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147


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.
PMCID:10827928
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.
PMCID:10665946
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.
PMCID:10617467
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.
PMCID:10575587
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

Structure of an endogenous mycobacterial MCE lipid transporter

Chen, James; Fruhauf, Alice; Fan, Catherine; Ponce, Jackeline; Ueberheide, Beatrix; Bhabha, Gira; Ekiert, Damian C
To replicate inside macrophages and cause tuberculosis, Mycobacterium tuberculosis must scavenge a variety of nutrients from the host1,2. The mammalian cell entry (MCE) proteins are important virulence factors in M. tuberculosis1,3, where they are encoded by large gene clusters and have been implicated in the transport of fatty acids4-7 and cholesterol1,4,8 across the impermeable mycobacterial cell envelope. Very little is known about how cargos are transported across this barrier, and it remains unclear how the approximately ten proteins encoded by a mycobacterial mce gene cluster assemble to transport cargo across the cell envelope. Here we report the cryo-electron microscopy (cryo-EM) structure of the endogenous Mce1 lipid-import machine of Mycobacterium smegmatis-a non-pathogenic relative of M. tuberculosis. The structure reveals how the proteins of the Mce1 system assemble to form an elongated ABC transporter complex that is long enough to span the cell envelope. The Mce1 complex is dominated by a curved, needle-like domain that appears to be unrelated to previously described protein structures, and creates a protected hydrophobic pathway for lipid transport across the periplasm. Our structural data revealed the presence of a subunit of the Mce1 complex, which we identified using a combination of cryo-EM and AlphaFold2, and name LucB. Our data lead to a structural model for Mce1-mediated lipid import across the mycobacterial cell envelope.
PMID: 37495693
ISSN: 1476-4687
CID: 5594732

Compilation of reported protein changes in the brain in Alzheimer's disease

Askenazi, Manor; Kavanagh, Tomas; Pires, Geoffrey; Ueberheide, Beatrix; Wisniewski, Thomas; Drummond, Eleanor
Proteomic studies of human Alzheimer's disease brain tissue have potential to identify protein changes that drive disease, and to identify new drug targets. Here, we analyse 38 published Alzheimer's disease proteomic studies, generating a map of protein changes in human brain tissue across thirteen brain regions, three disease stages (preclinical Alzheimer's disease, mild cognitive impairment, advanced Alzheimer's disease), and proteins enriched in amyloid plaques, neurofibrillary tangles, and cerebral amyloid angiopathy. Our dataset is compiled into a searchable database (NeuroPro). We found 848 proteins were consistently altered in 5 or more studies. Comparison of protein changes in early-stage and advanced Alzheimer's disease revealed proteins associated with synapse, vesicle, and lysosomal pathways show change early in disease, but widespread changes in mitochondrial associated protein expression change are only seen in advanced Alzheimer's disease. Protein changes were similar for brain regions considered vulnerable and regions considered resistant. This resource provides insight into Alzheimer's disease brain protein changes and highlights proteins of interest for further study.
PMCID:10368642
PMID: 37491476
ISSN: 2041-1723
CID: 5592142