to code for novel variants that may confer different biological functions. The homeodomain transcription factor Nanog acts collaboratively with core factors Oct4 and Sox2 to govern the maintenance of pluripotency. We have discovered that Nanog is regulated by alternative splicing. Two novel exons and six subexons have been identified that extend the known Nanog gene structure and protein coding capacity. Alternative splicing results in two novel Nanog protein variants with attenuated capacities for self-renewal and pluripotency in ES cells. Our previous results have implicated the C-terminal domain, including the tryptophan-rich (WR) domain of Nanog, to be important for the function of Nanog (Wang, J., Levasseur, D. N., and Orkin, S. H. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 6326–6331). Using point mutation analyses, serine 2 (Ser-2) of Nanog has been identified as critical for ES cell self-renewal and for stabilizing a pluripotent gene signature. An inducible conditional knock-out was created to test the ability of new Nanog variants to genetically complement Nanog null ES cells. These results reveal for the first time an expanded Nanog protein coding capacity. We further reveal that a short region of the N-terminal domain and a single phosphorylatable Ser-2 is essential for the maintenance of self-renewal and pluripotency, demonstrating that this region of the protein is highly regulated.
Background: Nanog is a core factor that is required for the maintenance of embryonic stem (ES) cell pluripotency and self-renewal.
Results: Alternative splicing results in Nanog proteins with different capacities for maintaining the undifferentiated ES cell state.
Conclusion: The Nanog N-terminal domain is regulated by post-transcriptional modification.
Significance: Nanog protein variants either fully support the maintenance of pluripotency or facilitate differentiation.
Our previous studies demonstrated that SARS-CoV-2 spike protein could bind to primary hepatocytes and immortalized Hepatocyte-like cells (HLC) via the asialoglycoprotein receptor-1 (ASGR-1). The ...binding of biotinylated spike protein could be inhibited by Spike-neutralizing monoclonal antibodies, anti-ASGR-1 antibodies and unlabeled spike protein. The cells were unable to bind Spike S1 and Spike S1 was incapable of blocking labeled Spike protein, suggesting that the Receptor Binding Domain (RBD) was not involved in the binding event. This study was done to investigate the utility of these cells and immortalized alveolar type 2-like (AT-2) cells in studying the development of variant-specific antibodies post-vaccination.
Serum was collected from 10 individuals pre- and post-vaccination with the J&J, Moderna or Pfizer vaccines. The serum samples were quantified for variant-specific antibodies in a flow cytometry-based immunofluorescent assay utilizing beads coated with biotinylated variant spike proteins. Inhibition of spike protein binding to HLC and AT-2 cells by donor serum was analyzed by immunofluorescent confocal analysis.
All variant spike proteins bound to HLC and AT-2 cells. Post-vaccination serum samples demonstrated increases of SARS-CoV-2 antibody levels from 2 weeks to 2.5 months post-vaccination with associated increased spike-blocking capacity. It was also demonstrated that vaccination with all the available vaccines stimulated antibodies that inhibited binding of all the available variant spike proteins to both HLC and AT-2 cells.
HLC, along with AT-2 cells, provides a useful platform to study the development of neutralizing antibodies post-vaccination. Vaccination with the 3 available vaccines all elicited neutralizing serum antibodies that inhibited binding of each of the variant spike proteins to both AT-2 and HLC cells. This study suggests that inhibition of spike binding to target cells may be a more useful technique to assess immunity than gross quantitation of antibody.
While Inherited Retinal Diseases (IRDs) are typically considered rare diseases, Familial Exudative Vitreo-Retinopathy (FEVR) and Norrie Disease (ND) are more rare than retinitis pigmentosa. We wanted ...to determine if multigenic protein-altering variants are common in FEVR subjects within a set of FEVR-related genes. The potential occurrence of protein-altering variants in two different genes has been documented in a very small percentage of patients, but potential multigenic contributions to FEVR remain unclear. Genes involved in these orphan pediatric retinal diseases are not universally included in available IRD targeted-sequencing panels, and cost is also a factor limiting multigenic-sequence-based testing for these rare conditions. To provide an accurate solution at lower cost, we developed a targeted-sequencing protocol that includes seven genes involved in Familial Exudative Vitreo-Retinopathy (FEVR) and Norrie disease. Seventy-six DNA samples from persons refered to clinic with possible FEVR and some close relatives were sequenced using a novel Oakland-ERI orphan pediatric retinal disease panel (version 2) providing 900 times average read coverage. The seven genes involved in FEVR/ND were:
(ChrX),
(Chr3);
(Chr7);
(Chr10),
(Chr11),
(Chr11),
(Chr11). A total of 33 variants were found that alter protein sequence, with the following relative distribution:
13/33 (40%),
9/33 (27%),
6/33 (18%), (
3/33 (9%),
1/33 (3%),
1/33 (3%). Most protein-altering variants, 85%, were found in three genes:
,
, and
. Four previously known pathogenic variants were detected in five families and two unrelated individuals. Two novel, likely pathogenic variants were detected in one family (FZD4: Cys450ter), and a likely pathogenic frame shift termination variant was detected in one unrelated individual (LRP5: Ala919CysfsTer67). The average number of genes with protein-altering variants was greater in subjects with confirmed FEVR (1.46,
= 30) compared to subjects confirmed unaffected by FEVR (0.95,
= 20), (
= 0.009). Thirty-four percent of persons sequenced had digenic and trigenic protein-altering variants within this set of FEVR genes, which was much greater than expected in the general population (3.6%), as derived from GnomAD data. While the potential contributions to FEVR are not known for most of the variants in a multigenic context, the high multigenic frequency suggests that potential multigenic contributions to FEVR severity warrant future investigation. The targeted-sequencing format developed will support such exploration by reducing the testing cost to $250 (US) for seven genes and facilitating greater access to genetic testing for families with this very rare inherited retinal disease.
Expression of rdar (red, dry, and rough) colony morphology-based biofilm formation in Escherichia coli is highly variable. To investigate the molecular mechanisms of semi-constitutive rdar morphotype ...formation, we compared their cyclic di-GMP turnover protein content and variability to the highly regulated, temperature-dependent morphotype of the historical and modern ST10 isolates E. coli MG1655 and Fec10, respectively. Subsequently, we assessed the effects of cyclic di-GMP turnover protein variants of the EAL phosphodiesterases YcgG and YjcC and the horizontally transferred diguanylate cyclase DgcX on biofilm formation and motility. The two YcgG variants with truncations of the N-terminal CSS signaling domain were oppositely effective in targeting downregulation of rdar biofilm formation compared to the full-length reference protein. Expression of the C-terminal truncated variants YjcCFec67 and YjcCTob1 showed highly diminished apparent phosphodiesterase activity compared to the reference YjcCMG1655. For YjcCFec101, substitution of the C-terminus led to an apparently inactive enzyme. Overexpression of the diguanylate cyclase DgcX contributed to upregulation of cellulose biosynthesis but not to elevated expression of the major biofilm regulator csgD in the “classical” rdar-expressing commensal strain E. coli Fec10. Thus, the c-di-GMP regulating network is highly complex with protein variants displaying substantially different apparent enzymatic activities.
A recent global outbreak of COVID-19 caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) created a pandemic and emerged as a potential threat to humanity. The analysis of virus ...genetic composition has revealed that the spike protein, one of the major structural proteins, facilitates the entry of the virus to host cells.
The spike protein has become the main target for prophylactics and therapeutics studies. Here, we compared the spike proteins of SARS-CoV-2 variants using bioinformatics tools.
The spike protein sequences of wild-type SARS-CoV-2 and its 6 variants-D614G, alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2), gamma (P.1), and omicron (B.1.1.529)-were retrieved from the NCBI database. The ClustalX program was used to sequence multiple alignment and perform mutational analysis. Several online bioinformatics tools were used to predict the physiological, immunological, and structural features of the spike proteins of SARS-CoV-2 variants. A phylogenetic tree was constructed using CLC software. Statistical analysis of the data was done using jamovi 2 software.
Multiple sequence analysis revealed that the P681R mutation in the delta variant, which changed an amino acid from histidine (H) to arginine (R), made the protein more alkaline due to arginine's high pKa value (12.5) compared to histidine's (6.0). Physicochemical properties revealed the relatively higher isoelectric point (7.34) and aliphatic index (84.65) of the delta variant compared to other variants. Statistical analysis of the isoelectric point, antigenicity, and immunogenicity of all the variants revealed significant correlation, with
values ranging from <.007 to .04. The generation of a 2D gel map showed the separation of the delta spike protein from a grouping of the other variants. The phylogenetic tree of the spike proteins showed that the delta variant was close to and a mix of the
bat coronavirus and MERS-CoV.
The comparative analysis of SARS-CoV-2 variants revealed that the delta variant is more aliphatic in nature, which provides more stability to it and subsequently influences virus behavior.
The BRAF protein kinase is widely studied as a cancer driver and therapeutic target. However, the regulation of its expression is not completely understood.
Taking advantage of the RNA-seq data of ...more than 4800 patients belonging to 9 different cancer types, we show that BRAF mRNA exists as a pool of 3 isoforms (reference BRAF, BRAF-X1, and BRAF-X2) that differ in the last part of their coding sequences, as well as in the length (BRAF-ref: 76 nt; BRAF-X1 and BRAF-X2: up to 7 kb) and in the sequence of their 3'UTRs. The expression levels of BRAF-ref and BRAF-X1/X2 are inversely correlated, while the most prevalent among the three isoforms varies from cancer type to cancer type. In melanoma cells, the X1 isoform is expressed at the highest level in both therapy-naïve cells and cells with acquired resistance to vemurafenib driven by BRAF gene amplification or expression of the Δ3-10 splicing variant. In addition to the BRAF-ref protein, the BRAF-X1 protein (the full length as well as the Δ3-10 variant) is also translated. The expression levels of the BRAF-ref and BRAF-X1 proteins are similar, and together they account for BRAF functional activities. In contrast, the endogenous BRAF-X2 protein is hard to detect because the C-terminal domain is selectively recognized by the ubiquitin-proteasome pathway and targeted for degradation.
By shedding light on the repertoire of BRAF mRNA and protein variants, and on the complex regulation of their expression, our work paves the way to a deeper understanding of a crucially important player in human cancer and to a more informed development of new therapeutic strategies.
The stereoselective flavoenzyme D-amino acid oxidase (DAAO) catalyzes the oxidative deamination of neutral and polar D-amino acids producing the corresponding α-keto acids, ammonia, and hydrogen ...peroxide. Despite its peculiar and atypical substrates, DAAO is widespread expressed in most eukaryotic organisms. In mammals (and humans in particular), DAAO is involved in relevant physiological processes ranging from D-amino acid detoxification in kidney to neurotransmission in the central nervous system, where DAAO is responsible of the catabolism of D-serine, a key endogenous co-agonist of N-methyl-D-aspartate receptors. Recently, structural and functional studies have brought to the fore the distinctive biochemical properties of human DAAO (hDAAO). It appears to have evolved to allow a strict regulation of its activity, so that the enzyme can finely control the concentration of substrates (such as D-serine in the brain) without yielding to an excessive production of hydrogen peroxide, a potentially toxic reactive oxygen species (ROS). Indeed, dysregulation in D-serine metabolism, likely resulting from altered levels of hDAAO expression and activity, has been implicated in several pathologies, ranging from renal disease to neurological, neurodegenerative, and psychiatric disorders. Only one mutation in
gene was unequivocally associated to a human disease. However, several single nucleotide polymorphisms (SNPs) are reported in the database and the biochemical characterization of the corresponding recombinant hDAAO variants is of great interest for investigating the effect of mutations. Here we reviewed recently published data focusing on the modifications of the structural and functional properties induced by amino acid substitutions encoded by confirmed SNPs and on their effect on D-serine cellular levels. The potential significance of the different hDAAO variants in human pathologies will be also discussed.
Spider dragline is used by many members of the Araneae family not only as a proteinogenic safety thread but also for web construction. Spider dragline has been shown to possess high tensile strength ...in combination with elastic behavior. This high tensile strength can be attributed to the presence of antiparallel β-sheets within the thread; these antiparallel β-sheets are why the protein is classified as a silk. Due to the properties of spider silk and its technical and medical uses, including its use as a suture material and as a scaffold for tissue regeneration, spider dragline is a focus of the biotechnology industry. The production of sufficient amounts of spider silk is challenging, as it is difficult to produce large quantities of fibers because of the cannibalistic behavior of spiders and their large spatial requirements. In recent years, the heterologous expression of genes coding for spider silk analogs in various hosts, including plants such as
, has been established. We developed a simple and scalable method for the purification of a recombinant spider silk protein elastin-like peptide fusion protein (Q-/K-MaSp1-100× ELP) after heterologous production in tobacco leaves involving heat and acetone precipitation. Further purification was performed using centrifugal Inverse Transition Cycling (cITC). Up to 400 mg of highly pure spider silk protein derivatives can be isolated from six kilograms of tobacco leaves, which is the highest amount of silk protein derivatives purified from plants thus far.
The current study explored the potential links between breast cancer and human interferon α-2b (hIFNα-2b) gene mutations. The hIFNα-2b gene was amplified from breast cancer tumor tissue samples (N = ...60) by polymerase chain reaction (PCR) and the products were subjected to gene sequencing. A total of 38 (63.3%) samples showed positive PCR amplification results. Several of these also exhibited frequent alterations (mutations) after 400 bp and, in particular, adenine was replaced by other bases. A total of 19 selected mutated amino acids were analyzed for local/general fold pattern changes. Human IFNα-2b receptor (IFNAR): ligand (hIFNα-2b protein) interactions through a Z-DOCK (3.0.2) server were also evaluated to assess the binding patterns of each ligand to receptor to induce Janus-Kinase-signal transducer and activator of transcription antiproliferative signal transduction pathway inside the cancer cells. Certain local structural and conformational changes were predicted to be induced by mutations in the ligand. The variant models of the hIFNα-2b displayed structural and conformational changes that signified that changes to hIFNα-2b may be a risk factor in addition to other known factors associated with onset/progression of female breast carcinoma. It was hoped that others might build upon the research in this study evaluating protein structural models with mutations and their consequent interactions with receptors in the development of potent immune therapeutic drugs for breast cancer that are based on recombinant hIFNα-2b.
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