Analysis of convergent and parallel amino acid substitutions in the HSP90AA1 gene among high-elevation anurans

A significant amount of convergent and parallel amino acid substitutions in the HSP90AA1 gene has been detected among four species of high-elevation anurans: Bufo tibetanus, Scutiger boulengeri, Rana kukunoris, and Nanorana parkeri. As HSP proteins are involved in response to environmental stress, it is possible these mutations play a role in high-elevation adaptation. In this study, I investigated the functional consequences of these substitutions and inferred their potential links to adaptation. I examined HSP90AA1 sequences of 13 anuran species previously studied. Using PROVEAN, I isolated three deleterious mutations: P65S, K195A, and _199I, each shared between two of the high-elevation species. I further analyzed the protein structure, stability change, and structural damage using model predictions. Based on its buried location and cavity expansion, P65S was predicted to most likely alter protein function. Furthermore, I examined HSP90AA1 sequences of over 100 other animal species available from public databases and found that serine at site 65 is ubiquitously present in cold-water fish, suggesting the substitution is related to cold adaptation. Alanine at site 195 and isoleucine at site 199 were not found in any other species, but these substitutions also might impact protein function as they are predicted to be destabilizing and their ancestral residues have reported post-translational modifications in orthologs. Tests of protein function and an investigation of more sequences from high-elevation species would help to further link these substitutions to adaptation, particularly P65S. Identifying mutations that contribute to high-elevation adaptation would aid in uncovering the molecular mechanisms of adaptation.

In-silico analysis of mutations in ANK1, SPTB, SPTA1, SLC4A1, and EPB4.2 genes responsible for Hereditary Spherocytosis

Hereditary spherocytosis (HS) is a rare inherited disorder of red blood cells which are characterized by spherical, doughnut-shaped with increase deformability that lead to the gallstones and splenomegaly. The role of mutation in the genes responsible for the regulation of synthesis of proteins and stucture of RBC is well know studied. It was found that there are five genes whose mutation result in hereditary spherocytosis.Therefore, we aimed to study the consequences of ANK1, EPB4.2, SPTA1, SPTB, and SLC4A1 non-synonymous mutaion by using advanced inslico methods. Studied for nsSNPs using insilico techniques including OMIN, clinVar, SIFT, Polyphen, homology modelling. Misssence nsSNP were identified in all the gene selected and their effect on the protein structure, stability and functioning was studies. The result showed that 52 nsSNPs are responsible for the changes in the shape of RBCs. After identifying the nsSNPs the structure of proteins were modelled and their RMSD, relative solvent accessibility, and protein stability were studied. Protein stability analysis revealed significant change in free energy (ΔΔG) of the most identified nsSNPs variants. These finding may be helpful for genotype-phenotype research as well as development in pharmacogenetic studies. Finally, this study unveil a significance of inslico methods to figure out highly pathogenic genomic variants affected the structure and functional of HS causing protein

ER transmembrane protein TMTC3 contributes to O-mannosylation of E-cadherin, Cellular Adherence and Embryonic Gastrulation

Protein glycosylation plays essential roles in protein structure, stability and activity such as cell adhesion. The cadherin superfamily of adhesion molecules carry O-linked mannose glycans at conserved sites and it was recently demonstrated that the TMTC1-4 genes contribute to the addition of these O-linked mannoses. Here, biochemical, cell biological and organismal analysis was used to determine that TMTC3 supports the O-mannosylation of E-cadherin, cellular adhesion and embryonic gastrulation. Using genetically engineered cells lacking all four TMTC genes, overexpression of TMTC3 rescued O-linked glycosylation of E-cadherin and cell adherence. The knockdown of the Tmtcs in Xenopus laevis embryos caused a delay in gastrulation that was rescued by the addition of human TMTC3. Mutations in TMTC3 have been linked to neuronal cell migration diseases including Cobblestone lissencephaly. Analysis of TMTC3 mutations associated with Cobblestone lissencephaly found that three of the variants exhibit reduced stability and missence mutations were unable to complement TMTC3 rescue of gastrulation in Xenopus embryo development. Our study demonstrates that TMTC3 regulates O-linked glycosylation and cadherin-mediated adherence, providing insight into its effect on cellular adherence and migration, as well the basis of TMTC3-associated Cobblestone lissencephaly.

Human Milk Proteins and Their Glycosylation Exhibit Quantitative Dynamic Variations during Lactation

ABSTRACTBackgroundProteins in human milk are essential and known to support the growth, development, protection, and health of the newborn. These proteins are highly modified by glycans that are currently being recognized as vital to protein structure, stability, function, and health of the intestinal mucosa. Although milk proteins have been studied, the quantitative changes in milk proteins and their respective site-specific glycosylation are unknown.ObjectiveThis study expanded the analytical tools for milk proteins and their site-specific glycosylation and applied these tools to a large cohort to determine changes in individual protein concentrations and their site-specific N-glycosylation across lactation.DesignA tandem mass spectrometry method was applied to 231 breast-milk samples from 33 mothers in Davis, California, obtained during 7 different periods of lactation. Dynamic changes in the absolute abundances of milk proteins, as well as variation in site-specific N-glycosylation of individual proteins, were quantified.Resultsα-Lactalbumin, β-casein, k-casein, and α-antitrypsin were significantly increased from colostrum to transitional milk (4.37 ± 1.33 g/L to 6.41 ± 0.72 g/L, 2.25 ± 0.86 g/L to 2.59 ± 0.78 g/L, 1.33 ± 0.44 g/L to 1.60 ± 0.39 g/L, and 0.09 ± 0.10 g/L to 0.11 ± 0.04 g/L, respectively; P < 0.002). α-Lactalbumin (37%), β-casein (9%), and lysozyme (159%) were higher in mature milk than in colostrum. Glycans exhibited different behavior. Fucosylated glycans of lactoferrin and high-mannose, undecorated, fucosylated, sialylated, and combined fucosylated + sialylated glycans of secretory immunoglobulin A increased during lactation even when the concentrations of the parent proteins decreased.ConclusionsProteins in healthy mothers vary dynamically through lactation to support the development of infants. Individual milk proteins carried unique glycan modifications that varied systematically in structure even with site specificity. The role of glycosylation in human milk proteins will be important in understanding the functional components of human milk. This trial was registered at clinicaltrials.gov as NCT01817127.

Seven perspectives on GPCR H/D-exchange proteomics methods

Recent research shows surging interest to visualize human G protein-coupled receptor (GPCR) dynamic structures using the bottom-up H/D-exchange (HDX) proteomics technology. This opinion article clarifies critical technical nuances and logical thinking behind the GPCR HDX proteomics method, to help scientists overcome cross-discipline pitfalls, and understand and reproduce the protocol at high quality. The 2010 89% HDX structural coverage of GPCR was achieved with both structural and analytical rigor. This article emphasizes systematically considering membrane protein structure stability and compatibility with chromatography and mass spectrometry (MS) throughout the pipeline, including the effects of metal ions, zero-detergent shock, and freeze-thaws on HDX result rigor. This article proposes to view bottom-up HDX as two steps to guide choices of detergent buffers and chromatography settings: (I) protein HDX labeling in native buffers, and (II) peptide-centric analysis of HDX labels, which applies (a) bottom-up MS/MS to construct peptide matrix and (b) HDX MS to locate and quantify H/D labels. The detergent-low-TCEP digestion method demystified the challenge of HDX-grade GPCR digestion. GPCR HDX proteomics is a structural approach, thus its choice of experimental conditions should let structure lead and digestion follow, not the opposite.