Biology subjects

Trujillo, M.

Publications and source records attributed to Trujillo, M..

11 recordsLinked to original sources

bioRxiv2026

Biochemical regulation of Arabidopsis PUB33: a receptor-like cytoplasmic kinase with an integrated U-box domain that ubiquitinates Ralstonia pseudosolanacearum effector protein RipV1

Plant immunity relies on the detection of microbes and the rapid activation of intracellular defense pathways. Catalyzed by protein kinases and E3 ubiquitin ligases, respectively, phosphorylation and ubiquitination are among the most abundant post-translational modifications that regulate immune pathways. It has been well established that members of the receptor-like cytoplasmic kinase (RLCK) and plant U-box E3 ligase (PUB) families are critical components of plant immune signaling. Interestingly, a group of proteins that contain both an RLCK domain and a PUB domain has been conserved throughout plant evolution, referred to as subgroups RLCK-IXb and PUB-VI within their respective families. While very little is known about these proteins, evidence from multiple independent studies indicates that orthologous PUB-VI/RLCK-IXb proteins in potato, tomato, Nicotiana benthamiana, and Arabidopsis thaliana associate with diverse pathogen effectors from the oomycete pathogen Phytophthora infestans, bacterial pathogen Ralstonia pseudosolanacearum, and the mirid bug Apolygus lucorum, suggesting that they may be critical virulence targets or components of the immune response. However, the biochemical activities of these proteins and how they contribute to plant health remain poorly defined. Here, we introduce the PUB-VI/RLCK-IXb clade in Arabidopsis, focusing on PUB32, PUB33, and PUB50. We show that PUB33 exhibits dual kinase and E3 ubiquitin ligase activities that are inversely regulated by autophosphorylation at Thr333. PUB33 forms homomers and heteromers with PUB32 which attenuate PUB33 catalytic activity. Although we did not observe clear defects in innate immune signaling in pub32, pub33, or pub50 mutants, we found that overexpression of PUB33 can suppress cell death triggered by the R. pseudosolanacearum effector RipV1 in N. benthamiana. Moreover, PUB33 directly ubiquitinates RipV1 in vitro and reduces RipV1 accumulation in planta, suggesting that it functions as part of the immune response against R. pseudosolanacearum.

plant biology↗

bioRxiv2025

A Chemostat-Based Model for Growing Bacterial Biofilms

Biofilms are groups of microbes that live together in dense communities, often attached to a surface. They play an outsized role in all aspects of microbial life, from chronic infections to biofouling to dental decay. In recent decades, appreciation for the diversity of roles that biofilms play in the environment has grown. Yet, most bacterial studies still rely upon approaches developed in the 19th century and center on planktonic populations alone. Here we present a chemostat-based experimental platform to investigate not only biofilms themselves, but how they interact with their surrounding environments. Our results show that biofilms grow to larger sizes in chemostats as opposed to flasks. In addition, we show that biofilms may be a consistent source of migrants into planktonic populations. We also show that secondary biofilms rapidly develop, although these may be more susceptible to environmental conditions. Taken together, our data suggest that chemostats may be a flexible and insightful platform for the study of biofilms in vitro. IMPORTANCEBiofilms are the predominant way that bacteria live in natural environments and are characterized by three emergent properties: ubiquity, resilience, and impact. They can be found across all environments, both natural and human-made, and across all of recorded time, dating back at least 3.5 billion years. Biofilms also represent a major economic impact of over $5 trillion annually. Yet, most of what is known about bacteria is the result of studies using planktonically growing liquid cultures under laboratory conditions. Here, we propose a comprehensive experimental platform that allows for study of biofilms on the molecular, organismal, and community levels using chemostats.

microbiology↗

bioRxiv2025

Growth Hormone Excess Drives Liver Aging via increased Glycation stress

Growth hormone (GH) plays a crucial role in various physiological functions, with its secretion tightly regulated by complex endocrine mechanisms. Pathological conditions such as acromegaly or pituitary tumors result in elevated circulating GH levels, which have been implicated in a spectrum of metabolic disorders, potentially by regulating liver metabolism. In this study, we focused on the liver, a key organ in metabolic regulation and a primary target of GH, to investigate the impact of high circulating GH on liver metabolism. We used bovine GH overexpressing transgenic (bGH-Tg) mice to conduct a comprehensive transcriptomic analysis of hepatic tissues. The bGH-Tg mouse livers exhibit dysregulated fatty acid metabolism and heightened inflammatory responses. Notably, the transcriptomic profile of young bGH-Tg mouse livers resembled that of aged livers and displayed markers of increased cellular senescence. Furthermore, these mice exhibited a significant accumulation of advanced glycation end products (AGEs). Intervention with glycation-lowering compounds effectively reversed the insulin resistance and aberrant transcriptomic signatures in the liver that are associated with elevated GH levels. These findings underscore the potential therapeutic value of glycation-lowering agents in mitigating the deleterious effects of chronic GH overexpression. HighlightsO_LIOverexpression of bovine growth hormone impacts transcriptional changes in liver fat metabolism and inflammatory response in mice. C_LIO_LIHigh circulating growth hormone leads to transcriptional changes that suggest enhanced liver aging and induce cellular senescence. C_LIO_LIDetoxification pathways in bGH-Tg mice are inhibited, leading to the accumulation of Advanced Glycation End (AGE) products. C_LIO_LIGlycation-lowering compounds can mitigate pathologies associated with high GH levels. C_LI

physiology↗

bioRxiv2025

Methylglyoxal-induced glycation stress promotes aortic stiffening: Putative mechanistic roles of oxidative stress and cellular senescence

BackgroundHere, we assessed the role of the advanced glycation end-product (AGE) precursor methylglyoxal (MGO) and its non-crosslinking AGE MGO-derived hydroimidazolone (MGH)-1 in aortic stiffening and explored the potential of a glycation stress-lowering compound (Gly-Low) to mitigate these effects. MethodsYoung (3-6 month) C57BL/6 mice were supplemented with MGO (in water) and Gly-Low (in chow). Aortic stiffness was assessed in vivo via pulse wave velocity (PWV) and ex vivo through elastic modulus. Putative mechanisms underlying MGO- and MGH-1-induced aortic stiffening were explored using complementary experimental approaches in aortic tissue and cultured human aortic endothelial cells (HAECs). Moreover, aortic stiffness was assessed in old (24 month) mice after consumption of Gly-Low-enriched chow. ResultsMGO-induced glycation stress increased PWV in young mice by 21% (P<0.05 vs. control), which was prevented with Gly-Low (P=0.93 vs. control). Ex vivo, MGO increased aortic elastic modulus 2-fold (P<0.05), superoxide production by [~]40% (P<0.05), and MGH-1 expression by 50% (P<0.05), which were all mitigated by Gly-Low. Chronic MGO exposure elevated biomarkers of cellular senescence in HAECs, comparable to a known senescence inducer Doxorubicin, an effect partially blocked by Gly-Low. Moreover, elevated aortic elastic modulus induced by Doxorubicin (P<0.05 vs. control) was prevented with Gly-Low (P=0.71 vs. control). Aortic RNA sequencing implicated preservation of endogenous cellular detoxification pathways with Gly-Low following exposure to MGH-1. Old mice supplemented with Gly-Low had lower PWV (P<0.05) relative to old control mice. ConclusionsMGO-induced glycation stress contributes to aortic stiffening and glycation stress lowering compounds hold promise for mitigating these effects. What Is New?This study provides the first comprehensive line of evidence that methylglyoxal (MGO)-induced glycation stress directly contributes to aortic stiffening and does so through mechanisms involving oxidative stress and cellular senescence. Using complementary in vivo, ex vivo, and in vitro experimental models, we establish that MGO-mediated glycation stress independently induces aortic stiffening. Furthermore, we demonstrate that the glycation-lowering compound, Gly-Low, mitigates MGO-induced aortic stiffening by mitigating excessive oxidative stress and cellular senescence, and can lower aortic stiffness in old mice. Mechanistically, activation of the detoxification enzyme, glyoxalase-1 (Glo-1), is a novel pathway by which Gly-Low mediates its therapeutic effects on aortic stiffening. Lastly, we show that Gly-Low holds promise for lowering aortic stiffness in old age. What Is Relevant?Aortic stiffening is a major risk factor for cardiovascular diseases (CVD) and a significant predictor of CV-related morbidity and mortality. Yet, the underlying mechanisms driving this process remain incompletely understood. This study identifies MGO-derived glycation stress as a critical and modifiable factor contributing to aortic stiffening through pathways involving excessive oxidative stress and cellular senescence. By establishing the efficacy of Gly-Low in mitigating these effects, our findings underscore the importance of targeting glycation stress in the context of aging, and likely in other settings of glycation stress, to improve arterial health and reduce CVD risk. Clinical/Pathophysiological ImplicationsThese findings have significant clinical implications, as they demonstrate that glycation stress is a viable and modifiable therapeutic target for the prevention and treatment of aortic stiffening. Gly-Low offers a promising therapeutic approach to ameliorate glycation stress- and age-related aortic stiffening, by directly targeting excess glycation stress, oxidative stress, and cellular senescence. Additionally, the involvement of the Glo-1 detoxification pathway suggests a specific molecular target for future interventions aimed at improving arterial health and mitigating the progression of CVD.

molecular biology↗

bioRxiv2023

A Novel Interaction Between Aquaporin 1 and Caspase-3 in Pulmonary Arterial Smooth Muscle Cells

Pulmonary arterial hypertension (PAH) is a disease in which remodeling of the precapillary pulmonary vasculature leads to hyperplasia and hypertrophy of the muscular vascular wall, and the formation of vaso-occlusive lesions. These pathologic changes are predominantly due to abnormal proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), enhanced cellular functions that have been linked to increases in the cell membrane protein aquaporin-1 (AQP1). However, the mechanisms underlying increased AQP1 abundance have not been fully elucidated. Here we present data that establishes a novel interaction between AQP1 and the proteolytic enzyme caspase-3. In silico analysis of the AQP1 protein reveals two caspase-3 cleavage sites on its c-terminal tail, proximal to known ubiquitin sites. Using biotin proximity ligase techniques, we establish that AQP1 and caspase-3 interact in both HEK293A cells and rat PASMCs. Furthermore, we demonstrate that AQP1 levels increase and decrease with enhanced caspase-3 activity and inhibition respectively. Ultimately, further work characterizing this interaction could provide the foundation for novel PAH therapeutics.

cell biology↗

bioRxiv2022

New algorithms for accurate and efficient de-novo genome assembly from long DNA sequencing reads

Producing de-novo genome assemblies for complex genomes is possible thanks to long-read DNA sequencing technologies. However, maximizing the quality of assemblies based on long reads is a challenging task that requires the development of specialized data analysis techniques. In this paper, we present new algorithms for assembling long-DNA sequencing reads from haploid and diploid organisms. The assembly algorithm builds an undirected graph with two vertices for each read based on minimizers selected by a hash function derived from the k-mers distribution. Statistics collected during the graph construction are used as features to build layout paths by selecting edges, ranked by a likelihood function that is calculated from the inferred distributions of features on a subset of safe edges. For diploid samples, we integrated a reimplementation of the ReFHap algorithm to perform molecular phasing. The phasing procedure is used to remove edges connecting reads assigned to different haplotypes and to obtain a phased assembly by running the layout algorithm on the filtered graph. We ran the implemented algorithms on PacBio HiFi and Nanopore sequencing data taken from bacteria, yeast, Drosophila, rice, maize, and human samples. Our algorithms showed competitive efficiency and contiguity of assemblies, as well as superior accuracy in some cases, as compared to other currently used software. We expect that this new development will be useful for researchers building genome assemblies for different species.

bioinformatics↗

bioRxiv2020

Phosphorylation-dependent sub-functionalization of the calcium-dependent protein kinase CPK28

Calcium (Ca2+)-dependent protein kinases (CDPKs or CPKs) are a unique family of Ca2+-sensor/kinase-effector proteins with diverse functions in plants. In Arabidopsis thaliana, CPK28 contributes to immune homeostasis by promoting degradation of the key immune signaling receptor-like cytoplasmic kinase BOTRYTIS-INDUCED KINASE 1 (BIK1), and additionally functions in vegetative-to-reproductive stage transition. How CPK28 controls these seemingly disparate pathways is unknown. Here, we identify a single phosphorylation site in the kinase domain of CPK28 (Ser318) that is differentially required for its function in immune homeostasis and stem elongation. We show that CPK28 undergoes intra- and inter-molecular auto-phosphorylation on Ser318 and can additionally be trans-phosphorylated on this residue by BIK1. Analysis of several other phosphorylation sites demonstrates that Ser318 phosphorylation is uniquely required to prime CPK28 for Ca2+ activation at physiological concentrations of Ca2+, possibly through stabilization of the Ca2+-bound active state as indicated by intrinsic fluorescence experiments. Together, our data indicate that phosphorylation of Ser318 is required for the activation of CPK28 at low intracellular [Ca2+] to prevent initiation of an immune response in the absence of infection. By comparison, phosphorylation of Ser318 is not required for stem-elongation, indicating pathway specific requirements for phosphorylation-based Ca2+-sensitivity priming. We additionally provide evidence for a conserved function for Ser318 phosphorylation in related group IV CDPKs which holds promise for biotechnological applications by generating CDPK alleles that enhance resistance to microbial pathogens without consequences to yield.

plant biology↗

bioRxiv2019

The Pleiades cluster of fungal effector genes inhibit 1 host defenses

SummaryBiotrophic plant pathogens secrete effector proteins to manipulate the host physiology. Effectors suppress defenses and induce an environment favorable to disease development. Sequence-based prediction of effector function is difficulted by their rapid evolution rate. In the maize pathogen Ustilago maydis, effector-coding genes frequently organize in clusters. Here we describe the functional characterization of the pleiades, a cluster of ten symplastic effectors. Deletion of the pleiades leads to strongly impaired virulence and accumulation of reactive oxygen species (ROS) in infected tissue. Eight of the Pleiades suppress the production of ROS upon perception of pathogen associated molecular patterns (PAMPs). Although genetically redundant, the Pleiades target different host components. The paralogs Taygeta1 and Merope1 suppress ROS production in either the cytoplasm or nucleus, respectively. Merope1 targets and promotes the autoubiquitination activity of RFI2, a conserved family of E3 ligases that regulates the production of PAMP-triggered ROS burst and influences flowering time in plants.

microbiology↗

bioRxiv2018

Phosphorylation of the exocyst subunit Exo70B2 contributes to the regulation of its function

The exocyst is a conserved hetero-octameric complex that mediates early tethering of post-Golgi vesicles during exocytosis. Its Exo70 subunit functions as a spatiotemporal regulator by mediating numerous interactions with proteins and lipids. However, a molecular understanding of the exocyst functions remains challenging. Exo70B2 localized to dynamic foci at the plasma membrane and transited through Brefeldin A (BFA)-sensitive compartments, indicating that it participates in conventional secretion. Conversely, treatment with the immunogenic peptide flg22 or the salicylic acid (SA) defence hormone analogue Benzothiadiazole (BTH), induced Exo70B2 transport into the vacuole where it colocalized with autophagic markers AUTOPHAGY-RELATED PROTEIN 8 (ATG8) and NEIGHBOR OF BRCA1 GENE 1 (NBR1). According with its role in immunity, we discovered that Exo70B2 interacts with and is phosphorylated by the MITOGEN-ACTIVATED PROTEIN KINASE 3 (MPK3). Mimicking phosphorylation inhibited Exo70B2 localization at sites of active secretion. By contrast, lines expressing phosphonull variants displayed higher Effector-Triggered Immunity and were hypersensitive to BTH, conditions known to induce the secretory pathway. Our results suggest a molecular mechanism by which phosphorylation of Exo70B2 regulates interaction with the plasma membrane, and couples the secretory pathway with cellular signalling.

cell biology↗

bioRxiv2017

Wine yeast phenomics: a standardized fermentation method for assessing quantitative traits of Saccharomyces cerevisiae strains in enological conditions.

This work describes the set up of a small scale fermentation methodology for measuring quantitative traits of hundreds of samples in an enological context. By using standardized screw cap vessels, the alcoholic fermentation kinetics of Saccharomyces cerevisiae strains were measured by following the weight loss over time. Preliminary results showed that the kinetic parameters measured are in agreement with those observed in larger scale vats. The small volume used did not impair any analysis of the strain performance. Indeed, this fermentation system was coupled with robotized enzymatic assays and 8 end-point metabolites of enological interest were measured accurately. Moreover, the vessel used offers the possibility to assay 32 volatiles compounds using a headspace solid-phase microextraction coupled to gas chromatography-mass spectrometry approach. Data presented demonstrates that the shaking conditions significantly impacted the mean and the variance of kinetic parameters, primary metabolites, and the production of volatile compounds. This effect was likely due to an enhanced transfer of dissolved oxygen during the first hours of the alcoholic fermentation. To test the efficiency of this experimental design, the phenotypic response of 35 wine industrial starters was measured in five grape juices from the Bordeaux area. A multivariate analysis illustrated that strains were better discriminated by some grape must, than others. The technological performances as well as the phenotypic robustness of these strains was measured and discussed. The optimized methodology developed allows investigating multiple fermentation traits for numerous yeast strains and conditions and greatly contributes in achieving quantitative genetics studies as well as yeast selection programs.

microbiology↗

bioRxiv2017

The partial duplication of an E3-ligase gene in Triticeaespecies mediates resistance to powdery mildew fungi

In plant-pathogen interactions, components of the plant ubiquitination machinery are preferred targets of pathogen-encoded effectors suppressing defense responses or co-opting host cellular functions for accommodation. Here, we employed transient and stable gene silencing-and over-expression systems in Hordeum vulgare (barley) to study the function of HvARM1 (for H. vulgare Armadillo 1), a partial gene duplicate of the U-box/armadillo-repeat E3 ligase HvPUB15 (for H. vulgare Plant U-Box 15). The partial ARM1 gene was derived from an ancient gene-duplication event in a common ancestor of the Triticeae tribe of grasses comprising the major crop species H. vulgare, Triticum aestivum and Secale cereale. The barley gene HvARM1 contributed to quantitative host as well as nonhost resistance to the biotrophic powdery mildew fungus Blumeria graminis, and allelic variants were found to be associated with powdery mildew-disease severity. Both HvPUB15 and HvARM1 proteins interacted in yeast and plant cells with the susceptibility-related, plastid-localized barley homologs of THF1 (for Thylakoid formation 1) and of ClpS1 (for Clp-protease adaptor S1) of Arabidopsis thaliana. The results suggest a neo-functionalization HvARM1 to increase resistance against powdery mildew and provide a link to plastid function in susceptibility to biotrophic pathogen attack.

plant biology↗