Journal of Biological Chemistry

Intrinsically disordered regions (IDRs) of proteins are thought to be inherently sensitive to proteolysis and considered one of the key components constituting an efficient degron. Here we report that IDRs can also suppress protein degradation. Our recent study showed that yeast ribosomal proteins, while posttranslationally stable, are subject to cotranslational protein degradation (CTPD). In mapping the degron responsible for CTPD of ribosomal protein Rpl8A, we found that its N-terminal IDR suppresses CTPD, whereas the adjacent structured domain acts as a degron. We further assessed the N-terminal IDRs of 9 other yeast proteins and found that they all inhibit CTPD. These results suggest that suppression of CTPD is likely a generic function of N-terminal IDRs. Moreover, we showed that the N-terminal IDR of human ribosomal protein hRpl7A also functions as a stabilizer against CTPD in human cells. When transplanted to the N-terminus of cystic fibrosis transmembrane conductance regulator (CFTR), the N-terminal IDR of hRpl7A reduces CTPD of CFTR by more than 80%. Thus, the stabilizer function of N-terminal IDRs is conserved from yeast to human. Using mass spectrometry, we demonstrated that HSP70 chaperone proteins Ssa and Ssb bind to the N-terminal IDR of Rpl8A. These data suggest that N-terminal IDRs may inhibit CTPD through recruiting HSP70 chaperone proteins to nascent chains, thereby facilitating cotranslational folding. Our study unveils a new role for IDRs in suppressing CTPD.

Protein misfolding and aggregation, including amyloid fibril formation, underlie a large class of human diseases including prominent neurological disorders such as Alzheimer's and Parkinson's disease. A small number of human proteins have been identified that inhibit amyloidogenesis. One such protein is sTREM2, a soluble receptor liberated from microglia, the resident macrophages of the central nervous system. The extracellular domain of TREM2 is shed upon proteolytic cleavage to create sTREM2, which has previously been shown to inhibit amyloid-{beta} aggregation in vitro. TREM2 is also expressed by intestinal macrophages, which are known to directly bind the bacterial amyloid curli and mount cytokine responses upon exposure. Here we show that sTREM2 is a sub-stoichiometric inhibitor of CsgA amyloidogenesis, CsgA being the major protein component of curli that drives biofilm formation in uropathogenic Escherichia coli and many other proteobacteria. In vitro, sTREM2 potently and sub-stoichiometrically inhibited CsgA amyloidogenesis in a dose-dependent manner. Kinetic modeling indicated that sTREM2 slowed primary and secondary nucleation, rather than altering fiber elongation. When added exogenously to bacterial growth medium, sTREM2 significantly suppressed curli-dependent pellicle biofilm formation without affecting bacterial growth. These findings establish sTREM2 as a member of the small group of human proteins capable of inhibiting bacterial functional amyloidogenesis, suggesting that gut-resident TREM2-expressing macrophages, which are already known to interact with curli, may employ sTREM2 as a physiologically relevant defense against bacterial amyloid formation.

N-linked glycoprotein processing critically depends on the trimming of -1,2 mannose residues, a key step required both for glycoprotein maturation along the secretory pathway and for targeting defective glycoproteins to endoplasmic reticulum-associated degradation (ERAD). Mammalian cells express seven Class I -1,2 mannosidases, yet their individual roles remain poorly defined, particularly for ManIA, ManIB, and ManIC, which were originally considered Golgi-resident maturation enzymes. Here, we re-evaluated the subcellular localization and functional contributions of these three mannosidases to glycoprotein quality control and maturation. We found that ManIA, ManIB, and ManIC localize predominantly to quality control vesicles (QCVs), previously identified by our group, whereas only ManIC displays a substantial Golgi population. Surprisingly, each enzyme is confined to a different vesicular population. All three enzymes promote ERAD targeting of misfolded model glycoproteins, albeit with different substrate preferences. In addition, they redundantly support the maturation and cell-surface delivery of a model glycoprotein. Most strikingly, in vitro analyses revealed that ManIA, ManIB, and ManIC preferentially trim a properly folded model glycoprotein rather than its denatured form. This is the opposite of the substrate preference that we previously observed for ERManI, EDEM1, and EDEM2. These findings support a model in which ERManI and the EDEMs selectively process misfolded glycoproteins to promote their recognition by the proximal lectin OS-9 and subsequent ERAD. In contrast, ManIA, ManIB and ManIC can slowly process misfolded glycoproteins but act rapidly on properly folded glycoprotein molecules, releasing them from ER-Golgi lectin-mediated retention or retrieval pathways, thereby promoting forward trafficking and maturation in the Golgi.

The SNARE proteins syntaxin/SNAP-25B (t-SNAREs) and synaptobrevin (v-SNARE) contain motifs that assemble into four-helix bundles to drive synaptic vesicle exocytosis; SNAP-25B contributes two helices, D1 and D2. The sequence in which these motifs interact remains unresolved. To address this, we used fluorescence anisotropy of SNARE motifs to conduct real-time order-of-addition experiments and found that the order in which components are mixed can determine whether on- or off-pathway complexes are formed. Beginning with soluble SNARE fragments alone, the first step in assembly is the binding of D1 to syntaxin, followed by the binding of synaptobrevin and D2, where the latter motif acts as a gatekeeper to control v-SNARE*t-SNARE interactions. We then examined the impact of two regulatory factors, Munc18 and the MUN domain of Munc13-1. Strikingly, in the presence of Munc18, all four isolated SNARE motifs must be present at the same time for assembly to occur, revealing a concerted mechanism, while the Munc13-1 fragment was without effect. We created C-SCORE, which reports assembly of the two SNARE motifs of SNAP-25B via FRET, and confirmed that in the presence of Munc18, SNARE assembly becomes concerted. Munc18 also disaggregated syntaxin, potentially contributing to its activation. Finally, our findings regarding SNARE motif folding were well-correlated with function using full-length SNAREs in in vitro lipid mixing assays. Hence, Munc18 acts as a molecular chaperone that directly promotes the concurrent assembly of SNARE proteins into functional fusion machines.

Cellular redox homeostasis plays a critical role in regulating protein function, including chaperone activity, through reversible oxidation of cysteine and methionine residues. Previously, we found that budding yeast cells experiencing redox imbalance due to inactivated thioredoxin reductase (trr1{Delta}) activate the heat shock response and induce hyperaccumulation of the small heat shock protein/sequestrase Hsp42 with misfolded proteins. Building on that finding, this study identified cysteine 127 (C127) within Hsp42 as a redox-active residue that becomes oxidized in trr1{Delta} cells, upon treatment with the powerful oxidant hydrogen peroxide, or by exposure to the cysteine crosslinker divinyl sulfone (DVSF). In trr1{Delta} cells, C127 oxidation promoted intermolecular disulfide bond formation and contributed to Hsp42 homo-oligomerization. We show that stable oligomerization requires both the prion-like domain (PrLD) and C127 oxidation. While Hsp42-GFP formed prominent persistent foci in trr1{Delta} cells, replacement of C127 with non-thiol reactive serine decreased foci formation. Furthermore, the C127S mutation diminished Hsp42 oligomerization and sedimentability. Immunoprecipitation coupled with mass spectrometry analysis revealed that Hsp42 in trr1{Delta} cells preferentially associated with mitochondrial precursor proteins accumulated in the cytoplasm, as well as oxidation-reduction enzymes. The observed client selectivity was altered by the C127S mutation that diversified the spectrum of Hsp42-associated proteins. Collectively, these findings identify Cys127 as a redox-active switch that regulates Hsp42 assembly, foci formation, stability, and client specificity in response to oxidative stress.

Glycogen storage disease type 1a (GSD1a) is an autosomal recessive Mendelian disorder that can be caused by missense variants in glucose-6-phosphatase catalytic subunit 1 (G6PC1). Although hundreds of missense variants have been identified, the vast majority are of unknown clinical significance, and the molecular mechanism(s) of bona fide pathogenic variants are ill-defined. We combine bioinformatic data and clinical associations with the protein language model AlphaMissense to guide mechanistic exploration of 78 missense variants at 55 residue positions using robust biochemical and biophysical assays to distill general principles of enzyme dysfunction. Correlation analysis established a strong linear relationship between folded G6PC1 abundance and catalytic capacity for most variants. Pathogenic variants within this paradigm were linked to compromised stability and activation of the unfolded protein response. However, outliers characterized by relatively high abundance, yet low activity clustered to a network of sidechains adjacent to the active site that allosterically modulate catalysis. Contextualized by recent high-resolution structures and AlphaFold modeling, our holistic analysis of G6PC1 in vitro metrics facilitates clinical (re)classification of variants according to explicit molecular phenotypes and identifies therapeutic design directions. Moreover, our approach illustrates a blueprint for variant characterization that integrates computational prediction with experimental validation to discover disease etiology.

AMP deaminase (AMPD) plays an integral role in fructose metabolism via its regulation by ATP, GTP and phosphate (Pi). The fructose catabolic pathway consumes ATP, producing ADP, which is further metabolized to AMP, triggering a cascade of reactions initiated by AMPD. This degradative pathway results in the final product uric acid, which is associated with metabolic acidosis, mitochondrial dysfunction, and gout. Understanding the regulation of the human liver AMPD isozyme (hAMPD2-2) under physiological conditions and under fructose consumption conditions will enable the design of targeted therapeutics to block the accumulation of uric acid. We report the first successful expression and purification from Escherichia coli of both the full-length and catalytic domains of hAMPD2-2. Steady-state kinetics confirmed allosteric activation by ATP of both the full-length and catalytic domains of hAMPD2-2 at physiological ATP concentrations (2-5 mM), suggesting that the allosteric ATP-binding site is located in the catalytic domain. Competitive inhibition by GTP of the ATP-activated enzyme, with Ki values of 74 and 101 M for the full-length and catalytic domains, respectively, was also consistent with this regulatory model. Pi, previously described in yeast AMPD as a competitive inhibitor, was shown to play a more nuanced role, that of enhancing inhibition of hAMPD2-2 when the enzyme is complexed to GTP, via competition at the ATP allosteric site. Pi binding thus further inhibits the pathway under normal physiological conditions, limiting production of cellular uric acid unless and until Pi and GTP levels are low.

Bi-allelic mutations in MSTO1 are linked to clinical disease phenotypes characteristic of mitochondrial dysfunction, including ataxia and muscular dystrophy. Consistent with this, MSTO1 patient-derived fibroblasts have fragmented mitochondria and a striking loss of mtDNA. Although MSTO1 has been implicated in regulating mitochondrial fusion, the molecular function of this cytosolic protein in vertebrate cells remains unclear. Using the auxin-inducible degradation (AID) system we demonstrate that MSTO1-FLAG-AID protein is rapidly depleted to almost undetectable levels. Importantly, these cells recapitulate the fragmented mitochondrial phenotype observed in patients and thus are a valuable model of disease. Surprisingly, prior to any changes in mitochondria, we show that MSTO1-depleted cells have a significant decrease in TRiC levels, an essential cytosolic ATP-dependent chaperone required to fold diverse substrates, including actin and tubulin. We reveal that TRiC is also reduced in MSTO1 patient-derived fibroblasts, indicating that loss of TRiC may contribute to disease pathophysiology. We further demonstrate that knockdown of TRiC leads to a decrease in MSTO1 protein levels and remarkably, was sufficient to induce a fragmented mitochondrial phenotype, independent of changes in tubulin or actin. This reveals a previously unrecognized connection between TRiC and mitochondrial homeostasis. Using co-immunoprecipitation we found that MSTO1 interacts with the TRiC chaperone. We also observe accumulation of early TRiC assembly subcomplexes in the absence of MSTO1 suggesting that MSTO1 facilitates assembly of TRiC. Together, our findings identify MSTO1 as a TRiC assembly factor and connect mitochondrial defects caused by MSTO1-depletion to the loss of TRiC. SignificanceMSTO1, a protein linked to myopathy and ataxia, has been thought to control mitochondrial fusion, although the molecular mechanism is unknown. Using rapid depletion of MSTO1, we found that mitochondrial fragmentation appears only after six days. Significantly, the levels of the essential cytosolic chaperonin TRiC are reduced within two days of MSTO1 depletion. Directly depleting TRiC reproduces the fragmented mitochondrial phenotype seen with loss of MSTO1, consistent with a model where mitochondrial dysfunction is a downstream consequence of impaired protein folding rather than a direct effect of MSTO1 loss. We show that MSTO1 is required for assembly of TRiC, identifying it as a long-sought assembly factor for this macromolecular protein complex.

Weak, transient molecular interactions are ubiquitous amongst biological molecules. Regulatability by intracellular concentrations, localization, physical properties of the cytoplasm, and other factors is central to their importance. Here, we measure co-localization between fluorescently labeled proteins, Miro2 and {beta}-Pix, to assess their effective binding affinity and its dependence on protein concentration. Co-localization between the two is quantifiable due to the mitochondrial localization of Miro2, which allows the otherwise cytosolic {beta}-Pix to be drawn to mitochondria. Determining the fraction of overexpressed {beta}-Pix co-localizing with mitochondria as a function of overexpressed Miro2 levels allows calculation of an effective dissociation constant from a mass-action binding model. Both the fit to the model and the effective dissociation constant for Miro2 and {beta}-Pix binding depend on the concentration of {beta}-Pix, which suggests that one or more model assumptions do not hold. Additional experiments reveal that competitive binding and/or {beta}-Pix sequestration by Git1 are possible explanations for the {beta}-Pix concentration dependence. Our modeling strategy is readily extendable to ectopic localization of proteins to mitochondria and other compartments for the assessment of co-localization with a binding partner. This reveals fundamental properties of protein-protein interactions and how they may be regulated by protein concentration and other intracellular conditions.

The role of tau fibril structure in seeding and propagation of aggregation remains a central unresolved question in tauopathy biology. While non-proteinaceous cofactors are increasingly observed in patient-derived tau filaments, whether they actively determine fibril structure and function is not well understood. Here, we show that zinc, a divalent cation dysregulated in Alzheimers disease (AD), can drive fundamentally different aggregation and seeding outcomes depending on tau sequence context. Using heparin-free conditions, we compared full-length 2N4R tau (residues 1-441) with an AD-tau fragment (residues 304-380) corresponding to the ordered fibril core. Strikingly, Zn2+ exerted opposite effects on these constructs: it accelerated aggregation, increased fibril length, and enhanced cellular seeding for AD-tau, while slowing aggregation, shortening fibrils, and suppressing seeding for full-length tau. These findings demonstrate that cofactor effects are not intrinsic properties of the cofactor itself, but emerge from its interplay with tau sequence and conformational constraints. More broadly, our results support a model in which small-molecule cofactors act as active architects of fibril structure and function, suggesting that chemically distinct environments could generate structurally and biologically distinct tau strains in disease.

Poly (ADP-ribose) Polymerase 1 (PARP1) is an abundant nuclear enzyme that dynamically engages chromatin in diverse cellular scenarios. In the context of DNA repair, PARP1 becomes enzymatically activated and subsequently attaches ADP-ribose units onto various proteins, including histones, to signal and coordinate the DNA damage response. In the absence of DNA damage, PARP1 modulates chromatin structure by directly binding to nucleosomes, however, the molecular basis of this interaction is unknown. Here, we define a distinct, enzymatically inactive mode of PARP1 chromatin binding, in which the Zn1, Zn2, Zn3, and BRCT domains cooperatively bind nucleosomal linker DNA and drive compaction of undamaged chromatin. This binding mode does not trigger catalytic activation and therefore is insensitive to PARP inhibitors (PARPi). Together, our results support a model in which PARP1 associates with the genome in an inactive state to compact chromatin and to surveil for DNA lesions. SummaryPARP1 engages undamaged chromatin in a distinct binding mode that results in chromatin compaction but does not lead to enzymatic activation.

Analysis of RNA-binding proteome data from different bacterial species revealed many cell wall metabolic enzymes cross-linking to RNA in vivo, hinting that these proteins directly bind RNA. Surprisingly, penicillin-binding proteins (PBPs) were also abundantly identified as putative RNA-binding proteins. The cell surface localisation properties of many of these proteins therefore beg the question at what stage of their cellular life cycle these proteins interact with RNA and what the functional significance is. Here, we characterised the RNA-binding activity of PBP2a, the alternative transpeptidase that confers {beta}-lactam resistance in MRSA. Using in vivo RNA-binding assays, we show that PBP2a interacts with hundreds of transcripts without apparent sequence specificity. Computational analyses identified a possible RNA-binding cleft in PBP2a proximal to its active site. Mutation of only two predicted positively charged residues located in this cleft substantially reduced cross-linking in vivo, implying that RNA recognition is largely dictated by RNA backbone interactions. While PBP2a does not regulate RNA steady-state levels, RNA-binding appears important for proper protein function: an RNA-binding deficient mutant exhibits reduced oxacillin resistance. These findings establish PBP2a as an RNA-binding protein in vivo and provide a framework to investigate how this non-canonical interaction may relate to cell wall biogenesis and {beta}-lactam resistance.

Matrix metalloproteinase 13 (MMP13) is a zinc-dependent protease that plays key roles in extracellular matrix remodeling. Like several other MMPs, MMP13 has been shown to interact with heparan sulfate (HS), a highly sulfated glycosaminoglycan found at the cell surface and in the extracellular matrix, but the significance of the interaction remains unknown. Here we report that while zymogen and mature forms of MMP13 both bind HS with high affinity, their interactions with HS display markedly different characteristics in terms of preferred HS structure and binding kinetics. By structure-guided mutagenesis, we identified a large HS-binding site of MMP13 consists of 10 residues in the hemopexin domain, 3 residues in the catalytic domain, and 2 residues in the linker region. While these basic residues participate in binding to both zymogen and mature forms of MMP13, the relative contribution of many residues differs substantially between the two forms, which likely contributes to their distinct HS-binding characteristics. Binding of HS to mature MMP13 resulted in selective inhibition of the collagenase activity of MMP13 in a length- and sulfation-dependent manner, but the binding had no effect on degradation of non-collagen substrates. Mechanistically, the inhibitory effect of HS likely results from reduced interdomain flexibility after binding of HS, and/or HS-induced dimerization of MMP13. In sum, our study establishes HS as a multifaceted regulator of MMP13 activity, and discovers that the HS-binding site of MMP13 is a novel exosite that can be targeted to inhibits its collagenase activity.

AbstractPhospholipid asymmetry is a hallmark of mammalian cell membranes and reflects the selective distribution of distinct phospholipid species between the two leaflets of the lipid bilayer. Although this asymmetry is tightly maintained, the membrane proteins whose functions depend on it remain largely unknown. To perturb phospholipid asymmetry experimentally, we expressed a constitutively active phospholipid scramblase and thereby identified transient receptor potential melastatin 8 (TRPM8) as an ion channel regulated by this membrane property. Activation of TRPM8 by both l-menthol and innocuous cold was markedly suppressed following disruption of phospholipid asymmetry. Likewise, selective depletion of phosphatidylserine (PS), a phospholipid enriched in the cytoplasmic leaflet, using a cytosolically targeted PS decarboxylase attenuated TRPM8 activation, indicating that cytoplasmic PS is required for proper TRPM8 function. Mechanistically, our findings suggest that cytoplasmic PS supports efficient TRPM8 activation by maintaining the biochemical state of the channel. Together, these findings identify TRPM8 as a phospholipid asymmetry-dependent ion channel and establish an experimental strategy for systematically identifying membrane proteins regulated by phospholipid asymmetry. This work provides a foundation for future studies investigating the biological significance of this fundamental membrane property.

Phosphatidic acid (PA) phosphatase (PAP) is an enzyme that plays a major role in lipid signaling by controlling the cellular levels of two lipid secondary messengers: its substrate, PA, and its product, diacylglycerol. Two types of mammalian PAPs have been reported to date. Type 1 PAP (PAP1) is an Mg2+-dependent, N-ethylmaleimide (NEM)-sensitive cytosolic enzyme (EC 3.1.3.4), whereas type 2 PAP (PAP2), also known as phospholipid phosphate (PLPP) (EC 3.1.3.113), is an Mg2+-independent, NEM-insensitive transmembrane protein. PAP2 also hydrolyzes other bioactive lipids such as lyso-PA (LPA), sphingosine-1-phosphate (S1P), and ceramide-1-phosphate (C1P). Here, we purified human phosphatase orphan 2 (PHOSPHO2), a putative cytosolic phosphatase containing a haloacid dehalogenase-like domain, and characterized its enzymological properties in vitro. Purified PHOSPHO2 displays Mg2+-dependent, NEM-sensitive phosphatase activities toward PA, LPA, S1P, C1P, and glycerol-3-phosphate (G3P) in vitro. Moreover, PHOSPHO2 showed substrate selectivity for PA molecular species containing shorter saturated fatty acids such as lauric acid and myristic acid, or polyunsaturated fatty acids such as docosahexaenoic acid and arachidonic acid. The PAP activity of PHOSPHO2, but not its other phosphatase activities, was strongly enhanced in the presence of phosphatidylcholine and phosphatidylethanolamine, major components of the cell membranes. These results indicate that mammalian PHOSPHO2 is a novel cytosolic PLPP that primarily functions as a PAP on cytoplasm-facing membranes.

Transthyretin (TTR) is a secreted protein associated with cardiac and other amyloid diseases via misfolding. We have previously shown that agitation of human TTR solutions at neutral pH results in aggregation and fibril formation. Here we report that agitation-induced aggregation of TTR from species with very different heart rates (Annas hummingbird, hbTTR, and African elephant, aeTTR) differs from that of human TTR (huTTR). Aggregation of hbTTR is slow and favors formation of smaller, fibrillar aggregates, while aeTTR aggregation is rapid and favors larger, more amorphous particles. Spherical, early-stage oligomeric intermediates were found for all variants by mass photometry and electron microscopy. The slow aggregation of hbTTR matches its resistance to denaturation by 8 M urea. The widely different aggregation behavior exhibited by these naturally occurring TTR variants in response to mechanical agitation under close to physiological conditions provides insight into how small sequence differences can contribute to the evolutionary fitness of different animals.

SH2 domains are phosphotyrosine-binding modules that play a critical role in cell signaling by mediating protein-protein interactions. While tyrosine phosphorylation has been shown to impact SH2 domain function in signaling, the specific effects of phosphorylation at different sites within the domain remain poorly understood. In this study, we selected two conserved regions of tyrosine phosphorylation within SH2 domains, near conserved binding interface residues, and developed approaches to evaluate the impact of those sites on ligand binding. Using a modified dot blot assay to screen phosphomimic mutations, we studied specific tyrosine residues within the PTPN11-N, LYN, and SYK-C SH2 domains, finding that the PTPN11 N-terminal site (Y63) modulates the specificity, reducing binding of physiologically relevant substrates. Our findings provide new insights into the regulatory mechanisms governing SH2 domain function and highlight the importance of site-specific phosphorylation in modulating protein-protein interactions in cell signaling pathways.

Rifamycins inhibit bacterial transcription by targeting RNA polymerase (RNAP), but their clinical effectiveness is limited by the rapid emergence of resistance caused by mutations within the rifamycin-binding pocket. Rifamycin B (Rif B), one of the earliest discovered members of this antibiotic family and a precursor of clinically used derivatives, has remained poorly characterized because of its chemical instability and relatively weak antibacterial activity. Here, we revisit Rif B using biochemical and structural approaches. We show that Rif B remains sufficiently stable under assay conditions and retains inhibitory activity against RNAP variants carrying clinically relevant rifampicin-resistance mutations. We report the first crystal structure of Rif B and determine the structure of Rif B bound to bacterial RNAP. The structures reveal that the distinctive C-4 O-carboxymethyl substituent of Rif B forms an intramolecular interaction in the free molecule but establishes a salt bridge with fork loop 2 of the RNAP {beta}-subunit upon binding. This additional interaction explains the reduced sensitivity of Rif B to resistance-associated substitutions and identifies the C-4 position as an underexplored site for rational rifamycin modification. These findings redefine Rif B as a mechanistically distinct rifamycin scaffold and provide new insights for developing inhibitors targeting rifampicin-resistant RNAP.

Altered cholesterol metabolism is a recognized hallmark of cancer, but systemic modulation has not yet delivered significant clinical results. Accumulating evidence shows that cholesterol plays distinct roles across diverse cellular membranes, suggesting that site-specific modulation may produce superior therapeutic outcomes. Cholesterol is associated with gastric cancer (GC), but the mechanistic link is complex and no effective cholesterol-targeted therapy has been developed. Here, we report that cholesterol levels in GC cells are site-specifically elevated in the inner leaflet of the plasma membrane (IPM). This elevated IPM cholesterol constitutively activates Wnt-{beta}-catenin signaling to drive cell survival and proliferation. Mechanistically, Niemann-Pick C1-like 1 (NPC1L1), which is highly expressed in GC patient tissues and cell lines, acts as a cholesterol flippase to raise IPM cholesterol levels, facilitating ligand-independent {beta}-catenin signalosome formation. Ezetimibe, a clinically approved NPC1L1 inhibitor, blocks this flippase activity, lowers IPM cholesterol levels, and suppresses {beta}-catenin signaling. Ezetimibe treatment induces apoptosis in GC cells while sparing normal primary gastric epithelial cells, which exhibit low levels of NPC1L1 and IPM cholesterol. Collectively, these results suggest that site-specific modulation of cellular cholesterol is a viable approach to developing safe and effective therapies for cancers linked to local cholesterol elevation.

Infections caused by the opportunistic fungal pathogen Aspergillus fumigatus pose a serious public health system burden. The inherent limitations in existing antifungal drugs in conjunction with a rising emergence of antifungal resistance emphasizes an urgent need to identify and target alternative pathways crucial to survival and virulence. Targeting the fungal mannitol biosynthesis enzymes provides a promising avenue in the development of new antifungals due to the multifaceted roles mannitol fulfils in the fungal life cycle. However, a distinct lack of available structural information for these enzymes has hindered drug discovery efforts. We report the first crystal structure of mannitol-2-dehydrogenase from A. fumigatus in an unbound monomeric state (1.8 [A]) and bound to its co-factor NADH (2.1 [A]), via. a large, central cavity lined with positively charged residues that readily accommodates NADH. This interaction is further stabilised by a network of hydrogen bond interactions and {pi}-{pi} stacking between Phe45 and the nicotinamide ring of NADH. Furthermore, rigorous kinetic characterisation of A. fumigatus mannitol-2-dehydrogenase demonstrates the dose-dependent inhibitory activity of 1,4-benzoquinone, a cysteine-modifying small molecule inhibitor (IC50 = 1.2 {+/-} 0.2 nM). In addition, intact MS and proteomic analysis further reveal that 1,4-benzoquinone modifies up to five cysteine residues of mannitol-2-dehydrogenase and displays antifungal activity against A. fumigatus, which is enhanced in combination with a front-line antifungal voriconazole. From this work, we have established the foundations for a novel antifungal drug discovery avenue that targets the fungal mannitol biosynthesis pathway to better treat aspergillosis and related pathogenic infections.