Biology subjects

Kay, M. S.

Publications and source records attributed to Kay, M. S..

9 recordsLinked to original sources

bioRxiv2025

A Divergent Cytochrome c in Malaria Parasites with an Anomalously Low Redox Potential

Eukaryotic cytochrome (cyt) c is a highly conserved mitochondrial protein central to cellular respiration, featuring a covalently attached hexacoordinate heme whose redox potential is tuned by axial His/Met ligands and surrounding residues to support electron transport chain (ETC) function. We have identified an unrecognized lineage of eukaryotic cyt c homologs in Apicomplexa, a phylum of intracellular pathogens that includes Plasmodium falciparum malaria parasites. P. falciparum cyt c-2 (Pfcyt c-2) exemplifies this divergent lineage and has an unusual pentacoordinate heme despite conservation of His/Met ligands. We determined that Pfcyt c-2 has a redox potential of -278 mV that is over 500 mV lower than canonical cyt c homologs (+250 mV) and contradicts a conserved ETC role. This anomalous redox potential is lower than any natural monoheme c-type cyt. Nevertheless, Pfcyt c-2 displays canonical thermostability and low-level peroxidase activity, while showing signs of elevated structural heterogeneity. These results reveal a new clade of eukaryotic cyt c variants with divergent biochemical properties and biological roles, opening new scaffolds for mechanistic discovery and redox engineering.

biochemistry↗

bioRxiv2024

Electron tomography visualization of HIV-1 virions trapped by fusion inhibitors to host cells in infected tissues

HIV-1 delivers its genetic material to infect a cell after fusion of the viral and host cell membranes, which takes place after the viral envelope (Env) binds host receptor and co-receptor proteins. Binding of host receptor CD4 to Env results in conformational changes that allow interaction with a host co-receptor (CCR5 or CXCR4). Further conformational rearrangements result in an elongated pre-hairpin intermediate structure in which Env is anchored to the viral membrane by its transmembrane region and to the host cell membrane by its fusion peptide. Although budding virions can be readily imaged by electron tomography (ET) of HIV-1-infected tissues and cultured cells, virions that are fusing (attached to host cells via pre-hairpin intermediates) are not normally visualized, perhaps because the process of membrane fusion is too fast to capture by EM. To image virions during fusion, we used fusion inhibitors to prevent downstream conformational changes in Env that lead to membrane fusion, thereby trapping HIV-1 virions linked to target cells by prehairpin intermediates. ET of HIV-1 pseudovirions bound to CD4+/CCR5+ TZM-bl cells revealed presumptive pre-hairpin intermediates as 2-4 narrow spokes linking a virion to the cell surface. To extend these results to a more physiological setting, we used ET to image tissues and organs derived from humanized bone marrow, liver, thymus (BLT) mice infected with HIV-1 and then treated with CPT31, a high-affinity D-peptide fusion inhibitor linked to cholesterol. Trapped HIV-1 virions were found in all tissues studied (small intestine, mesenteric lymph nodes, spleen, and bone marrow), and spokes representing pre-hairpin intermediates linking trapped virions to cell surfaces were similar in structure and number to those seen in the previous pseudovirus and cultured cell ET study. IMPORTANCETrapped and untrapped HIV-1 virions, both mature and immature, were distinguished by localizing spokes via 3D tomographic reconstructions of HIV-1 infected and fusion-inhibitor treated tissues of humanized mice. The finding of trapped HIV-1 virions in all tissues examined demonstrates a wide distribution of the CPT31 inhibitor, a desirable property for a potential therapeutic. In addition, the presence of virions trapped by spokes, particularly in vascular endothelial cells, demonstrates that fusion inhibitors can be used as markers for potential HIV-1-target cells within tissues, facilitating the mapping of HIV-1 target cells within the complex cellular milieu of infected tissues.

microbiology↗

bioRxiv2024

A stabilized MERS-CoV spike ferritin nanoparticle vaccine elicits robust and protective neutralizing antibody responses

Middle East respiratory syndrome coronavirus (MERS-CoV) was first identified as a human pathogen in 2012 and causes ongoing sporadic infections and outbreak clusters. Despite case fatality rates (CFRs) of over 30% and the pandemic potential associated with betacoronaviruses, a safe and efficacious vaccine has not been developed for prevention of MERS in at-risk individuals. Here we report the design, in vitro characterization, and preclinical evaluation of MERS-CoV antigens. Our lead candidate comprises a stabilized spike ectodomain displayed on a self-assembling ferritin nanoparticle that can be produced from a high-expressing, stable cell pool. This vaccine elicits robust antibody titers in BALB/c mice as measured by MERS- CoV pseudovirus and live-virus neutralization assays. Immunization of non-human primates (NHPs) with a single dose of Alhydrogel-adjuvanted vaccine elicits >103 geometric mean titer (GMT) of pseudovirus neutralizing antibodies that can be boosted with a second dose. These antibody levels are durable, with GMTs that surpass the post-prime levels for more than 5 months post-boost. Importantly, sera from these NHPs exhibit broad cross-reactivity against lentiviruses pseudotyped with spike proteins from MERS-CoV clades A, B, and C as well as a more distant pangolin merbecovirus. In transgenic mice expressing human DPP4, immunization provided dose-dependent protection against MERS-CoV lethal challenge, and in an established alpaca challenge model, immunization fully protected against MERS-CoV infection. This protein-based MERS-CoV nanoparticle vaccine is a promising candidate for advancement to clinical development to protect at-risk individuals and for future use in a potential outbreak setting.

biochemistry↗

bioRxiv2024

A bipartite interaction with the processivity clamp potentiates Pol IV-mediated TLS

Processivity clamps mediate polymerase switching for translesion synthesis (TLS). All three E. coli TLS polymerases interact with the {beta}2 processivity clamp through a conserved clamp-binding motif (CBM), which is indispensable for TLS. Notably, Pol IV also makes a unique secondary contact with the clamp through non-CBM residues. However, the role of this "rim contact" in Pol IV-mediated TLS remains poorly understood. Here we show that the rim contact is critical for TLS past strong replication blocks. In in vitro reconstituted Pol IV-mediated TLS, ablating the rim contact compromises TLS past 3-methyl dA, a strong block, while barely affecting TLS past N2-furfuryl dG, a weak block. Similar observations are also made in E. coli cells bearing a single copy of these lesions in the genome. Within lesion-stalled replication forks, the rim interaction and ssDNA binding protein cooperatively poise Pol IV to better compete with Pol III for binding to a cleft through its CBM. We propose that this bipartite clamp interaction enables Pol IV to rapidly resolve lesion-stalled replication through TLS at the fork, which reduces damage induced mutagenesis.

biochemistry↗

bioRxiv2023

A synthetic delivery vector for mucosal vaccination

The success of mRNA-based vaccines during the Covid-19 pandemic has highlighted the value of this new platform for vaccine development against infectious disease. However, the CD8+ T cell response remains modest with mRNA vaccines, and these do not induce mucosal immunity, which would be needed to prevent viral spread in the healthy population. To address this drawback, we developed a dendritic cell targeting mucosal vaccination vector, the homopentameric STxB. Here, we describe the highly efficient chemical synthesis of the protein, and its in vitro folding. This straightforward preparation led to a synthetic delivery tool whose biophysical and intracellular trafficking characteristics were largely indistinguishable from recombinant STxB. The chemical approach allowed for the generation of new variants with bioorthogonal handles. Selected variants were chemically coupled to several types of antigens derived from the mucosal viruses SARS-CoV-2 and type 16 human papillomavirus. Upon intranasal administration in mice, mucosal immunity, including resident memory CD8+ T cells and IgA antibodies was induced against these antigens. Our study thereby identifies a novel synthetic antigen delivery tool for mucosal vaccination with an unmatched potential to respond to an urgent medical need.

biochemistry↗

bioRxiv2020

Electron tomography visualization of HIV-1 fusion with target cells using fusion inhibitors to trap the pre-hairpin intermediate

Fusion of HIV-1 with the membrane of its target cell, an obligate first step in virus infectivity, is mediated by binding of the viral envelope (Env) spike protein to its receptors, CD4 and CCR5/CXCR4, on the cell surface. The process of viral fusion appears to be fast compared with viral egress and has not been visualized by electron microscopy (EM). To capture fusion events for EM, the process must be slowed or stopped by trapping Env-receptor binding at an intermediate stage. Here we describe using fusion inhibitors to trap HIV-1 virions attached to target cells by Envs in an extended pre-hairpin intermediate state. Electron tomography revealed HIV-1 virions bound to TZM-bl cells by 2-4 narrow spokes, with slightly more spokes present when evaluated with mutant virions that lacked the Env cytoplasmic tail. These results represent the first direct visualization of the hypothesized pre-hairpin intermediate and improve our understanding of Env-mediated HIV-1 fusion and infection of host cells.

cell biology↗

bioRxiv2020

Prevention and Treatment of SHIVAD8 Infection in Rhesus Macaques by a Potent D-peptide HIV Entry Inhibitor

Cholesterol-PIE12-trimer (CPT31) is a potent D-peptide HIV entry inhibitor that targets the highly conserved gp41 N-peptide pocket region. CPT31 exhibited strong inhibitory breadth against diverse panels of primary virus isolates. In a SHIV macaque model, CPT31 prevented infection from a single high-dose rectal challenge. In chronically infected animals, CPT31 monotherapy rapidly reduced viral load by ~2 logs before rebound occurred due to the emergence of drug resistance. In chronically infected animals with viremia initially controlled by combination antiretroviral therapy (cART), CPT31 monotherapy prevented viral rebound after discontinuation of cART. These data establish CPT31 as a promising new candidate for HIV prevention and treatment.

microbiology↗

bioRxiv2020

Conflicts with diarrheal pathogens trigger rapid evolution of an intestinal signaling axis

The pathogenesis of infectious diarrheal diseases is largely attributed to enterotoxin proteins that disrupt intestinal water absorption, causing severe dehydration. Despite profound health consequences, the impacts of diarrhea-causing microbes on the evolutionary history of host species are largely unknown. We investigated patterns of genetic variation in mammalian Guanylate Cyclase-C (GC-C), an intestinal receptor frequently targeted by bacterial enterotoxins, to determine how hosts might adapt in response to diarrheal infections. Under normal conditions, GC-C interacts with endogenous guanylin peptides to promote water secretion in the intestine, but signaling can be hijacked by bacterially-encoded heat-stable enterotoxins (STa) during infection, which leads to overstimulation of GC-C and diarrhea. Phylogenetic analysis in mammals revealed evidence of recurrent positive selection in the GC-C ligand-binding domain in primates and bats, consistent with selective pressures to evade interactions with STa. Using in vitro assays and transgenic intestinal organoids to model STa-mediated diarrhea, we show that GC-C diversification in these lineages results in substantial variation in toxin susceptibility. In bats, we observe a unique pattern of compensatory coevolution in the endogenous GC-C ligand uroguanylin, reflecting intense bouts of positive selection at the receptor-ligand interface. These findings demonstrate control of water physiology as a previously unrecognized interface for genetic conflict and reveal diarrheal pathogens as a source of selective pressure among diverse mammals.

evolutionary biology↗

bioRxiv2019

Characterization of resistance to a potent D-peptide HIV entry inhibitor

BackgroundPIE12-trimer is a highly potent D-peptide HIV-1 entry inhibitor that broadly targets group M isolates. It specifically binds the three identical conserved hydrophobic pockets at the base of the gp41 N-trimer with sub-femtomolar affinity. This extremely high affinity for the transiently exposed gp41 trimer provides a reserve of binding energy (resistance capacitor) to combat resistance via stepwise accumulation of modest affinity-disrupting mutations. Viral passaging in the presence of escalating PIE12-trimer concentrations ultimately selected for PIE12-trimer resistant populations, but required an extremely extended timeframe (>1 year) in comparison to other entry inhibitors. Eventually, HIV developed resistance to PIE12-trimer by mutating Q577 in the gp41 pocket.\n\nResultsUsing deep sequence analysis, we identified three mutations at Q577 (R, N and K) in our two PIE12-trimer resistant pools. Each point mutant is capable of conferring the majority of PIE12-trimer resistance seen in the polyclonal pools. Surface plasmon resonance studies demonstrated substantial affinity loss between PIE12-trimer and the Q577R-mutated gp41 pocket. A high-resolution x-ray crystal structure of PIE12 bound to the Q577R pocket revealed the loss of two hydrogen bonds, the repositioning of neighboring residues, and a small decrease in buried surface area. The Q577 mutations in an NL4-3 backbone decreased viral growth rates. Fitness was ultimately rescued in resistant viral pools by a suite of compensatory mutations in gp120 and gp41, of which we identified seven candidates from our sequencing data.\n\nConclusionsThese data show that PIE12-trimer exhibits a high barrier to resistance, as extended passaging was required to develop resistant virus with normal growth rates. The primary resistance mutation, Q577R/N/K, found in the conserved gp41 pocket, substantially decreases inhibitor affinity but also damages viral fitness. Identified candidate compensatory mutations in gp160 will be the focus of future mechanistic studies.

microbiology↗