Biology subjects

Cid, V. J.

Publications and source records attributed to Cid, V. J..

6 recordsLinked to original sources

bioRxiv2026

In vivo functional classification of PTEN variants reveals context-dependent oncogenicity and interindividual variability

Gene variants, secondary mutations, and stochastic individual variability complicate cancer diagnosis, prognosis, and treatments. Here, we systematically assess the functional impact of PTEN cancer-related missense mutations in mammalian cell lines, yeast, and Caenorhabditis elegans. While cell-based assays revealed alterations in lipid phosphatase activity, CRISPR-based engineering of orthologous mutations in C. elegans enabled classification of variants based on organismal phenotypes and transcriptional profiles, providing a rapid framework to predict oncogenic potential. We further show that secondary mutations, such as gain-of-function of cdc-25.1/CDC25A, can enhance the phenotypic impact of specific daf-18/PTEN variants, revealing context-dependent oncogenicity. Finally, single-worm transcriptomic analyses uncovered substantial interindividual variability among isogenic animals with identical cdc-25.1 and daf-18 mutations, linking transcriptional states to divergent phenotypic outcomes. Together, our results establish C. elegans as a powerful in vivo platform to integrate genetic, functional, and transcriptional information for the interpretation of cancer-associated variants.

cancer biology↗

bioRxiv2025

Human IRAK kinases differentially alter metabolic regulation and mitochondrial function in Saccharomyces cerevisiae

Interleukin-1 receptor-associated kinases (IRAKs) are Ser/Thr protein kinases characterized by an N-terminal Death domain (DD). Upon stimulation of Toll-like receptors (TLRs) or the interleukin-1 receptor (IL-1R), IRAKs are recruited to supramolecular signalling complexes, known as myddosomes, through interactions between their DDs and the adaptor protein MyD88. Myddosomes are essential for the activation of nuclear factor kappa B (NF-{kappa}B) in response to diverse pathogen- and damage-associated molecular patterns (PAMPs and DAMPs), and they contribute to inflammation, cell survival, and proliferation. In the hierarchical assembly of the myddosome, MyD88 first recruits IRAK4, which serves as a scaffold for the subsequent binding of IRAK1 and/or IRAK2. To explore alternative models for studying IRAK function, we expressed human IRAK1, IRAK2 and IRAK4 individually in Saccharomyces cerevisiae and performed a comparative analysis. Heterologous expression of these kinases, especially IRAK4, impaired yeast growth; an effect dependent on its kinase activity. Transcriptomic and biochemical assays revealed that IRAK1 and IRAK4, but not IRAK2, differentially impacted metabolic regulation. Notably, IRAK4 induced mitochondrial fragmentation and mitochondrial membrane potential depolarization, whereas IRAK1 had the opposite effect. Additionally, IRAK4 led to actin depolarization and vacuole fragmentation. Based on these findings, we develop two yeast-based bioassays to screen for IRAK4 kinase inhibitors: one based on growth recovery and another using a fluorescent reporter. We provide proof-of-concept that both assays are suitable for evaluating IRAK4 function and its pharmacological inhibition. ImportanceIRAK kinases are essential components of the myddosome signalling complex, a key mediator of the innate immune response, with IRAK4 playing a pivotal role in the recruitment of IRAK1 and/or IRAK2. Although the precise cellular functions of IRAK-dependent phosphorylation remain incompletely understood, IRAK inhibitors are emerging as promising therapeutic candidates for the treatment of autoimmune disorders, such as rheumatoid arthritis, and various cancers, including acute myeloid leukemia. To date, most insights into IRAK function have been derived from studies in animal models, particularly mice. Our work establishes Saccharomyces cerevisiae as a convenient and genetically tractable platform for in vivo analyses of human IRAKs. We demonstrate that IRAK kinases induce metabolic deregulation and growth inhibition in yeast. Engineered S. cerevisiae strains could therefore be exploited for the preclinical screening of anti-inflammatory and antitumor compounds targeting IRAK signalling, potentially contributing to the development of therapies for severe human diseases.

cell biology↗

bioRxiv2022

Human Gasdermin D and MLKL disrupt mitochondria, endocytic traffic and TORC1 signaling in budding yeast

Gasdermin D (GSDMD) and mixed lineage kinase domain-like protein (MLKL) are the pore-forming effectors of pyroptosis and necroptosis, respectively, with the capacity to disturb plasma membrane selective permeability and induce programmed cell death. The budding yeast Saccharomyces cerevisiae has long been used as a simple eukaryotic model for the study of proteins associated with human diseases by heterologous expression. In this work, we expressed in yeast both GSDMD and its N-terminal domain [GSDMD(NT)] to characterize their cellular effects, and compare them to those of MLKL. GSDMD(NT) and MLKL inhibited yeast growth, formed cytoplasmic aggregates, and fragmented mitochondria. Loss-of-function point mutants of GSDMD(NT) showed affinity for this organelle. Besides, GSDMD(NT) and MLKL caused an irreversible cell cycle arrest through TORC1 inhibition, and disrupted endosomal and autophagic vesicular traffic. Our results provide a basis for a humanized yeast platform to study GSDMD and MLKL, a useful tool for structure-function assays and drug discovery.

cell biology↗

bioRxiv2021

Heterologous expression of human pro-inflammatory Caspase-1 in Saccharomyces cerevisiae and comparison to pro-apoptotic Caspase-8

Caspases are a family of cysteine proteases that play an essential role in inflammation, apoptosis, cell death, and development. Here we delve into the effects caused by heterologous expression of human Caspase-1 in the yeast Saccharomyces cerevisiae and compare them to those of Caspase-8. Overexpression of both caspases in the heterologous model led to their activation, and caused mitochondrial depolarization, ROS production, damage to different organelles, and cell death. All these effects were dependent on their protease activity, and Caspase-8 was more aggressive than Caspase-1. Growth arrest could be at least partially explained by dysfunction of the actin cytoskeleton as a consequence of the processing of the yeast Bni1 formin, which we identify here as a likely direct substrate of both caspases. Through the modulation of the GAL1 promoter by using different galactose:glucose ratios in the culture medium, we have established a scenario in which Caspase-1 is sufficiently expressed to become activated while yeast growth is not impaired. Finally, we used the yeast model to explore the role of death-fold domains (DD) of both caspases in their activity. Peculiarly, the DDs of either caspase showed an opposite involvement in its intrinsic activity, as the deletion of the caspase activation and recruitment domain (CARD) of Caspase-1 enhanced its activity, while the deletion of the death effector domain (DED) of Caspase-8 diminished it. We propose the yeast model as a useful and manageable tool to explore Caspase-1 structure-function relationships, the impact of mutations or the activity of putative inhibitors or regulators.

molecular biology↗

bioRxiv2019

The TIR-domain containing effectors BtpA and BtpB from Brucella abortus block energy metabolism

Brucella species are facultative intracellular Gram-negative bacteria relevant to animal and human health. Their ability to establish an intracellular niche and subvert host cell pathways to their advantage depends on the delivery of bacterial effector proteins through a type IV secretion system. Brucella Toll/Interleukin-1 Receptor (TIR)-domain-containing proteins BtpA (also known as TcpB) and BtpB are among such effectors. Although divergent in primary sequence, they interfere with Toll-like receptor (TLR) signaling to inhibit the innate immune responses. However, the molecular mechanisms implicated still remain unclear. To gain insight into the functions of BtpA and BtpB, we expressed them in the budding yeast Saccharomyces cerevisiae as a eukaryotic cell model. We found that both effectors were cytotoxic and that their respective TIR domains were necessary and sufficient for yeast growth inhibition. Growth arrest was concomitant with actin depolymerization, endocytic block and a general decrease in kinase activity in the cell, suggesting a failure in energetic metabolism. Indeed, levels of ATP and NAD+ were low in yeast cells expressing BtpA and BtpB TIR domains, consistent with the recently described enzymatic activity of some TIR domains as NAD+ hydrolases. In human epithelial cells, both BtpA and BtpB expression reduced intracellular total NAD levels. In infected cells, both BtpA and BtpB contributed to reduction of total NAD, indicating that their NAD+ hydrolase functions are active intracellularly during infection. Overall, combining the yeast model together with mammalian cells and infection studies our results show that BtpA and BtpB modulate energy metabolism in host cells through NAD+ hydrolysis, assigning a novel role for these TIR domain-containing effectors in Brucella pathogenesis.

microbiology↗

bioRxiv2019

Klebsiella pneumoniae type VI secretion system-mediated microbial competition is PhoPQ controlled and reactive oxygen species dependent

Klebsiella pneumoniae is recognized as an urgent threat to human health due to the increasing isolation of multidrug resistant strains. Hypervirulent strains are a major concern due to their ability to cause life-threating infections in healthy hosts. The type VI secretion system (T6SS) is widely implicated in microbial antagonism, and it mediates interactions with host eukaryotic cells in some cases. In silico search for genes orthologous to T6SS component genes and T6SS effector genes across 700 K. pneumoniae genomes shows extensive diversity in T6SS genes across the K. pneumoniae species. Temperature, oxygen tension, pH, osmolarity, iron levels, and NaCl regulate the expression of the T6SS encoded by a hypervirulent K. pneumoniae strain. Polymyxins and human defensin 3 also increase the activity of the T6SS. A screen for regulators governing T6SS uncover the correlation between the transcription of the T6SS and the ability to kill E. coli prey. Whereas H-NS represses the T6SS, PhoPQ, PmrAB, Hfq, Fur, RpoS and RpoN positively regulate the T6SS. K. pneumoniae T6SS mediates intra and inter species bacterial competition. This antagonism is only evident when the prey possess an active T6SS. The PhoPQ two component system governs the activation of K. pneumoniae T6SS in bacterial competitions. Mechanistically, PhoQ periplasmic domain, and the acid patch within, is essential to activate K. pneumoniae T6SS. Klebsiella T6SS also mediates anti-fungal competition. We have delineated the contribution of each of the individual VgrGs in microbial competition, and identified VgrG4 as a T6SS effector. Structurally, domain DUF2345 of VgrG4 is sufficient to intoxicate bacteria and yeast. ROS generation mediates the antibacterial effects of VgrG4, and the antitoxin Sel1E protects against the toxic activity of VgrG4. Our findings provide a better understanding of the regulation of the T6SS in bacterial competitions, and place ROS as an early event in microbial competition.\n\nAUTHOR SUMMARYKlebsiella pneumoniae has been singled out as an \"urgent threat to human health\" due to extremely drug resistant strains. Numerous studies investigate the molecular mechanisms underlying antibiotic resistance in K. pneumoniae, while others dissect the virulence strategies of this pathogen. However, there is still limited knowledge on the fitness of Klebsiella in the environment, and particularly the competition of Klebsiella with other species. Here, we demonstrated that Klebsiella exploits the type VI secretion system (T6SS) nanoweapon to kill bacterial competitors and fungi. K. pneumoniae perceives T6SS attacks from bacterial competitors, resulting in retaliation against the aggressive cell. The perception of the attack involved the sensor PhoPQ and led to the up-regulation of the T6SS. We identified one of the toxins deployed by the T6SS to antagonize other microbes, and revealed how Klebsiella protects itself from this toxin. Our findings provide a better understanding of the T6SS role in microbial competition and uncover new aspects on how bacteria regulate T6SS-mediated microbial antagonism.

microbiology↗