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Proteomics from compartment-specific APEX2 labeling in Mycobacterium tuberculosis reveals Type VII secretion substrates in the cell wall

Jaisinghani, Neetika; Previti, Mary L; Andrade, Joshua; Askenazi, Manor; Ueberheide, Beatrix; Seeliger, Jessica C
The cell wall of mycobacteria plays a key role in interactions with the environment. Its ability to act as a selective filter is crucial to bacterial survival. Proteins in the cell wall enable this function by mediating the import and export of diverse metabolites, from ions to lipids to proteins. Identifying cell wall proteins is an important step in assigning function, especially as many mycobacterial proteins lack functionally characterized homologues. Current methods for protein localization have inherent limitations that reduce accuracy. Here we showed that although chemical labeling of live cells did not exclusively label surface proteins, protein tagging by the engineered peroxidase APEX2 within live Mycobacterium tuberculosis accurately identified the cytosolic and cell wall proteomes. Our data indicate that substrates of the virulence-associated Type VII ESX secretion system are exposed to the periplasm, providing insight into the currently unknown mechanism by which these proteins cross the mycobacterial cell envelope.
PMID: 37967559
ISSN: 2451-9448
CID: 5644222

Using Constellation Pharmacology to Characterize a Novel α-Conotoxin from Conus ateralbus

Neves, Jorge L B; Urcino, Cristoval; Chase, Kevin; Dowell, Cheryl; Hone, Arik J; Morgenstern, David; Chua, Victor M; Ramiro, Iris Bea L; Imperial, Julita S; Leavitt, Lee S; Phan, Jasmine; Fisher, Fernando A; Watkins, Maren; Raghuraman, Shrinivasan; Tun, Jortan O; Ueberheide, Beatrix M; McIntosh, J Michael; Vasconcelos, Vitor; Olivera, Baldomero M; Gajewiak, Joanna
The venom of cone snails has been proven to be a rich source of bioactive peptides that target a variety of ion channels and receptors. α-Conotoxins (αCtx) interact with nicotinic acetylcholine receptors (nAChRs) and are powerful tools for investigating the structure and function of the various nAChR subtypes. By studying how conotoxins interact with nAChRs, we can improve our understanding of these receptors, leading to new insights into neurological diseases associated with nAChRs. Here, we describe the discovery and characterization of a novel conotoxin from Conus ateralbus, αCtx-AtIA, which has an amino acid sequence homologous to the well-described αCtx-PeIA, but with a different selectivity profile towards nAChRs. We tested the synthetic αCtx-AtIA using the calcium imaging-based Constellation Pharmacology assay on mouse DRG neurons and found that αCtx-AtIA significantly inhibited ACh-induced calcium influx in the presence of an α7 positive allosteric modulator, PNU-120596 (PNU). However, αCtx-AtIA did not display any activity in the absence of PNU. These findings were further validated using two-electrode voltage clamp electrophysiology performed on oocytes overexpressing mouse α3β4, α6/α3β4 and α7 nAChRs subtypes. We observed that αCtx-AtIA displayed no or low potency in blocking α3β4 and α6/α3β4 receptors, respectively, but improved potency and selectivity to block α7 nAChRs when compared with αCtx-PeIA. Through the synthesis of two additional analogs of αCtx-AtIA and subsequent characterization using Constellation Pharmacology, we were able to identify residue Trp18 as a major contributor to the activity of the peptide.
PMID: 38535458
ISSN: 1660-3397
CID: 5644892

Similar brain proteomic signatures in Alzheimer's disease and epilepsy

Leitner, Dominique; Pires, Geoffrey; Kavanagh, Tomas; Kanshin, Evgeny; Askenazi, Manor; Ueberheide, Beatrix; Devinsky, Orrin; Wisniewski, Thomas; Drummond, Eleanor
The prevalence of epilepsy is increased among Alzheimer's Disease (AD) patients and cognitive impairment is common among people with epilepsy. Epilepsy and AD are linked but the shared pathophysiological changes remain poorly defined. We aim to identify protein differences associated with epilepsy and AD using published proteomics datasets. We observed a highly significant overlap in protein differences in epilepsy and AD: 89% (689/777) of proteins altered in the hippocampus of epilepsy patients were significantly altered in advanced AD. Of the proteins altered in both epilepsy and AD, 340 were altered in the same direction, while 216 proteins were altered in the opposite direction. Synapse and mitochondrial proteins were markedly decreased in epilepsy and AD, suggesting common disease mechanisms. In contrast, ribosome proteins were increased in epilepsy but decreased in AD. Notably, many of the proteins altered in epilepsy interact with tau or are regulated by tau expression. This suggests that tau likely mediates common protein changes in epilepsy and AD. Immunohistochemistry for Aβ and multiple phosphorylated tau species (pTau396/404, pTau217, pTau231) showed a trend for increased intraneuronal pTau217 and pTau231 but no phosphorylated tau aggregates or amyloid plaques in epilepsy hippocampal sections. Our results provide insights into common mechanisms in epilepsy and AD and highlights the potential role of tau in mediating common pathological protein changes in epilepsy and AD.
PMID: 38289539
ISSN: 1432-0533
CID: 5627492

Systematic Fe(II)-EDTA Method of Dose-Dependent Hydroxyl Radical Generation for Protein Oxidative Footprinting

Chapman, Jessica R; Paukner, Max; Leser, Micheal; Teng, Kai Wen; Koide, Shohei; Holder, Marlene; Armache, Karim-Jean; Becker, Chris; Ueberheide, Beatrix; Brenowitz, Michael
Correlating the structure and dynamics of proteins with biological function is critical to understanding normal and dysfunctional cellular mechanisms. We describe a quantitative method of hydroxyl radical generation via Fe(II)-ethylenediaminetetraacetic acid (EDTA)-catalyzed Fenton chemistry that provides ready access to protein oxidative footprinting using equipment commonly found in research and process control laboratories. Robust and reproducible dose-dependent oxidation of protein samples is observed and quantitated by mass spectrometry with as fine a single residue resolution. An oxidation analysis of lysozyme provides a readily accessible benchmark for our method. The efficacy of our oxidation method is demonstrated by mapping the interface of a RAS-monobody complex, the surface of the NIST mAb, and the interface between PRC2 complex components. These studies are executed using standard laboratory tools and a few pennies of reagents; the mass spectrometry analysis can be streamlined to map the protein structure with single amino acid residue resolution.
PMID: 38049117
ISSN: 1520-6882
CID: 5595392

Unlatching of the stem domains in the Staphylococcus aureus pore-forming leukocidin LukAB influences toxin oligomerization

Ilmain, Juliana K; Perelman, Sofya S; Panepinto, Maria C; Irnov, Irnov; Coudray, Nicolas; Samhadaneh, Nora; Pironti, Alejandro; Ueberheide, Beatrix; Ekiert, Damian C; Bhabha, Gira; Torres, Victor J
Staphylococcus aureus (S. aureus) is a serious global pathogen that causes a diverse range of invasive diseases. S. aureus utilizes a family of pore-forming toxins, known as bi-component leukocidins, to evade the host immune response and promote infection. Among these is LukAB (leukocidin A/leukocidin B), a toxin that assembles into an octameric β-barrel pore in the target cell membrane, resulting in host cell death. The established cellular receptor for LukAB is CD11b of the Mac-1 complex. Here, we show that hydrogen voltage-gated channel 1 is also required for the cytotoxicity of all major LukAB variants. We demonstrate that while each receptor is sufficient to recruit LukAB to the plasma membrane, both receptors are required for maximal lytic activity. Why LukAB requires two receptors, and how each of these receptors contributes to pore-formation remains unknown. To begin to resolve this, we performed an alanine scanning mutagenesis screen to identify mutations that allow LukAB to maintain cytotoxicity without CD11b. We discovered 30 mutations primarily localized in the stem domains of LukA and LukB that enable LukAB to exhibit full cytotoxicity in the absence of CD11b. Using crosslinking, electron microscopy, and hydroxyl radical protein footprinting, we show these mutations increase the solvent accessibility of the stem domain, priming LukAB for oligomerization. Together, our data support a model in which CD11b binding unlatches the membrane penetrating stem domains of LukAB, and this change in flexibility promotes toxin oligomerization.
PMID: 37802313
ISSN: 1083-351x
CID: 5614202

Gene recoding by synonymous mutations creates promiscuous intragenic transcription initiation in mycobacteria

Hegelmeyer, Nuri K; Parkin, Lia A; Previti, Mary L; Andrade, Joshua; Utama, Raditya; Sejour, Richard J; Gardin, Justin; Muller, Stephanie; Ketchum, Steven; Yurovsky, Alisa; Futcher, Bruce; Goodwin, Sara; Ueberheide, Beatrix; Seeliger, Jessica C
PMID: 37787543
ISSN: 2150-7511
CID: 5614172

The REEP5/TRAM1 complex binds SARS-CoV-2 NSP3 and promotes virus replication

Li, Jie; Gui, Qi; Liang, Feng-Xia; Sall, Joseph; Zhang, Qingyue; Duan, Yatong; Dhabaria, Avantika; Askenazi, Manor; Ueberheide, Beatrix; Stapleford, Kenneth A; Pagano, Michele
Generation of virus-host protein-protein interactions (PPIs) maps may provide clues to uncover SARS-CoV-2-hijacked cellular processes. However, these PPIs maps were created by expressing each viral protein singularly, which does not reflect the life situation in which certain viral proteins synergistically interact with host proteins. Our results reveal the host-viral protein-protein interactome of SARS-CoV-2 NSP3, NSP4, and NSP6 expressed individually or in combination. Furthermore, REEP5/TRAM1 complex interacts with NSP3 at ROs and promotes viral replication. The significance of our research is identifying virus-host interactions that may be targeted for therapeutic intervention.
PMID: 37768083
ISSN: 1098-5514
CID: 5614142

Mitochondrial DNA breaks activate an integrated stress response to reestablish homeostasis

Fu, Yi; Sacco, Olivia; DeBitetto, Emily; Kanshin, Evgeny; Ueberheide, Beatrix; Sfeir, Agnel
Mitochondrial DNA double-strand breaks (mtDSBs) lead to the degradation of circular genomes and a reduction in copy number; yet, the cellular response in human cells remains elusive. Here, using mitochondrial-targeted restriction enzymes, we show that a subset of cells with mtDSBs exhibited defective mitochondrial protein import, reduced respiratory complexes, and loss of membrane potential. Electron microscopy confirmed the altered mitochondrial membrane and cristae ultrastructure. Intriguingly, mtDSBs triggered the integrated stress response (ISR) via the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) by DELE1 and heme-regulated eIF2α kinase (HRI). When ISR was inhibited, the cells experienced intensified mitochondrial defects and slower mtDNA recovery post-breakage. Lastly, through proteomics, we identified ATAD3A-a membrane-bound protein interacting with nucleoids-as potentially pivotal in relaying signals from impaired genomes to the inner mitochondrial membrane. In summary, our study delineates the cascade connecting damaged mitochondrial genomes to the cytoplasm and highlights the significance of the ISR in maintaining mitochondrial homeostasis amid genome instability.
PMID: 37832546
ISSN: 1097-4164
CID: 5604282

A noncanonical function of SKP1 regulates the switch between autophagy and unconventional secretion

Li, Jie; Krause, Gregory J; Gui, Qi; Kaushik, Susmita; Rona, Gergely; Zhang, Qingyue; Liang, Feng-Xia; Dhabaria, Avantika; Anerillas, Carlos; Martindale, Jennifer L; Vasilyev, Nikita; Askenazi, Manor; Ueberheide, Beatrix; Nudler, Evgeny; Gorospe, Myriam; Cuervo, Ana Maria; Pagano, Michele
Intracellular degradation of proteins and organelles by the autophagy-lysosome system is essential for cellular quality control and energy homeostasis. Besides degradation, endolysosomal organelles can fuse with the plasma membrane and contribute to unconventional secretion. Here, we identify a function for mammalian SKP1 in endolysosomes that is independent of its established role as an essential component of the family of SCF/CRL1 ubiquitin ligases. We found that, under nutrient-poor conditions, SKP1 is phosphorylated on Thr131, allowing its interaction with V1 subunits of the vacuolar ATPase (V-ATPase). This event, in turn, promotes V-ATPase assembly to acidify late endosomes and enhance endolysosomal degradation. Under nutrient-rich conditions, SUMOylation of phosphorylated SKP1 allows its binding to and dephosphorylation by the PPM1B phosphatase. Dephosphorylated SKP1 interacts with SEC22B to promote unconventional secretion of the content of less acidified hybrid endosomal/autophagic compartments. Collectively, our study implicates SKP1 phosphorylation as a switch between autophagy and unconventional secretion in a manner dependent on cellular nutrient status.
PMID: 37831778
ISSN: 2375-2548
CID: 5604232

Antibody responses to dietary antigens are accompanied by specific plasma cells in the infant thymus

Cordero, Hector; Hess, Jacob; Nitschki, Elio; Kanshin, Evgeny; Roy, Poulomi; Shihab, Ronzon; Kalfa, David M; Bacha, Emile A; Ueberheide, Beatrix; Zorn, Emmanuel
BACKGROUND:Human infants develop IgG responses to dietary antigens during the first 2 years of life. Yet, the source of these antibodies is unclear. In previous studies we reported on the thymus as a unique functional niche for plasma cells (PCs) specific to environmental antigens. OBJECTIVE:We sought to examine whether PCs specific to dietary antigens are detected in the infant thymus. METHODS:We tested IgG reactivity to 112 food antigens and allergens in the serum of 20 neonates and infants using microarrays. The presence of PC-secreting IgG specific to the most prominent antigens was then assessed among thymocytes in the same cohort. Using an LC-MS proteomics approach, we looked for traces of these antigens in the thymus. RESULTS:Our studies first confirmed that cow's milk proteins are prevalent targets of serum IgG in early life. Subjects with the highest serum IgG titers to cow's milk proteins also harbored IgG-producing PCs specific to the same antigens in the thymic niche. Furthermore, we detected multiple peptide fragments of cow's milk antigens in the thymus. Lastly, we verified that both serum IgG and IgG secreted by thymic PCs recognized the peptide epitopes found in the thymus. CONCLUSIONS:Our studies reveal the presence of antibody-secreting PCs specific to common dietary antigens in the infant thymus. The presence of these antigens in the thymus suggested that activation and differentiation of specific PCs occurred in this organ. Further studies are now warranted to evaluate the possible implication of these cells in tolerance to dietary antigens.
PMID: 37406823
ISSN: 1097-6825
CID: 5539242