Biology subjects

Ahmad, J.

Publications and source records attributed to Ahmad, J..

6 recordsLinked to original sources

bioRxiv2025

Spike protein E2 of chikungunya virus: a plant-based vaccine exhibited potent immunogenicity in BALB/c mice

Over the last two decades, chikungunya virus (CHIKV) infections have surged worldwide, causing significant suffering. CHIKV E2 gene codes for spike protein E2, essential for virus-host interactions and thus serves as a potential vaccine candidate. We expressed a full-length E2 (S27 African prototype) in both E. coli and Nicotiana tabacum, which triggered an immune response in BALB/c mice. First, E2 was computationally analyzed for PTM patterns and prediction of B-cell and T-cell epitopes. Next, molecular docking of epitopes with MHCs (Class I and II) revealed high affinity, confirmed by Molecular Dynamics Simulation. Then, the chemically synthesized E2-6xHis tag was cloned and expressed in both E. coli and N. tabacum under T7 and CaMV promoters, respectively. E2 was cloned into the pUC57 vector (E. coli) and expressed using the pET28a(+) vector in BL21(DE3)pLysS cells, followed by cloning into the pCAMBIA1302 vector and transformation into Agrobacterium tumefaciens. RT-PCR and confocal visualization confirmed the formation of E2 transcripts. Recombinant E2 was purified on Ni-NTA columns and visualized as a protein of [~]49 kDa on SDS PAGE. Finally, E2 was injected into BALB/c mice; neutralizing antibodies, including IgG, were detected as positive in the indirect ELISA, with the highest levels observed at 3 days post-infiltration (3 dpi). Western blot also confirmed E2 expression in E coli and tobacco, and induction of E2-specific antibodies in BALB/c mice. This study presents a promising approach to developing a safe and effective vaccine against chikungunya fever in plants.

molecular biology↗

bioRxiv2024

Genome-wide association study of glucocerebrosidase activity modifiers.

One of the most common genetic risk factors for Parkinsons disease (PD) are variants in GBA1, which encodes the lysosomal enzyme glucocerebrosidase (GCase). GCase deficiency has been associated with an increased PD risk, but not all individuals with low GCase activity are carriers of GBA1 mutations, suggesting other factors may be acting as modifiers. We aimed to discover common variants associated with GCase activity, as well as replicate previously reported associations, by performing a genome-wide association study using two independent cohorts: a Columbia University cohort consisting of 697 PD cases and 347 controls and the Parkinsons Progression Markers Initiative (PPMI) cohort consisting of 357 PD cases and 163 controls. As expected, GBA1 variants have the strongest association with decreased activity, led by p.N370S (beta = -4.36, se = 0.32, p = 5.05e-43). We also identify a novel association in the GAA locus (encoding for acid alpha-glucosidase, beta = -0.96, se = 0.17, p = 5.23e-09) that may be the result of an interaction between GCase and acid alpha-glucosidase based on various interaction analyses. Lastly, we show that several PD-risk loci are potentially associated with GCase activity. Further research will be needed to replicate and validate our findings and to uncover the functional connection between acid alpha-glucosidase and GCase.

genetics↗

bioRxiv2023

Experimental structures of antibody/MHC-I complexes reveal details of epitopes overlooked by computational prediction

Monoclonal antibodies (mAb) to major histocompatibility complex class I (MHC-I) molecules have proved to be crucial reagents for tissue typing and fundamental studies of immune recognition. To augment our understanding of epitopic sites seen by a set of anti-MHC-I mAb, we determined X-ray crystal structures of four complexes of anti-MHC-I antigen-binding fragments (Fab) bound to peptide/MHC-I/{beta}2m (pMHC-I). An anti-H2-Dd mAb, two anti-MHC-I 3 domain mAb, and an anti-{beta}2-microglobulin ({beta}2m) mAb bind pMHC-I at sites consistent with earlier mutational and functional experiments, and the structures explain allelomorph specificity. Comparison of the experimentally determined structures with computationally derived models using AlphaFold Multimer (AF-M) showed that although predictions of the individual pMHC-I heterodimers were quite acceptable, the computational models failed to properly identify the docking sites of the mAb on pMHC-I. The experimental and predicted structures provide insight into strengths and weaknesses of purely computational approaches and suggest areas that merit additional attention. ONE SENTENCE SUMMARYX-ray structures of anti-MHC-I/MHC-I complexes define epitopes overlooked by computational prediction.

immunology↗

bioRxiv2023

The Parkinson's disease risk gene cathepsin B promotes fibrillar alpha-synuclein clearance, lysosomal function and glucocerebrosidase activity in dopaminergic neurons

Variants in the CTSB gene encoding the lysosomal hydrolase cathepsin B (catB) are associated with increased risk of Parkinsons disease (PD). However, neither the specific CTSB variants driving these associations nor the functional pathways that link catB to PD pathogenesis have been characterized. CatB activity contributes to lysosomal protein degradation and regulates signaling processes involved in autophagy and lysosome biogenesis. Previous in vitro studies have found that catB can cleave monomeric and fibrillar alpha-synuclein, a key protein involved in the pathogenesis of PD that accumulates in the brains of PD patients. However, truncated synuclein isoforms generated by catB cleavage have an increased propensity to aggregate. Thus, catB activity could potentially contribute to lysosomal degradation and clearance of pathogenic alpha synuclein from the cell, but also has the potential of enhancing synuclein pathology by generating aggregation-prone truncations. Therefore, the mechanisms linking catB to PD pathophysiology remain to be clarified. Here, we conducted genetic analyses of the association between common and rare CTSB variants and risk of PD. We then used genetic and pharmacological approaches to manipulate catB expression and function in cell lines and induced pluripotent stem cell-derived dopaminergic neurons and assessed lysosomal activity and the handling of aggregated synuclein fibrils. We find that catB inhibition impairs autophagy, reduces glucocerebrosidase (encoded by GBA1) activity, and leads to an accumulation of lysosomal content. In cell lines, reduction of CTSB gene expression impairs the degradation of pre-formed alpha-synuclein fibrils, whereas CTSB gene activation enhances fibril clearance. In midbrain organoids and dopaminergic neurons treated with alpha-synuclein fibrils, catB inhibition potentiates the formation of inclusions which stain positively for phosphorylated alpha-synuclein. These results indicate that the reduction of catB function negatively impacts lysosomal pathways associated with PD pathogenesis, while conversely catB activation could promote the clearance of pathogenic alpha-synuclein.

neuroscience↗

bioRxiv2022

Uncovering the evolutionary tail of GZMM: An NSP4 related protease

Serine proteases are the most predominant class performing a number of activities in organisms. Undergoing several mutations in their sequence over a span of a billion years yet S1 chymotrypsin/trypsin family has maintained a common fold. Granule Associated Serine Peptidases of Immune Defense (GASPIDS) belonging to the S1 class, found in the granules of immune cells are explicitly involved in the regulation of immune-related functions possessing a conserved catalytic triad Ser-Asp-His. The neutrophils along with other cells express four serine proteases (ELA2, PR3, CTSG and NSP4) sharing certain common characteristics. Similarly, CTLs and NK cells express a set of proteases, Granzymes. This study infers an evolutionary relationship among GASPIDs. We employed computational strategies and found that a higher degree of similarity existed between NSP4 and GZMM as compared to their members i.e. NSPs and granzymes, respectively. Similarly, GZMM a protease of NK cells and t cells lineage is found in the Met-ase locus consisting of NSPs genes i.e., Ela2, Prtn3 and Ctsg. The evolutionary relationship of Prss57/NSP4 and gzmm/GZMM was reconstructed through empirical phylogenetic analysis which revealed Prss57/NSP4 to be closely related to gzmm/GZMM. Additional co-expression analysis was carried out to determine the regulatory role of Prss57, inducing Gzmm. From this work, we inferred that Prss57/NSP4 is closely related to Gzmm/GZMM.

bioinformatics↗

bioRxiv2021

Synthetic nanobody-SARS-CoV-2 receptor-binding domain structures identify distinct epitopes

The worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands unprecedented attention. We report four X-ray crystal structures of three synthetic nanobodies (sybodies) (Sb16, Sb45 and Sb68) bind to the receptor-binding domain (RBD) of SARS-CoV-2: binary complexes of Sb16-RBD and Sb45-RBD; a ternary complex of Sb45-RBD-Sb68; and Sb16 unliganded. Sb16 and Sb45 bind the RBD at the ACE2 interface, positioning their CDR2 and CDR3 loops diametrically. Sb16 reveals a large CDR2 shift when binding the RBD. Sb68 interacts peripherally at the ACE2 interface; steric clashes with glycans explain its mechanism of viral neutralization. Superposing these structures onto trimeric spike (S) protein models indicates these sybodies bind conformations of the mature S protein differently, which may aid therapeutic design. One Sentence SummaryX-ray structures of synthetic nanobodies complexed with the receptor-binding domain of the spike protein of SARS-CoV-2 reveal details of CDR loop interactions in recognition of distinct epitopic sites.

immunology↗