A Conserved Tryptophan in the Envelope Cytoplasmic Tail Regulates HIV-1 Assembly and Spread

The HIV-1 envelope (Env) is an essential determinant of viral infectivity, tropism and spread between T cells. Lentiviral Env contain an unusually long 150 amino acid cytoplasmic tail (EnvCT), but the function of the EnvCT and many conserved domains within it remain largely uncharacterised. Here, we identified a highly conserved tryptophan motif at position 757 (W757) in the LLP-2 alpha helix of the EnvCT as a key determinant for HIV-1 replication and spread between T cells. Alanine substitution at this position potently inhibited HIV-1 cell–cell spread (the dominant mode of HIV-1 dissemination) by preventing recruitment of Env and Gag to sites of cell–cell contact, inhibiting virological synapse (VS) formation and spreading infection. Single-molecule tracking and super-resolution imaging showed that mutation of W757 dysregulates Env diffusion in the plasma membrane and increases Env mobility. Further analysis of Env function revealed that W757 is also required for Env fusion and infectivity, which together with reduced VS formation, result in a potent defect in viral spread. Notably, W757 lies within a region of the EnvCT recently shown to act as a supporting baseplate for Env. Our data support a model in which W757 plays a key role in regulating Env biology, modulating its temporal and spatial recruitment to virus assembly sites and regulating the inherent fusogenicity of the Env ectodomain, thereby supporting efficient HIV-1 replication and spread.

Exogenous Production of N-acetylmuramyl-L Alanine Amidase (LysM2) from Siphoviridae Phage Affecting Anti-Gram-Negative Bacteria: Evaluation of Its Structure and Function

Background: To obtain endolysin with impact(s) on gram-negative bacteria as well as gram-positive bacteria, N-acetylmuramyl L-alanine-amidase (MurNAc-LAA) from a Bacillus subtilis-hosted Siphoviridae phage (SPP1 phage, Subtilis Phage Pavia 1) was exogenously expressed in Escherichia coli (E. coli). Methods: The sequences of MurNAc-LAA genes encoding peptidoglycan hydrolases were obtained from the Virus-Host database. The sequence of MurNAc-LAA was optimized by GenScript software to generate MurNAc-LAA-MMI (LysM2) for optimal expression in E. coli. Furthermore, the structure and function of LysM2 was evaluated in silico. The optimized gene was synthesized, subcloned in the pET28a, and expressed in E. coli BL21(DE3). The antibacterial effects of the protein on the peptidoglycan substrates were studied. Results: LysM2, on 816 bp gene encoding a 33 kDa protein was confirmed as specific SPP1 phage enzyme. The enzyme is composed of 271 amino acids, with a half-life of 10 hr in E. coli. In silico analyses showed 34.2% alpha-helix in the secondary structure, hydrophobic N-terminal, and lysine-rich C-terminal, and no antigenic properties in LysM2 protein. This optimized endolysin revealed impacts against Proteus (sp) by turbidity, and an antibacterial activity against Klebsiella pneumoniae, Salmonella typhi-murium, and Proteus vulgaris in agar diffusion assays. Conclusion: Taken together, our results confirmed that LysM2 is an inhibiting agent for gram-negative bacteria.

In-silico characterization and structure-based functional annotation of a hypothetical protein from Campylobacter jejuni involved in propionate catabolism

Campylobacter jejuni is one of the most prevalent organisms associated with foodborne illness across the globe causing campylobacteriosis and gastritis. Many proteins of C. jejuni are still unidentified. The purpose of this study was to determine the structure and function of a non-annotated hypothetical protein (HP) from C. jejuni. A number of properties like physiochemical characteristics, 3D structure, and functional annotation of the HP (accession No. CAG2129885.1) were predicted using various bioinformatics tools followed by further validation and quality assessment. Moreover, the protein-protein interactions and active site were obtained from the STRING and CASTp server, respectively. The hypothesized protein possesses various characteristics including an acidic pH, thermal stability, water solubility, and cytoplasmic distribution. While alpha-helix and random coil structures are the most prominent structural components of this protein, most of it is formed of helices and coils. Along with expected quality, the 3D model has been found to be novel. This study has identified the potential role of the HP in 2-methylcitric acid cycle and propionate catabolism. Furthermore, protein-protein interactions revealed several significant functional partners. The in-silico characterization of this protein will assist to understand its molecular mechanism of action better. The methodology of this study would also serve as the basis for additional research into proteomic and genomic data for functional potential identification.

In Silico Study of Secondary Structure of Hemoglobin Protein

Protein structure prediction is one of the important goals in the area of bioinformatics and biotechnology. Prediction methods include structure prediction of both secondary and tertiary structures of protein. Protein secondary structure prediction infers knowledge related to presence of helixes, sheets and coils in a polypeptide chain whereas protein tertiary structure prediction infers knowledge related to three dimensional structures of proteins. Protein secondary structures represent the possible motifs or regular expressions represented as patterns that are predicted from primary protein sequence in the form of alpha helix, betastr and and coils. The secondary structure prediction is useful as it infers information related to the structure and function of unknown protein sequence. There are various secondary structure prediction methods used to predict about helixes, sheets and coils. Based on these methods there are various prediction tools under study. This study includes prediction of hemoglobin using various tools. The results produced inferred knowledge with reference to percentage of amino acids participating to produce helices, sheets and coils. PHD and DSC produced the best of the results out of all the tools used.

Characterisation of chalcone isomerase gene isolated from Pueraria montana var.lobata plant

Chalcone isomerase (CHI) is well-known as an important enzyme in the biosynthetic pathways such as flavonoid, isoflavonoid, and anthocyanin biosynthesis. The enzyme was investigated in some kinds of plants in Fabaceae but no research was conducted about the CHI gene of Pueraria montana var. lobata (P. lobata) in Vietnam. In order to provide more information and characterisation of the gene, our study isolated the CHI gene by RT-PCR and Sangersequencing. The sequence of the CHIgene was analysed with nucleotide and deduced amino acid sequences to find the main domains. A full-length CDS of CHI gene from P. lobata is 672 bp encoded 224 amino acids. By using bioinformatic tools to compare, the isolated gene shared 99.7% homology with the same species reference (code D63577.1). Two different nucleotides in the gene were altered the amino acids in the protein, but the differences have not happened in active sites. Additionally, the conserved amino acids related to active catalysis of a hydrogen bond network also appeared in the P. lobataCHI gene. SWISS-MODEL was used to build the complete protein modeling showing that P. lobataCHI protein was the most similar with CHI of Medicago sativa - was defined structure in which all alpha-helix and beta-helix were completelyhomologies.

Implementing Complementary Approaches to Shape the Mechanism of α-Synuclein Oligomerization as a Model of Amyloid Aggregation

The aggregation of proteins into amyloid fibers is linked to more than forty still incurable cellular and neurodegenerative diseases such as Parkinson’s disease (PD), multiple system atrophy, Alzheimer’s disease and type 2 diabetes, among others. The process of amyloid formation is a main feature of cell degeneration and disease pathogenesis. Despite being methodologically challenging, a complete understanding of the molecular mechanism of aggregation, especially in the early stages, is essential to find new biological targets for innovative therapies. Here, we reviewed selected examples on α-syn showing how complementary approaches, which employ different biophysical techniques and models, can better deal with a comprehensive study of amyloid aggregation. In addition to the monomer aggregation and conformational transition hypothesis, we reported new emerging theories regarding the self-aggregation of α-syn, such as the alpha-helix rich tetramer hypothesis, whose destabilization induce monomer aggregation; and the liquid-liquid phase separation hypothesis, which considers a phase separation of α-syn into liquid droplets as a primary event towards the evolution to aggregates. The final aim of this review is to show how multimodal methodologies provide a complete portrait of α-syn oligomerization and can be successfully extended to other protein aggregation diseases.

Omicron and Delta Variant of SARS-CoV-2: A Comparative Computational Study of Spike protein

Emerging SARS-CoV-2 variants, especially those of concern, may have an impact on the virus’s transmissibility and pathogenicity, as well as diagnostic equipment performance and vaccine effectiveness. Even though the SARS-CoV-2 Delta variant (B.1.617.2) emerged during India’s second wave of infections, Delta variants have grown dominant internationally and are still evolving. On November 26, 2021, WHO identified the variant B.1.1.529 as a variant of concern, naming it Omicron, based on evidence that Omicron contains numerous mutations that may influence its behaviour. However, the mode of transmission and severity of the Omicron variant remains unknown. We used computational studies to examine the Delta and Omicron variants in this work and found that the Omicron variant had a higher affinity for human ACE2 than the Delta variant due to a significant number of mutations in the SARS-CoV-2 receptor binding domain, indicating a higher potential for transmission. Based on docking studies, the Q493R, N501Y, S371L, S373P, S375F, Q498R, and T478K mutations contribute significantly to high binding affinity with human ACE2. In comparison to the Delta variant, both the entire spike protein and the RBD in Omicron include a high proportion of hydrophobic amino acids such as leucine and phenylalanine. These amino acids are located within the protein’s core and are required for structural stability. Omicron has a higher percentage of alpha-helix structure than the Delta variant in both whole spike protein and RBD, indicating that it has a more stable structure. We observed a disorder-order transition in the Omicron variant between spike protein RBD regions 468-473, and it may be significant in the influence of disordered residues/regions on spike protein stability and binding to ACE2. A future study might investigate the epidemiological and biological consequences of the Omicron variant.

Case Report: A Novel In-Frame Deletion of GLIS2 Leading to Nephronophthisis and Early Onset Kidney Failure

Variants in the GLIS family zinc finger protein 2 (GLIS2) are a rare cause of nephronophthisis-related ciliopathies (NPHP-RC). A reduction in urinary concentration and a progressive chronic tubulointerstitial nephropathy with corticomedullary cysts are the major characteristic features of NPHP. NPHP demonstrates phenotypic and genetic heterogeneity with at least 25 different recessive genes associated with the disease. We report a female, from a consanguineous family, who presented age 9 years with echogenic kidneys with loss of cortico-medullary differentiation and progressive chronic kidney disease reaching kidney failure by 10 years of age. A novel homozygous in-frame deletion (NM_032,575.3: c.560_574delACCATGTCAACGATT, p.H188_Y192del) in GLIS2 was identified using whole exome sequencing (WES) that segregated from each parent. The five amino acid deletion disrupts the alpha-helix of GLIS2 zinc-finger motif with predicted misfolding of the protein leading to its predicted pathogenicity. This study broadens the variant spectrum of GLIS2 variants leading to NPHP-RC. WES is a suitable molecular tool for children with kidney failure suggestive of NPHP-RC and should be part of routine diagnostics in kidney failure of unknown cause, especially in consanguineous families.

Cryo-EM structure of hexameric yeast Lon (PIM1) highlights importance of conserved structural elements

Lon protease is a conserved ATP-dependent serine protease composed of an AAA+ domain that mechanically unfolds substrates and a serine protease domain that degrades unfolded substrates. In yeast, dysregulation of Lon protease (PIM1) attenuates lifespan and leads to gross mitochondrial morphologic perturbations. Although structures of bacterial and human Lon protease reveal a hexameric assembly, PIM1 was speculated to form a heptameric assembly, and is uniquely characterized by a $\sim$50 residue insertion between the ATPase and protease domains. To understand the yeast-specific properties of PIM1, we determined a high-resolution cryo-EM structure of PIM1 in a substrate-translocating state. Here, we reveal that PIM1 forms a hexamer, conserved with that of bacterial and human Lon proteases, wherein the ATPase domains form a canonical closed spiral that enables pore loop residues to translocate substrate to the protease chamber. In the substrate-translocating state, PIM1 protease domains form a planar protease chamber in an active conformation and are uniquely characterized by a $\sim$15 residue C-terminal extension. These additional C-terminal residues form an alpha-helix that is located along the base of the protease domain. Finally, we did not observe density for the yeast-specific insertion between the ATPase and protease domains, likely due to high conformational flexibility. Biochemical studies to investigate the insertion using constructs that truncated or replaced the insertion with a glycine-serine linker suggest that the yeast-specific insertion is dispensable for PIM1 enzymatic function. Altogether, our structural and biochemical studies highlight unique components of PIM1 machinery and demonstrate evolutionary conservation of Lon protease function.

Conserved tandem arginines for PbgA/YejM allow Salmonella Typhimurium to regulate LpxC and control lipopolysaccharide biogenesis during infection.

Enterobacteriaceae use the periplasmic domain of the conserved inner membrane protein, PbgA/YejM, to regulate lipopolysaccharide (LPS) biogenesis. Salmonella enterica serovar Typhimurium ( S. Typhimurium) relies on PbgA to cause systemic disease in mice and this involves functional interactions with LapB/YciM, FtsH, and LpxC. Escherichia coli PbgA interacts with LapB, an adaptor for the FtsH protease, via the transmembrane segments. LapB and FtsH control proteolysis of LpxC, the rate-limiting LPS biosynthesis enzyme. Lipid A-core, the hydrophobic anchor of LPS molecules, co-crystallizes with PbgA and interacts with residues in the basic region. The model predicts that PbgA-LapB detects periplasmic LPS molecules and prompts FtsH to degrade LpxC. However, the key residues and critical interactions are not defined. We establish that S. Typhimurium uses PbgA to regulate LpxC and define the contribution of two pairs of arginines within the basic region. PbgA R215 R216 form contacts with lipid A-core in the structure and R231 R232 exist in an adjacent alpha helix. PbgA R215 R216 are necessary for S . Typhimurium to regulate LpxC, control lipid-A core biogenesis, promote survival in macrophages, and enhance virulence in mice. In contrast, PbgA R231 R232 are not necessary to regulate LpxC or to control lipid A-core levels, nor are they necessary to promote survival in macrophages or mice. However, residues R231 R232 are critical for infection lethality, and the persistent infection phenotype requires mouse Toll-like receptor four, which detects lipid A. Therefore, S. Typhimurium relies on PbgA’s tandem arginines for multiple interconnected mechanisms of LPS regulation that enhance pathogenesis.