Biology subjects

Kazmi, M. A.

Publications and source records attributed to Kazmi, M. A..

4 recordsLinked to original sources

bioRxiv2024

Sampling Techniques and Genomic Analysis of Biological Material from Artworks

The genomic analysis of biological material obtained from artwork or cultural heritage artifacts can be used to guide curation, preservation and restoration. However, the recovery of biological samples from artworks is dependent on the sampling technique used and the media from which the biological materials are recovered. The ideal sampling method should be non-invasive, yet robust. Only rare examples of direct comparisons among sampling techniques have been reported. To develop a framework for the recovery of biological samples for subsequent microbial DNA analysis, we studied five artworks on paper and compared three sampling methods, each with increasing degrees of invasiveness. Minimally invasive swabbing techniques collect samples from the surface, whereas more aggressive techniques such as wet vacuuming were expected to yield more biological material from within the support media. We report a comparison of collection techniques to generate microbial DNA sequence data from artworks on paper. We observed that wet vacuuming resulted in higher DNA recovery than double swabbing and core punches; however, all three techniques yielded satisfactory microbial DNA sequences. Diverse microbial populations existed to varying degrees on the corners and centers of the five artworks studied, but the distribution of the total biomass was relatively even across the surfaces of the works sampled. Studies of peripheral regions, where sampling is less likely to cause alterations to the artwork, could thus yield useful results in microbiome studies. These results provide a framework for sampling artworks on paper to obtain biological material for microbial genomic analysis. The methods described may provide microbiome identification to facilitate restoration and preservation, and might also contribute to the determination of provenance.

genetics↗

bioRxiv2021

Entry Pathway for the Inverse Agonist Ligand in the G Protein-Coupled Receptor Rhodopsin

While the number of high-resolution structures of ligand-bound G protein-coupled receptors (GPCRs) has been steadily climbing, ligand binding and unbinding pathways remain largely undefined. The visual photoreceptor rhodopsin (Rho) represents a curious case among GPCRs because its primary ligand 11-cis-retinal (11CR) is an inverse agonist, which partitions into the bilayer and is likely to enter its orthosteric binding pocket through an intermembranous pathway. Light activates Rho by converting 11CR to all-trans-retinal (ATR), which serves as an agonist ligand. The light-triggered switch from the inactive to the active conformation creates two openings in the transmembrane region, suggesting pathways for exit of ATR and subsequent entry of 11CR to regenerate Rho. However, stabilization of an active ligand-free opsin conformation has been found to inhibit 11CR binding. Here we address this paradox of opsin regeneration with 11CR. We used genetic code expansion to engineer Rho mutants that serve as fluorescence sensors for measuring 11CR binding kinetics and energetics. We found mutations that alter a channel between transmembrane helices 5 and 6 (TM5/6) dramatically affect 11CR binding kinetics, but not ATR release kinetics. Our data provide direct experimental evidence for 11CR entry between TM5/6 in Rho that involves dynamic allosteric control of the ligand entry channel. Our findings can be extended to other visual pigments and a wide range of GPCRs with hydrophobic ligands that are hypothesized to enter their binding pockets through transmembrane pores.

biophysics↗

bioRxiv2019

Uveal Melanoma Oncogene CYSLTR2 Encodes a Constitutively Active GPCR Highly Biased Toward Gq Signaling

The G protein-coupled receptor (GPCR) cysteinyl-leukotriene receptor 2 (CysLTR2) with a single amino acid mutation at position 3.43 (Leu replaced with Gln at position 129 in transmembrane helix 3) causes uveal melanoma in humans. The ability of CysLTR2-L129Q to cause malignant transformation has been hypothesized to result from constitutive activity. We show that CysLTR2-L129Q is a constitutively active mutant (CAM) that strongly drives Gq/11 signaling pathways in melan-a melanocytes and in HEK293T cells in culture. However, the mutant receptor only very weakly recruits beta-arrestins 1 and 2. The mutant receptor displays profound signaling bias while avoiding arrestin-mediated downregulation. The mechanism of the signaling bias results from the creation of a hydrogen-bond network that stabilizes the active G protein signaling state through novel interactions with the highly-conserved NPxxY motif on helix 7. Furthermore, the mutation destabilizes a putative allosteric sodium-binding site that usually stabilizes the inactive state of GPCRs. Thus, the mutation has a dual role of promoting the active state while destabilizing inactivating allosteric networks. The high degree of constitutive activity renders existing orthosteric antagonist ligands of CysLTR2 ineffective as inverse agonists of the mutant. CysLTR2 is the first example of a GPCR oncogene that encodes a GPCR with constitutive highly biased signaling that can escape cellular downregulation mechanisms.

cancer biology↗

bioRxiv2019

High-Affinity Binding of Chemokine Analogs that Display Ligand Bias at the HIV-1 Co-receptor CCR5

The chemokine receptor CCR5 is a drug target to prevent transmission of HIV/AIDS. We studied four analogs of the native chemokine RANTES (CCL5) that have anti-HIV potencies of around 25 pM, which is more than four orders-of-magnitude higher than that of RANTES itself. It has been hypothesized that the ultra-high potency of the analogs is due to their ability to bind populations of receptors not accessible to native chemokines. To test this hypothesis, we developed a homogeneous dual-color fluorescence cross-correlation spectroscopy (FCCS) assay for saturation and competition binding experiments. The FCCS assay has the advantage that it does not rely on competition with radioactively labeled native chemokines used in conventional assays. We prepared site-specifically labeled fluorescent analogs using native chemical ligation of synthetic peptides, followed by bioorthogonal fluorescent labeling. We engineered a mammalian cell expression construct to provide fluorescently labeled CCR5, which was purified using a tandem immunoaffinity and size-exclusion chromatography approach to obtain monomeric fluorescent CCR5 in detergent solution. We found subnanomolar binding affinities for the two analogs 5P12-RANTES and 5P14-RANTES, and about twenty-fold reduced affinities for PSC-RANTES and 6P4-RANTES. Using homologous and heterologous competition experiments with unlabeled chemokine analogs, we conclude that the analogs all bind at the same binding site; whereas, the native chemokines (RANTES and MIP1) fail to displace bound fluorescent analogs even at tens of micromolar concentrations. Our results can be rationalized with de novo structural models of the N-terminal tails of the synthetic chemokines that adopt a different binding mode as compared to the parent compound.

biophysics↗