Biology subjects

Gonzalez, D. J.

Publications and source records attributed to Gonzalez, D. J..

15 recordsLinked to original sources

bioRxiv2026

Mapping bacterial bile amidation across the bile salt hydrolase family

Evaluating enzyme activity and specificity in complex microbiomes is challenging. The recent discovery that bacterial bile salt hydrolases (BSHs) also function as transaminases in the human gut microbiome highlights what remains to be disentangled, even within one of the most extensively studied microbiomes. BSHs as transaminases can conjugate bile acids with bioavailable amines to produce bacterial bile amidates (BBAs). The implications of BBAs for human health are rapidly evolving, but our understanding of which BSHs produce which specific BBAs remains limited. Here, we connect BSH sequence and function by evaluating the transaminase activities of 115 BSHs and reveal distinct amine specificities using molecular networking. Additionally, we identify enzymatically generated BBAs distributed across life-stage, tissue-, and disease-specific human samples by mining public metabolomics repositories, highlighting the previously unrecognized catalytic diversity of BSHs and their potential relevance to human health.

biochemistry↗

bioRxiv2026

Cardioselective β-blocker Therapy Resolves Staphylococcus aureus Toxin-Induced Innate Immunoparalysis

During Staphylococcus aureus sepsis, the pore-forming -toxin (Hla) triggers lytic platelet death and provokes the release of catecholamines that modulate leukocyte effector function. The resulting surge in catecholaminergic action paralyzes host antimicrobial capacity by upregulating interleukin-10 (IL-10), profoundly suppressing the protective interferon (IFN) response to orchestrate innate immunoparalysis. Here, we deconvolute a multi-cellular regulatory pathway in the blood and highlight a pivotal immunological checkpoint, where pharmacological intervention with cardioselective {beta}-blockers uncouples the immunosuppressive cascade, restores host antimicrobial function, and significantly improves survival outcomes.

microbiology↗

bioRxiv2026

Selective knockout of PKA regulatory subunits reveal opposite catalytic and metabolic consequences with implications for Alzheimer's disease

cAMP-dependent Protein Kinase A (PKA) is a master regulator of cell signaling involved in energy metabolism, synaptic plasticity, and stress response. Dysregulated PKA signaling is implicated in diseases including neurodegeneration and cancer. PKA catalytic activity is regulated by two nonredundant regulatory subunits, Type I (RI/RI{beta}) and Type II (RII/RII{beta}), whose divergent functions are not fully understood. We generated double-knockout (KO) cell lines of RI/RI{beta} and RII/RII{beta} subunits and performed multiplexed MS-based proteomic and phosphoproteomic profiling under basal and glucose-perturbed conditions. We found that RI and RII loss drives distinct, and often opposite, remodeling of the cellular proteome and phosphoproteome. While both mutants blunted metabolic flexibility to glycolytic stressors and stimuli, RI and RII KO cells exhibited elevated and depressed glycolytic signaling, respectively. Interestingly, RI KO increased the abundance and kinase activity of the PKA catalytic subunit C isoform, leading to an increase in PKA substrate phosphorylation, whereas RII KO decreased the abundance, kinase activity, and substrate phosphorylation by the catalytic subunit C{beta} isoform. Notably, one of the most differentially affected PKA sites between RI and RII KOs maps to Tau, whose hyperphosphorylation is a hallmark of Alzheimers disease. Loss of RI increased Tau phosphorylation, which was not only caused by increased PKA catalytic activity, but also a higher binding affinity of Tau to RII subunits on the negatively-charged flexible linker region. Overall, the present study demonstrates that PKA RI and RII subunits play nonredundant roles in modulating PKA activity, metabolic flexibility, and phospho-regulation of key disease-associated substrates such as Tau.

biochemistry↗

bioRxiv2026

Rapid optogenetic manipulation of autophagy reveals that the nuclear pore complex is a robust autophagy substrate

Autophagy, a conserved recycling process, manages intracellular quality control to mitigate stress. To determine the rapid effects of autophagy perturbation, we developed the first optogenetic tool to rapidly inhibit autophagy, termed ASAP. Our approach leverages light-induced proteolytic cleavage to selectively inhibit autophagy within 5 minutes of light exposure, providing a precise and dynamic approach to study autophagy regulation. Proteomic profiling with ASAP revealed the most tightly regulated autophagy substrates along with novel, previously unidentified substrates, including nuclear pore complex (NPC) proteins. Interestingly, autophagy regulates quality control of cytoplasmic complexes of nucleoporins via specific LC3-interacting regions (LIRs), sparing nuclear pore complex proteins embedded in the nuclear envelope. Upon rapid autophagy inhibition, incomplete nucleoporin complexes accumulate and instead of undergoing autophagic degradation, cytoplasmic nucleoporin complexes aggregate in processing bodies (P bodies). Using ASAP, we demonstrate rapid and specific inhibition of autophagy, revealing that nuclear pore complex proteins are tightly regulated autophagy substrates.

cell biology↗

bioRxiv2025

mspms: A Comprehensive R Package and Graphic Interface for Multiplex Substrate Profiling by Mass Spectrometry Analysis

Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS) is a powerful method for determining the substrate specificity of proteolytic enzymes, knowledge key for developing protease inhibitors, diagnostics, and protease-activated therapeutics. However, the complex datasets generated by MSP-MS pose significant analytical challenges. To address this, we developed mspms, a Bioconductor R package complemented by an intuitive graphical interface. Mspms streamlines MSP-MS data analysis by standardizing workflows for data preparation, processing, statistical analysis, and visualization. Designed for accessibility, it serves both advanced users via the R package and broader audiences through the web interface. We validated mspms by profiling the substrate specificity of four well-characterized cathepsins (A-D), demonstrating its ability to reliably capture expected substrate specificities. As the first publicly available platform for MSP-MS data analysis, mspms delivers comprehensive functionality, transparency, and ease of use, making it a valuable resource for the protease research community. Access to mspms is available through the Bioconductor project at https://bioconductor.org/packages/mspms, and a graphic interface is available at https://gonzalezlab.shinyapps.io/mspms_shiny/. Author SummaryWe developed mspms, an easy-to-use tool that helps researchers analyze data from a proteomics technique called Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS). This software improves on previous methods of analyzing MSP-MS data, which required the user to navigate a confusing mix of R scripts, manual manipulation of spreadsheets, and third-party tools--an approach that was daunting for collaborators and new graduate students alike. Mspms streamlines the process, enabling faster, more reliable, and reproducible data analysis. We tested the tool using well-known proteases and found that it accurately identifies their known targets. As the first comprehensive tool for MSP-MS analysis, mspms makes this method approachable to a wider audience. Its available for free through the Bioconductor project at https://bioconductor.org/packages/mspms, and a graphical interface is available at https://gonzalezlab.shinyapps.io/mspms_shiny/.

bioinformatics↗

bioRxiv2024

Behavioral impairments are linked to neuroinflammation in mice with Cerebral Cavernous Malformation disease

BackgroundCerebral Cavernous Malformations (CCMs) are neurovascular abnormalities in the central nervous system (CNS) caused by loss of function mutations in KRIT1 (CCM1), CCM2, or PDCD10 (CCM3) genes. One of the most common symptoms in CCM patients is associated with motor disability, weakness, seizures, stress, and anxiety, and the extent of the symptom or symptoms may be due to the location of the lesion within the CNS or whether multiple lesions are present. Previous studies have primarily focused on understanding the pathology of CCM using animal models. However, more research has yet to explore the potential impact of CCM lesions on behavioral deficits in animal models, including effects on short-term and long-term memory, motor coordination, and function. MethodsWe used the accelerating RotaRod test to assess motor and coordination deficits. We also used the open field test to assess locomotor activity and pathology-related behavior and Pavlovian fear conditioning to assess short--and long-term memory deficits. Our behavioral studies were complemented by proteomics, histology, immunofluorescence, and imaging techniques. We found that neuroinflammation is crucial in behavioral deficits in male and female mice with neurovascular CCM lesions (Slco1c1-iCreERT2; Pdcd10fl/fl; Pdcd10BECKO). ResultsFunctional behavior tests in male and female Pdcd10BECKO mice revealed that CCM lesions cause sudden motor coordination deficits associated with the manifestation of profound neuroinflammatory lesions. Our findings indicate that maturation of CCM lesions in Pdcd10BECKO mice also experienced a significant change in short- and long-term memory compared to their littermate controls, Pdcd10fl/fl mice. Proteomic experiments reveal that as CCM lesions mature, there is an increase in pathways associated with inflammation, coagulation, and angiogenesis, and a decrease in pathways associated with learning and plasticity. Therefore, our study shows that Pdcd10BECKO mice display a wide range of behavioral deficits due to significant lesion formation in their central nervous system and that signaling pathways associated with neuroinflammation and learning impact behavioral outcomes. ConclusionsOur study found that CCM animal models exhibited behavioral impairments such as decreased motor coordination and amnesia. These impairments were associated with the maturation of CCM lesions that displayed a neuroinflammatory pattern.

animal behavior and cognition↗

bioRxiv2024

Proteome profiling identifies a link between the mitochondrial pathways and host-microbial sensor ELMO1 following Salmonella infection.

The host EnguLfment and cell MOtility protein 1 (ELMO1) is a cytosolic microbial sensor that facilitates bacterial sensing, internalization, clearance, and inflammatory responses. We have shown previously that ELMO1 binds bacterial effector proteins, including pathogenic effectors from Salmonella and controls host innate immune signaling. To understand the ELMO1-regulated host pathways, we have performed liquid chromatography Multinotch MS3-Tandem Mass Tag (TMT) multiplexed proteomics to determine the global quantification of proteins regulated by ELMO1 in macrophages during Salmonella infection. Comparative proteome analysis of control and ELMO1-depleted murine J774 macrophages after Salmonella infection quantified more than 7000 proteins with a notable enrichment in mitochondrial-related proteins. Gene ontology enrichment analysis revealed 19 upregulated and 11 downregulated proteins exclusive to ELMO1-depleted cells during infection, belonging to mitochondrial functions, metabolism, vesicle transport, and the immune system. By assessing the cellular energetics via Seahorse analysis, we found that Salmonella infection alters mitochondrial metabolism, shifting it from oxidative phosphorylation to glycolysis. Importantly, these metabolic changes are significantly influenced by the depletion of ELMO1. Furthermore, ELMO1 depletion resulted in a decreased ATP rate index following Salmonella infection, indicating its importance in counteracting the effects of Salmonella on immunometabolism. Among the proteins involved in mitochondrial pathways, mitochondrial fission protein DRP1 was significantly upregulated in ELMO1-depleted cells and in ELMO1-KO mice intestine following Salmonella infection. Pharmacological Inhibition of DRP1 revealed the link of the ELMO1-DRP1 pathway in regulating the pro-inflammatory cytokine TNF- following infection. The role of ELMO1 has been further characterized by a proteome profile of ELMO1-depleted macrophage infected with SifA mutant and showed the involvement of ELMO1-SifA on mitochondrial function, metabolism and host immune/defense responses. Collectively, these findings unveil a novel role for ELMO1 in modulating mitochondrial functions, potentially pivotal in modulating inflammatory responses. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=149 SRC="FIGDIR/small/592405v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@72862org.highwire.dtl.DTLVardef@1ec4a5borg.highwire.dtl.DTLVardef@5b805org.highwire.dtl.DTLVardef@17466d7_HPS_FORMAT_FIGEXP M_FIG C_FIG Significance StatementHost microbial sensing is critical in infection and inflammation. Among these sensors, ELMO1 has emerged as a key regulator, finely tuning innate immune signaling and discriminating between pathogenic and non-pathogenic bacteria through interactions with microbial effectors like SifA of Salmonella. In this study, we employed Multinotch MS3-Tandem Mass Tag (TMT) multiplexed proteomics to determine the proteome alterations mediated by ELMO1 in macrophages following WT and SifA mutant Salmonella infection. Our findings highlight a substantial enrichment of host proteins associated with metabolic pathways and mitochondrial functions. Notably, we validated the mitochondrial fission protein DRP1 that is upregulated in ELMO1-depleted macrophages and in ELMO1 knockout mice intestine after infection. Furthermore, we demonstrated that Salmonella-induced changes in cellular energetics are influenced by the presence of ELMO1. This work shed light on a possible novel link between mitochondrial dynamics and microbial sensing in modulating immune responses.

microbiology↗

bioRxiv2024

Giant polyketide synthase enzymes biosynthesize a giant marine polyether biotoxin.

Prymnesium parvum are harmful haptophyte algae that cause massive environmental fish-kills. Their polyketide polyether toxins, the prymnesins, are amongst the largest nonpolymeric compounds in nature, alongside structurally-related health-impacting "red-tide" polyether toxins whose biosynthetic origins have been an enigma for over 40 years. Here we report the PKZILLAs, massive P. parvum polyketide synthase (PKS) genes, whose existence and challenging genomic structure evaded prior detection. PKZILLA-1 and -2 encode giant protein products of 4.7 and 3.2 MDa with 140 and 99 enzyme domains, exceeding the largest known protein titin and all other known PKS systems. Their predicted polyene product matches the proposed pre-prymnesin precursor of the 90-carbon-backbone A-type prymnesins. This discovery establishes a model system for microalgal polyether biosynthesis and expands expectations of genetic and enzymatic size limits in biology.

genomics↗

bioRxiv2023

Ligand-Dependent Mechanisms of C-C Chemokine Receptor 5 (CCR5) Trafficking Revealed by APEX2 Proximity Labeling Proteomics

CC chemokine receptor 5 (CCR5) promotes inflammatory responses by driving cell migration and scavenging chemokine to shape directional chemokine gradients. A CCR5 inhibitor has been approved for blocking HIV entry into cells. However, targeting CCR5 for the treatment of other diseases has had limited success, likely because of the complexity of CCR5 pharmacology and biology. CCR5 is activated by natural and engineered chemokines that elicit distinct signaling and trafficking responses, including receptor sequestration inside the cell. Intracellular sequestration may be therapeutically exploitable as a strategy for receptor inhibition, but the mechanisms by which different ligands promote receptor retention in the cell versus presence on the cell membrane are poorly understood. We employed live cell ascorbic acid peroxidase (APEX2) proximity labeling and quantitative mass spectrometry proteomics for unbiased discovery of temporally resolved protein neighborhoods of CCR5 following stimulation with its endogenous agonist, CCL5, and two CCL5 variants that promote intracellular retention of the receptor. Along with targeted pharmacological assays, the data reveal distinct ligand-dependent CCR5 trafficking patterns with temporal and spatial resolution. All three chemokines internalize CCR5 via {beta}-arrestin-dependent, clathrin-mediated endocytosis but to different extents, with different kinetics and varying dependencies on GPCR kinase subtypes. The agonists differ in their ability to target the receptor to lysosomes for degradation, as well as to the Golgi compartment and the trans-Golgi network, and these trafficking patterns translate into distinct levels of ligand scavenging. The results provide insight into the cellular mechanisms behind CCR5 intracellular sequestration and suggest how trafficking can be exploited for the development of functional antagonists of CCR5. Significance StatementCCR5 plays a crucial role in the immune system and is important in numerous physiological and pathological processes such as inflammation, cancer and transmission of HIV. It responds to different ligands with distinct signaling and trafficking behaviors; notably some ligands induce retention of the receptor inside the cell. Using time-resolved proximity labeling proteomics and targeted pharmacological experiments, this study reveals the cellular basis for receptor sequestration that can be exploited as a therapeutic strategy for inhibiting CCR5 function.

cell biology↗

bioRxiv2023

A bacterial sialidase mediates early life colonization by a pioneering gut commensal

The early microbial colonization of the gastrointestinal tract can lead to long-term impacts in development and overall human health. Keystone species, including Bacteroides spp., play a crucial role in maintaining the structure, diversity, and function of the intestinal ecosystem. However, the process by which a defined and resilient community is curated and maintained during early life remains inadequately understood. Here, we show that a single sialidase, NanH, in Bacteroides fragilis mediates stable occupancy of the intestinal mucosa and regulates the commensal colonization program during the first weeks of life. This program is triggered by sialylated glycans, including those found in human milk oligosaccharides and intestinal mucus. After examining the dynamics between pioneer gut Bacteroides species in the murine gut, we discovered that NanH enables vertical transmission from dams to pups and promotes B. fragilis dominance during early life. Furthermore, we demonstrate that NanH facilitates commensal resilience and recovery after antibiotic treatment in a defined microbial community. Collectively, our study reveals a co-evolutionary mechanism between the host and the microbiota mediated through host-derived glycans to promote stable intestinal colonization.

microbiology↗

bioRxiv2023

FBP1 is a nonenzymatic safety valve that curtails AKT activation to prevent insulin hyperresponsiveness

Insulin inhibits gluconeogenesis and stimulates glucose conversion to glycogen and lipids. How these activities are coordinated to prevent hypoglycemia and hepatosteatosis is not clear. Fructose-1,6 bisphosphatase (FBP1) is rate controlling for gluconeogenesis. However, inborn human FBP1 deficiency does not cause hypoglycemia unless accompanied by fasting or starvation, which also trigger paradoxical hepatomegaly, hepatosteatosis, and hyperlipidemia in affected individuals. Hepatocyte FBP1-ablated mice exhibit identical fasting-conditional pathologies along with AKT hyperactivation, whose inhibition reversed hepatomegaly, hepatosteatosis and hyperlipidemia but not hypoglycemia. Surprisingly, fasting-mediated AKT hyperactivation is insulin-dependent. FBP1 prevents insulin hyperresponsiveness, independently of its catalytic activity, by interacting with AKT, PP2A-C and Aldolase-B (ALDOB) to specifically accelerate AKT dephosphorylation. Enhanced by fasting and weakened by elevated insulin, FBP1:AKT:PP2A-C:ALDOB complex formation, which is disrupted by human FBP1 deficiency mutations or a C-terminal FBP1 truncation, prevents insulin-triggered liver pathologies and maintains lipid and glucose homeostasis. Conversely, a complex disrupting peptide reverses diet-induced insulin resistance.

molecular biology↗

bioRxiv2023

Proteome allocation is linked to transcriptional regulation through a modularized transcriptome

It has proved challenging to quantitatively relate the proteome to the transcriptome on a per-gene basis. Recent advances in data analytics have enabled a biologically meaningful modularization of the bacterial transcriptome. We thus investigated whether matched datasets of transcriptomes and proteomes from bacteria under diverse conditions could be modularized in the same way to reveal novel relationships between their compositions. We found that; 1) the modules of the proteome and the transcriptome are comprised of a similar list of gene products, 2) the modules in the proteome often represent combinations of modules from the transcriptome, 3) known transcriptional and post-translational regulation is reflected in differences between two sets of modules, allowing for knowledge-mapping when interpreting module functions, and 4) through statistical modeling, absolute proteome allocation can be inferred from the transcriptome alone. Quantitative and knowledge-based relationships can thus be found at the genome-scale between the proteome and transcriptome in bacteria.

bioengineering↗

bioRxiv2021

Antimicrobials from a feline commensal bacterium inhibit skin infection by drug-resistant S. pseudintermedius.

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is an important emerging zoonotic pathogen that causes severe skin infections. To combat infections from drug-resistant bacteria, the transplantation of commensal antimicrobial bacteria as a therapeutic has shown clinical promise. We screened a collection of diverse staphylococcus species from domestic dogs and cats for antimicrobial activity against MRSP. A unique strain (S. felis C4) was isolated from feline skin that inhibited MRSP and multiple gram-positive pathogens. Competition experiments in mice showed that S. felis significantly reduced MRSP skin colonization and an antimicrobial extract from S. felis significantly reduced necrotic skin injury from MRSP infection. Fluorescence and electron microscopy revealed that S. felis antimicrobials disrupted bacterial but not eukaryotic cell membranes. LC/MS identified several S. felis phenol-soluble modulin beta (PSM{beta}) peptides that exhibited antimicrobial and anti-inflammatory activity. These findings indicate a feline commensal bacterium that could be utilized in bacteriotherapy against difficult-to-treat animal and human skin infections.

microbiology↗

bioRxiv2020

Disruption of innate defense responses by endoglycosidase HPSE promotes cell survival

The drive to withstand environmental stresses and defend against invasion is a universal trait extant in all forms of life. While numerous canonical signaling cascades have been characterized in detail, it remains unclear how these pathways interface to generate coordinated responses to diverse stimuli. To dissect these connections, we follow heparanase (HPSE), a protein best known for its endoglycosidic activity at the extracellular matrix but recently recognized to drive various forms of late stage disease through unknown mechanisms. Using herpes simplex virus-1 (HSV-1) infection as a model cellular perturbation, we demonstrate that HPSE acts beyond its established enzymatic role to restrict multiple forms of cell-intrinsic defense and facilitate host cell reprogramming by the invading pathogen. We reveal that cells devoid of HPSE are innately resistant to infection and counteract viral takeover through multiple amplified defense mechanisms. With a unique grasp of the fundamental processes of transcriptional regulation and cell death, HPSE represents a potent cellular intersection with broad therapeutic potential.

cell biology↗

bioRxiv2019

Kinetic profiling of metabolic specialists demonstrates stability and consistency of in vivo enzyme turnover numbers

Enzyme turnover numbers (kcats) are essential for a quantitative understanding of cells. Because kcats are traditionally measured in low-throughput assays, they are often noisy, non-physiological, inconsistent, and labor-intensive to obtain.\n\nWe use a data-driven approach to estimate in vivo kcats using metabolic specialist E. coli strains that resulted from gene knockouts in central metabolism followed by metabolic optimization via laboratory evolution. By combining absolute proteomics with fluxomics data, we find that in vivo kcats are robust against genetic perturbations, suggesting that metabolic adaptation to gene loss is mostly achieved through other mechanisms, like gene-regulatory changes. Combining machine learning and genome-scale metabolic models, we show that the obtained in vivo kcats predict unseen proteomics data with much higher precision than in vitro kcats. The results demonstrate that in vivo kcats can solve the problem of noisy and inconsistent parameterizations of cellular models.

systems biology↗