Biology subjects

Loo, J. A.

Publications and source records attributed to Loo, J. A..

15 recordsLinked to original sources

bioRxiv2026

Cryo-EM provides insight into how the Staphylococcus aureus IsdH receptor removes hemin from the hemoglobin:haptoglobin complex

Staphylococcus aureus extracts hemin from human hemoglobin (Hb) to overcome host-imposed iron limitation. How it recovers Hb-bound hemin from the hemoglobin:haptoglobin (Hb:Hp) complex, the major circulating form of Hb outside red blood cells, remains unclear. Here we use cryo-electron microscopy, biophysical measurements, and solution kinetics to define how the S. aureus IsdH surface receptor extracts hemin from Hb:Hp. A 3.1 [A] cryo-EM structure of Hb:Hp bound by full-length IsdH reveals that its N-terminal NEAT domain (N1) anchors it to Hb, whereas its downstream N2N3 extraction unit engages {beta}Hb to remove its hemin. The receptor engages Hb:Hp differently than isolated Hb, because N-linked glycans on haptoglobin bias the extraction unit toward {beta}Hb, sterically occluding its access to Hb while still permitting engagement by N1. Kinetic assays show that IsdH actively accelerates hemin release from Hb:Hp. Three-dimensional variability analysis indicates that this likely occurs via a dynamic interface in which receptor motions reposition the extraction unit relative to {beta}Hb, collectively supporting a model in which IsdH transiently perturbs the F-helix to promote hemin extraction. Alignment of that model with a previously determined CD163:Hb:Hp structure shows how IsdH may disrupt Hb:Hp recognition by macrophage and monocyte CD163 receptors, helping to explain how it may hinder clearance of Hb:Hp from circulation. In aggregate, these results help define the structural basis for hemin extraction from Hb:Hp and how IsdH may subvert receptor-mediated clearance of the Hb:Hp complex. SignificanceStaphylococcus aureus scavenges hemin from host hemoglobin to proliferate, yet most extracellular hemoglobin is sequestered in hemoglobin:haptoglobin (Hb:Hp) complexes, which are rapidly cleared from circulation. To clarify how bacteria access hemin in this context, we now show that the IsdH surface receptor is structurally adapted to extract hemin from Hb:Hp. Our results indicate that IsdH uses distinct NEAT domains to anchor to Hb and selectively extract hemin from {beta}Hb. This {beta}Hb selectivity is shaped by haptoglobin N-glycans and enhances microbial access to iron. These findings provide a mechanistic framework for targeting heme acquisition as an anti-virulence strategy.

microbiology↗

bioRxiv2026

Cross-Platform Assessment of Sub-50 nm Nanopipette Emitters for Native Electrospray Ionization Mass Spectrometry

Native mass spectrometry (nMS) is well established for measuring protein masses and stoichiometries using nano-electrospray ionization (nESI), yet salt adduction and source activation energies can limit routine measurements. In this study, we benchmark submicron quartz nanopipette nESI emitters (<50 nm internal diameter) across three mass spectrometry platforms (quadrupole-time-of-flight, quadrupole-Orbitrap, and tribrid-Orbitrap platforms) and a wide protein range (14.5-800 kDa). We analysed the intrinsically disordered protein alpha synuclein ( S; 14.5 kDa) and holo-myoglobin (17 kDa) over a range of concentrations (10 M-1 nM) and capillary voltages to determine limits of detection and define a gentle operating regime. We additionally observed reduced Na adduction and preservation of the Zn 2+-bound metalloproteoform of carbonic anhydrase II (29 kDa). Proteins and protein complexes spanning the mid-to-high mass range including ovalbumin (~44 kDa), malate dehydrogenase (~70 kDa), transferrin (80 kDa), glutamate dehydrogenase (~350 kDa), {beta}-galactosidase (~465 kDa), and GroEL (~800 kDa), were readily detected using nanopipette emitters. Compared with conventional 1-2 m internal diameter borosilicate emitters, quartz nanopipettes provided higher signal-to-noise ratios and fewer adducts. Finally, direct analysis of clarified bacterial lysate expressing -synuclein yielded a clear monomeric charge-state distribution, demonstrating compatibility with complex biological matrices. Collectively, these results establish quartz nanopipette nESI as an instrument-portable, salt-tolerant approach suitable for routine nMS analysis across a broad range of protein molecular weights and sample complexities.

biochemistry↗

bioRxiv2026

Native Mass Spectrometry-Based Proteomics Reveals the Mechanism of Hemophore Release by Pathogenic Corynebacterium diphtheriae

Iron is an essential micronutrient for nearly all forms of life, including pathogenic microbes that must acquire it from their host during infection. At the host-pathogen interface, humans restrict microbial access to iron through nutritional immunity, which many pathogens overcome by secreting hemophores that scavenge extracellular heme (iron protoporphyrin IX). However, identifying hemophores and other ligand-binding proteins in complex proteomes remains challenging using conventional peptide-based bottom-up mass spectrometry (MS). Here, we introduce ProteoMIX (Proteome Analysis by Mixing), a function-based native top-down proteomics workflow that combines slow-mixing mode native MS with charge reduction to identify ligand-binding proteins directly from complex mixtures. Applying ProteoMIX to the Corynebacterium diphtheriae exoproteome identified ChtA30-314, an abundant soluble hemophore generated by proteolytic processing of the surface-exposed ChtA heme receptor. Using native top-down MS sequencing, cell fractionation, and gene deletion, we show that the protease DIP2069 releases ChtA30-314 by removing ChtAs transmembrane helix, producing a soluble proteoform that delivers heme to support microbial growth. In contrast, the related paralog ChtC is not processed, enabling C. diphtheriae to generate localization-specific heme-binding proteoforms from related gene products. Extending this approach to Staphylococcus aureus, ProteoMIX also revealed soluble IsdA hemophores that coexist with surface-anchored variants, demonstrating the generality of the method and suggesting that protease-mediated hemophore release may operate across gram-positive pathogens.

microbiology↗

bioRxiv2025

GFAP Proteolysis in TBI: Linking Novel Modified Cleavage Products to Astrocyte Pathology and Patient Outcomes

Glial fibrillary acidic protein (GFAP) is a significant clinical biomarker of traumatic brain injury (TBI), yet understanding the nature, timing, and impact of its degraded and modified products would inform clinical utility. We report novel GFAP breakdown products (BDPs) and post-translational modifications (PTMs) that are unique to TBI including fragment- and patient-specific citrullination signatures that destabilize GFAP filaments. GFAP and its fragments were sequenced by mass spectrometry (MS) from severe TBI patients cerebrospinal fluid (CSF) and sera, identifying two distinct TBI-specific jointly generated product sets within the rod-domain covering coil1 (20-26kDa) and coil2 (15-19kDa). Their endings differed from GFAP fragments described in Alzheimers and Alexander disease. Label-free quantification showed biofluid co-product abundance differences with coil1-BDPs enriched over coil2-BDPs. Coil imbalance was independently confirmed by immunoblot densitometry in twenty-three TBI patients. Measurements over ten days showed injury day peaks of full-length and end-clipped GFAP fragments, while proteolytic 37/39kDa and small fragments remained elevated. Six-month outcome (Extended Glasgow Outcome Scale) correlated with GFAP fragments, whereas levels of uncleaved and end-clipped GFAP did not. A human astrocyte culture trauma model provided mechanistic insights linking fluid GFAP levels, subcellular localization, and injury-induced astrocytopathy. A new cleavage site between the two coils was identified through selective epitope loss. Coil1-BDPs were fluid-released post-injury, while coil2-BDPs remained intracellular. Their cellular retention was explained by non-filamentous aggregation of citrulline-modified coil2-BDPs in pathological astrocytes. Trauma-triggered proteolysis involved calpains and caspases in specific astrocyte injury states using protease inhibitors and live-dye-reporter imaging. These novel data link TBI biofluid GFAP fragments with assembly-defective GFAP aggregation in pathological astrocytes. Clinical TBI outcome correlated with GFAP degradation rather than with overall GFAP release. These translational findings indicate that TBI biomarker GFAP undergoes degradation and modification alongside astrocyte pathology, providing a new conceptual framework for investigating biomarker-associated astrocyte proteinopathy potentially linked to post-traumatic neurodegeneration.

neuroscience↗

bioRxiv2025

Structural basis for L-isoaspartyl-containing protein recognition by the PCMTD1 cullin-RING E3 ubiquitin ligase

A major type of spontaneous protein damage that accumulates with age is the formation of kinked polypeptide chains with L-isoaspartyl residues. Mitigating this damage is necessary for maintaining proteome stability and prolonging organismal survival. While repair through methylation by PCMT1 has been previously shown to suppress L-isoaspartyl accumulation, we provide an additional mechanism for L-isoaspartyl maintenance through PCMTD1, a cullin-RING ligase (CRL). We combined cryo-EM, native mass spectrometry, and biochemical assays to provide insight on how the assembly and architecture of PCMTD1 in the context of a CRL complex fulfils this alternative mechanism. We show that the PCMTD1 CRL complex specifically binds L-isoaspartyl residues when bound to AdoMet. This work provides evidence for a growing class of E3 ubiquitin ligases that recognize spontaneous covalent modifications as potential substrates for ubiquitylation and subsequent proteasomal degradation. eTOC BlurbLimiting the accrual of L-isoaspartyl damaged proteins is essential during aging. While this is thought to be mediated solely by the repair activity of the protein, PCMT1, Pang et al. now demonstrate that a related protein, PCMTD1, functions as a cullin-RING ligase to selectively target L-isoaspartyl-damaged substrates for potential regulation by the ubiquitylation-proteosomal system. HighlightsO_LIAtomic cryo-EM structure of CRL5-PCMTD1 determined C_LIO_LIArchitecture of PCMTD1 when complexed as a CRL supports ubiquitylation activity C_LIO_LIPCMTD1 recognizes L-isoaspartyl residues as a recruitment motif for potential CRL activity C_LIO_LIRecognition of L-isoaspartyl residues is dependent on cofactor engagement C_LI

biochemistry↗

bioRxiv2025

Allosteric Modulation of Pathological Ataxin-3 Aggregation: A Path to Spinocerebellar Ataxia Type-3 Therapies

Spinocerebellar ataxia type 3 (SCA3) is a rare inherited neurodegenerative disease caused by the expansion of a polyglutamine repeat in the protease ataxin-3 (Atx3). Despite extensive knowledge of the downstream pathophysiology, no disease-modifying therapies are currently available to halt disease progression. The accumulation of protein inclusions enriched in the polyQ-expanded Atx3 in neurons suggests that inhibiting its self-assembly may yield targeted therapeutic approaches. Here it is shown that a supramolecular tweezer, CLR01, binds to a lysine residue on a positively charged surface patch of the Atx3 catalytic Josephin domain. At this site, the binding of CLR01 decreases the conformational fluctuations of the distal flexible hairpin. This results in reduced exposure of the nearby aggregation-prone region, which overlaps with the substrate ubiquitin binding site and primes Atx3 self-assembly, ultimately delaying Atx3 amyloid fibril formation and reducing the secondary nucleation rate, a process linked to fibril proliferation and toxicity. These effects translate into the reversal of synapse loss in a SCA3 cultured cortical neuron model, an improved locomotor function in a C. elegans SCA3 model, and a delay in disease onset, accompanied by reduced severity of motor symptoms in a SCA3 mouse model. This study provides critical insights into Atx3 self-assembly, revealing a novel allosteric site for designing CLR01-inspired therapies targeting pathological aggregation pathways while sparing essential functional sites. These findings emphasize that targeting allosteric sites in amyloid-forming proteins may offer unique opportunities to develop safe therapeutic strategies for various protein misfolding disorders.

biophysics↗

bioRxiv2024

The exoproteome and surfaceome of toxigenic Corynebacterium diphtheriae 1737 and its response to iron-restriction and growth on human hemoglobin

Toxin-producing Corynebacterium diphtheriae strains are the etiological agent of the severe upper respiratory disease, diphtheria. A global phylogenetic analysis revealed that biotype gravis is particularly lethal as it produces diphtheria toxin and a range of other virulence factors, particularly when it encounters low levels of iron at sites of infection. To gain insight into how it colonizes its host we have identified iron-dependent changes in the exoproteome and surfaceome of C. diphtheriae strain 1737 using a combination of whole-cell fractionation, intact cell surface proteolysis, and quantitative proteomics. In total, we identified 1,425 of the predicted 2,265 (63%) proteins encoded by its reference genome. For each protein we quantified its degree of secretion and surface-exposure, revealing that exoproteases and hydrolases predominate in the exoproteome, while the surfaceome is enriched with adhesins, particularly DIP2093. Our analysis provides insight into how components in the heme-acquisition system are positioned, showing pronounced surface-exposure of the strain-specific ChtA/ChtC paralogues and high secretion of the species-conserved heme-binding HtaA protein suggesting it functions as a hemophore. Profiling the response of the exoproteome and surfaceome after microbial exposure to human hemoglobin and iron limitation reveals potential virulence factors that may be expressed at sites of infection. Data are available via ProteomeXchange with identifier PXD051674.

biochemistry↗

bioRxiv2022

The metabolite alpha-ketobutyrate increases health and life spans by activating AMPK

Aging is a complex process that is directly related to human health and disease. The extraordinary finding that aging is malleable, as shown in model organisms whose life and health spans are extended by specific gene mutations or dietary or pharmacological perturbations 1-3, has offered enormous hope for our understanding and treatment of aging and related diseases. Although many molecules have been identified that can extend the lifespan of model organisms, few have been shown to alleviate age-related symptoms or illness in mammals 4. Here we show that supplementation with the endogenous metabolite -ketobutyrate (-KB) increases the lifespan of adult C. elegans. Using Gelfree DARTS-PROTOMAP, we identified microtubule-actin cross-linking factor (MACF1) that was protected against proteolysis in the presence of -KB. MACF1 belongs to the spectraplakin family of giant, evolutionarily conserved proteins with versatile functions 5, but their link to longevity regulation has not been explored. -KBs longevity effect in C. elegans is abrogated by loss-of-function mutation in vab-10, encoding the worm ortholog of mammalian MACF1 6. Like -KB treatment, vab-10 knockdown activates AMP-activated protein kinase (AMPK), and AMPK is required for -KB effects on longevity. The findings suggest a model in which -KB increases longevity by activating AMPK via VAB-10/MACF1 modulation. -KB also delays aging in mammals, increasing the lifespan of aged male mice and the healthspan of both male and female animals. Targeting of broadly expressed scaffolding proteins in connection to cellular energy homeostasis seems to be a clever way that nature has devised for metabolite signals to impinge upon multiple organ and tissue systems, which may have utility for controlling aging and related diseases.

physiology↗

bioRxiv2022

mTORC2 Interactome and Localization Determine Aggressiveness of High-Grade Glioma Cells through Association with Gelsolin

The mTOR complex 2 (mTORC2) has been implicated as a key regulator of glioblastoma cell migration. However, the roles of mTORC2 in the migrational control process have not been entirely elucidated. Here we elaborate that active mTORC2 is crucial for GBM cell motility. Inhibition of mTORC2 impaired cell movement and negatively affected microfilaments and microtubules functions. We also aimed to characterize important players involved in the regulation of cell migration and other mTORC2-mediated cellular processes in GBM cells through the proteomic and bioinformatic analyses. Therefore, we quantitatively characterized the alteration of mTORC2 interactome under selective conditions using affinity-purification mass spectrometry (AP-MS) in glioblastoma. We demonstrated that changes in cell migration ability specifically altered mTORC2-associated proteins. Gelsolin (GSN) was identified as one of the most dynamic proteins. The mTORC2-GSN linkage was mostly highlighted in high-grade glioma cells and shown to connect functional mTORC2 to multiple proteins responsible for directional cell movement in GBM. GSN also mediated membrane localization of mTORC2. Loss of GSN disconnected mTORC2 from numerous cytoskeletal proteins but did not affect mTORC2 integrity. In addition, we reported 86 stable mTORC2 interactome involved with diverse molecular functions, predominantly cytoskeletal remodeling in GBM. Our findings help expand future opportunities for predicting the highly migratory phenotype of brain cancers in the clinical investigation.

cancer biology↗

bioRxiv2022

Dynamic acylome reveals metabolite driven modifications in Syntrophomonas wolfei

Syntrophomonas wolfei is an anaerobic syntrophic microbe that degrades short-chain fatty acids to acetate, hydrogen, and/or formate. This thermodynamically unfavorable process proceeds through a series of reactive acyl-Coenzyme A species (RACS). In other prokaryotic and eukaryotic systems, the production of intrinsically reactive metabolites correlates with acyl-lysine modifications, which have been shown to play a significant role in metabolic processes. Analogous studies with syntrophic bacteria, however, are relatively unexplored and we hypothesize that highly abundant acylations could exist in S. wolfei proteins, corresponding to the RACS derived from degrading fatty acids. Here, by mass spectrometry-based proteomics (LC-MS/MS), we characterize and compare acylome profiles of two S. wolfei subspecies grown on different carbon substrates. Because modified S. wolfei proteins are sufficiently abundant for post-translational modification (PTM) analyses without antibody enrichment, we could identify types of acylations comprehensively, observing six types (acetyl-, butyryl-, 3-hydroxybutyryl-, crotonyl-, valeryl-, hexanyl-lysine), two of which have not been reported in any system previously. All of the acyl-PTMs identified correspond directly to RACS in fatty acid degradation pathways. A total of 369 sites of modification were identified on 237 proteins. Changing the carbon substrate altered the acylation profile. Moreover, structural studies and in vitro acylation assays of a heavily modified enzyme, acetyl-CoA transferase, provided insight on the possible impact of these acyl-protein modifications. Our findings link protein acylation by RACS to shifts in cellular metabolism.

microbiology↗

bioRxiv2022

Amyloid fibrils in frontotemporal lobar degeneration with TDP-43 inclusions are composed of TMEM106B, rather than TDP-43

FTLD is the third most common neurodegenerative condition, following only Alzheimers and Parkinsons diseases. FTLD typically presents in 45-64-year-olds with behavioral changes or progressive decline of language skills. The subtype FTLD-TDP is characterized by certain clinical symptoms and pathological neuronal inclusions detected by TDP-43 immunoreactivity. Here, we extracted amyloid fibrils from brains of four patients, representing four out of five FTLD-TDP subclasses and determined their near-atomic resolution structures by cryo-EM. Unexpectedly, all amyloid fibrils examined are composed of a 135-residue C-terminal fragment of TMEM106B, a lysosomal membrane protein previously implicated as a genetic risk factor for FTLD-TDP. In addition to TMEM106B fibrils, abundant non-fibrillar aggregated TDP-43 is present, as revealed by immunogold labeling. Our observations confirm that FTLD-TDP is an amyloid-involved disease and suggest that amyloid involvement in FTLD-TDP is of protein TMEM106B, rather than of TDP-43.

molecular biology↗

bioRxiv2020

Secreted and cell surface proteome analysis of pathogenic Corynebacterium diphtheriae reveals proteins relevant to virulence

Corynebacterium diphtheriae is responsible for the severe upper respiratory tract disease, diphtheria, which can be fatal in healthy individuals without proper treatment. Its interaction with the infected host is crucial to its virulence, secreting diphtheria toxin and a variety of machinery to acquire nutrients for further import into the cell. Additionally, it shares a conserved iron import mechanism with related pathogenic actinobacteria such as Mycobacterium tuberculosis, and contains sortase-mediated anchored cell wall proteins similar to other gram-positive bacteria. Information obtained from secreted and cell-surface proteomes are relevant for the study of diphtheria and related bacterial infections. Mass spectrometry-based proteomics measurements on samples of pathogenic Corynebacterium diphtheriae culture supernatant and cell-surface digested proteins identified greater than 3 times more proteins than a previous similar study. The diphtheria toxin was identified, as well as pathologically relevant proteins involved in iron-uptake and cell adhesion. For instance, 17 proteins predicted to be on the iron-regulated DtxR promoter were identified in cells grown under different iron concentrations, opening the door to performing comparative quantitative proteomics studies on samples where iron sources are modulated. Results of this study serve as a repository for future studies on pathogenic C. diphtheriae uptake.

microbiology↗

bioRxiv2020

Leveraging immonium ions for identifying and targeting acyl-lysine modifications in proteomic datasets

Acyl modifications vary greatly in terms of elemental composition and site of protein modification. Developing methods to identify these modifications more confidently can help assess the scope of these modifications in large proteomic datasets. Herein we analyze the utility of acyl-lysine immonium ions for identifying the modifications in proteomic datasets. We demonstrate that the cyclized immonium ion is a strong indicator of acyl-lysine presence when its rank or relative abundance compared to other ions within a spectrum is considered. Utilizing a stepped collision energy method in a shotgun experiment highlights the immonium ion strongly. Implementing an analysis that accounted for features within each MS2 spectra, this method allows peptides with short chain acyl-lysine modifications to be clearly identified in complex lysates. Immonium ions can also be used to validate novel acyl-modifications; in this study we report the first examples of 3-hydroxylpimelyl-lysine modification and validate them using immonium ions. Overall these results solidify the use of the immonium ion as a marker for acyl-lysine modifications in complex proteomic datasets. Statement of SignificanceAcyl-lysine modifications come in a variety of elemental compositions. There is increasing evidence that these modifications can have a functional effect on protein and are present in proteomes across all domains of life. Here we describe a new method that can allow for more confident identification of acyl modifications in proteomes by utilizing the immonium ion of these modifications. Our utilization of these ions allows for more comprehensive insight into the role of acyl modifications at the systems level.

biochemistry↗

bioRxiv2019

Characterization of protein-ligand binding interactions of enoyl-ACP reductase (FabI) by native MS reveals allosteric effects of coenzymes and the inhibitor triclosan

The enzyme enoyl-ACP reductase (also called FabI in bacteria) is an essential member of the fatty acid synthase II pathway in plants and bacteria. This enzyme is the target of the antibacterial drug triclosan and has been the subject of extensive studies for the past 20 years. Despite the large number of reports describing the biochemistry of this enzyme, there have been no studies that provided direct observation of the protein and its various ligands. Here we describe the use of native MS to characterize the protein-ligand interactions of FabI with its coenzymes NAD+ and NADH and with the inhibitor triclosan. Measurements of the gas-phase affinities of the enzyme for these ligands yielded values that are in close agreement with solution-phase affinity measurements. Additionally, FabI is a homotetramer and we were able to measure the affinity of each subunit for each coenzyme, which revealed that both coenzymes exhibit a positive homotropic allosteric effect. An allosteric effect was also observed in association with the inhibitor triclosan. These observations provide new insights into this well-studied enzyme and suggest that there may still be gaps in the existing mechanistic models that explain FabI inhibition.

biochemistry↗

bioRxiv2019

N-terminal autoprocessing and acetylation of MARTX Makes-Caterpillars Floppy-like effector is stimulated by ADP-Ribosylation Factor 1 in advance of Golgi fragmentation

Studies have successfully elucidated the mechanism of action of several effector domains that comprise the MARTX toxins of Vibrio vulnificus. However, the biochemical linkage between the cysteine proteolytic activity of Makes Caterpillars Floppy-like (MCF) effector and its cellular effects remains unknown. In this study, we identify the host cell factors that activate in vivo and in vitro MCF autoprocessing as ADP-Ribosylation Factor 1 (ARF1) and ADP-ribosylation Factor 3 (ARF3). Autoprocessing activity is enhanced when ARF1 is in its active (GTP-bound) form compared to the inactive (GDP-bound) form. Subsequent to auto-cleavage, MCF is acetylated on its exposed N-terminal glycine residue. Acetylation apparently does not dictate subcellular localization, as MCF is found localized throughout the cell. However, the cleaved form of MCF gains the ability to bind to the specialized lipid phosphatidylinositol 5-phosphate enriched in Golgi and other membranes necessary for endocytic trafficking, suggesting a fraction of MCF may be subcellular localized. Traditional thin-section electron microscopy, high-resolution cryoAPEX localization, and fluorescent microscopy show that MCF causes Golgi dispersal resulting in extensive vesiculation. In addition, host mitochondria are disrupted and fragmented. Surprisingly, ARF1 is not itself processed or post-translationally modified by MCF. Further, only catalytically inactive MCF stably associates with ARF1, thus serving as a substrate trap. Our data indicate that ARF1 is a cross-kingdom activator of MCF, but reveal that MCF mediates cytotoxicity likely by directly targeting another yet to be identified protein. This study begins to elucidate the biochemical activity of this important domain and gives insight into how it may promote disease progression.

microbiology↗