Biology subjects

Fahim, A.

Publications and source records attributed to Fahim, A..

4 recordsLinked to original sources

bioRxiv2025

Reconstitution of glycan-driven MHC I recycling reveals calreticulin as mediator between TAPBPR and tapasin

Protein folding in the endoplasmic reticulum (ER) is essential for about one-third of the mammalian proteome. N-linked glycosylation and subsequent glycan remodeling barcodes glycoproteins during their maturation in the ER. Major histocompatibility complex class I (MHC I) molecules, key for adaptive immunity, rely on a dedicated quality control cycle that involves specialized chaperones and glycan-modifying enzymes for their maturation and loading of immunogenic peptides. However, the functional interplay of the MHC I editors tapasin as part of the peptide-loading complex (PLC), TAP-binding protein-related (TAPBPR), the UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1), and calreticulin in glycan-dependent transfer of MHC I clients has not been determined in a reconstituted system. With isolated components, we show that transfer of peptide-receptive MHC I from the downstream quality control factor TAPBPR back to tapasin depends on the recognition of the monoglucosylated glycan of MHC I by calreticulin. While calreticulins C-terminal acidic helix is dispensable for disengaging reglucosylated MHC I from TAPBPR, it is essential for docking MHC I onto tapasin. Our data provide a mechanistic basis for glycan-surveillance by calreticulin necessary for retrograde trafficking of misfolded or suboptimally loaded MHC I that escaped the first quality control at the PLC and were trapped by TAPBPR. Such finetuned dynamic network of glycan-dependent and MHC I-specific chaperones guarantees maturation of MHC I molecules and highlight the fundamental processes driving ER protein quality control. TeaserOur study dissects the mechanistic network of dedicated chaperones and glycan modifiers that quality-control MHC I. Significance StatementThe immune system relies on major histocompatibility complex (MHC) class I molecules to present protein fragments from within cells, enabling detection of infection or disease. This study uncovers how a dynamic, glycan-dependent chaperone network in the endoplasmic reticulum (ER) orchestrates the recycling of misfolded or suboptimally loaded MHC I. Using isolated components, the work shows how monoglucosylation of MHC I glycans by UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1) allows calreticulin to mediate transfer from the post-ER chaperone TAPBPR back to the ER-resident chaperone tapasin as part of the peptide-loading complex. These findings illuminate the coordinated action of TAPBPR, UGGT1, calreticulin, and tapasin-ERp57 in MHC I quality control, offering new insights in immune surveillance and how its disruption may contribute to disease.

immunology↗

bioRxiv2025

Single-molecule dynamics reveal ATP binding alone powers substrate translocation by an ABC transporter

ATP-binding cassette (ABC) transporters are molecular machines involved in diverse physiological processes, including antigen processing by TAP, a key component of adaptive immunity. TAP and its bacterial homolog TmrAB use ATP to translocate peptides across membranes, yet the precise mechanism linking ATP binding to substrate movement remains unclear. Here, we employ a single-molecule FRET sensor to visualize single translocation events by individual ABC transporters, overcoming the limitations of ensemble averaging. This approach reveals that substrate transport is driven by a conformational switch from the inward-to the outward-facing state. Using a slow-turnover TmrAB variant, we demonstrate that ATP binding alone, even in the absence of Mg2+, is sufficient to drive a single round of peptide translocation. Cryo-EM structures of wild-type and slow-turnover TmrAB show that ATP binding induces the outward-facing conformation even without Mg2+. In wild-type TmrAB, this conformational transition supports a single translocation event, whereas Mg2+-dependent ATP hydrolysis is required to reset the transporter. These findings establish a direct mechanistic link between ATP binding and substrate translocation at single-molecule resolution, providing new insights into the catalytic cycle of ABC transporters.

biochemistry↗

bioRxiv2025

US6 hijacks the peptide-loading complex by trapping transporter-chaperone dynamics

Adaptive immune responses are initiated by major histocompatibility class I (MHC I) presentation of antigenic peptides on the cell surface. This process relies on the peptide-loading complex (PLC), a dynamic transporter-multichaperone assembly in the endoplasmic reticulum (ER), to ensure high-fidelity selection, editing, and loading of peptides onto MHC I heterodimers1. The PLC is the primary target for viral immune evasion2, elicited in particular by human cytomegalovirus (HCMV)3, causing lifelong infections with severe risks for immunocompromised individuals. While the overall architecture of the PLC is known4, how its activity is jeopardized by viral immune evasins remains unclear. Here, we present the 2.59-2.88 [A] cryogenic electron microscopy structure of native human PLC associated with the HCMV immune evasin US6. US6 inhibits the heterodimeric transporter associated with antigen processing (TAP1/2) by latching its transmembrane helix laterally onto TAP2 and using its central disulfide-rich domain to mimic a translocating peptide. This effectively plugs the ER-lumenal exit and locks TAP in an outward-facing open conformation with closed nucleotide-binding domains and asymmetrically occluded ATP and ADP. The structure also highlights the role of the unique N-terminal transmembrane domains of TAP as dynamic scaffolds that recruit the MHC I-specific chaperone tapasin by clamping its transmembrane helix to the core transmembrane domain of each transporter subunit. Our findings uncover the molecular mechanism of US6-mediated viral immune evasion and reveal potential targets for therapeutic modulation of antigen presentation in cancer and infectious diseases.

immunology↗

bioRxiv2019

Stochastic Modeling Reveals Kinetic Heterogeneity in Post-replication DNA Methylation

DNA methylation is a heritable epigenetic modification that plays an essential role in mammalian development. Genomic methylation patterns are dynamically maintained, with DNA methyltransferases mediating inheritance of methyl marks onto nascent DNA over cycles of replication. A recently developed experimental technique employing immunoprecipitation of bromodeoxyuridine labeled nascent DNA followed by bisulfite sequencing (Repli-BS) measures post-replication temporal evolution of cytosine methylation, thus enabling genome-wide monitoring of methylation maintenance. In this work, we combine statistical analysis and stochastic mathematical modeling to analyze Repli-BS data from human embryonic stem cells. We estimate site-specific kinetic rate constants for the restoration of methyl marks on >10 million uniquely mapped cytosines within the CpG (cytosine-phosphate-guanine) dinucleotide context across the genome using Maximum Likelihood Estimation. We find that post-replication remethylation rate constants span approximately two orders of magnitude, with half-lives of per-site recovery of steady-state methylation levels ranging from shorter than ten minutes to five hours and longer. Furthermore, we find that kinetic constants of maintenance methylation are correlated among neighboring CpG sites. Stochastic mathematical modeling provides insight to the biological mechanisms underlying the inference results, suggesting that enzyme processivity and/or collaboration can produce the observed kinetic correlations. Our combined statistical/mathematical modeling approach expands the utility of genomic datasets and disentangles heterogeneity in methylation patterns arising from replication-associated temporal dynamics versus stable cell-to-cell differences.

genomics↗