Biology subjects

Silva, G.

Publications and source records attributed to Silva, G..

12 recordsLinked to original sources

bioRxiv2026

Redox-modulated bacterial deubiquitinase ElaD: Target recognition and suppression of K63-linked polyubiquitin accumulation in yeast.

Bacterial deubiquitinases (DUBs) are important virulence effectors that manipulate host ubiquitin signaling during infection. ElaD, a CE-clan DUB expressed by enterohemorrhagic Escherichia coli, preferentially cleaves K63-linked ubiquitin chains, yet its effects on conserved cellular stress responses remain poorly understood. We demonstrate that ElaD exhibits redox-dependent DUB activity in vitro. In addition, we identified the molecular basis underlying the selective recognition of substrate proteins, ubiquitin and NEDD8 by ElaD. Structural and mutational analyses reveal that, beyond the conserved catalytic site, ElaD engages ubiquitin through a combination of electrostatic and hydrophobic interactions. Using Saccharomyces cerevisiae as a heterologous model system, we show that wild-type ElaD rescues the proteotoxic stress phenotype of ubp2{Delta} yeast cells, whereas specific ElaD mutants fail to confer a similar response. Furthermore, expression of ElaD suppresses oxidative stress-induced accumulation of K63-linked polyubiquitin and may perturb stress-associated translational regulation linked to K63 ubiquitin signaling. Consequently, cells expressing ElaD exhibit altered stress adaptation and diminished fitness during prolonged oxidative stress. Collectively, these findings indicate that ElaD perturbs ubiquitin-mediated stress signaling by counteracting K63-linked ubiquitination events that support adaptive cellular responses. Our study highlights how a bacterial DUB can reprogram conserved ubiquitin-dependent pathways and exploit host ubiquitin signaling networks to modulate cellular stress responses and protein homeostasis. These findings further suggest potential host targets of bacterial DUBs during infection.

biophysics↗

bioRxiv2025

3D co-cultures of primary human hepatocytes and Kupffer-like cells to address innate immune responses to rAAV

Recombinant adeno-associated viruses (rAAVs) are a platform of choice for gene therapy. However, liver-directed transduction has been hindered by immune responses unpredicted in the preclinical models, resulting in therapy failure. Liver immune responses are strictly regulated by the interactions between hepatocytes and non-parenchymal liver cells, such as Kupffer cells (the liver-resident macrophages) but how rAAVs induce such responses remains largely unknown. Therefore, human models recapitulating such interactions are required to address innate immune responses. Here, we developed a human 3D model to characterize the contribution of hepatocytes and Kupffer cells to the innate immune response. We developed a strategy for the differentiation of Kupffer-like cells from circulating monocytes based on cell-cell contact with primary human hepatocytes. We fine-tuned critical co-culture parameters to obtain a Kupffer-like phenotype while retaining hepatocyte viability and identity. Functional assessment of the differentiated 3D co-cultures showed that the model is responsive to classical pathogen-associated molecular pattern molecules, at a gene expression and secretory level. Moreover, we observed increased proinflammatory cytokine expression and secretion when challenged with a rAAV vector. Our data indicate the suitability of the novel model to investigate hepatocyte-Kupffer cell interactions and address innate immune responses within a human liver microenvironment.

immunology↗

bioRxiv2025

Innate immunocompetent iNSpheroids: A hiPSC-derived 3D model to study the central nervous system captures an early CNS response to rAAV

Gene therapies using adeno-associated viruses (AAVs) for central nervous system (CNS) disorders face challenges due to host immune responses not represented in classical preclinical models. Here, we present a human-induced pluripotent stem cell (hiPSC)-derived innate immunocompetent 3D CNS model that recapitulates neuroinflammatory hallmarks, serving as a platform for preclinical gene therapy development. Utilizing various scales of stirred-tank bioreactor systems, we generated (neurospheroids) iNSpheroids composed of neurons, astrocytes, and oligodendrocytes, alongside microglial cells (iMGL) to mimic the neuro-immune axis. These systems enabled large-scale production of iNSpheroids and subsequent miniaturization for co-culture experiments and screening of inflammatory stimuli, while maintaining a highly controlled environment. The iMGL-iNSpheroids demonstrated active neuron-microglia crosstalk and exhibited distinct inflammatory responses to a series of neuroinflammatory factors. iMGL-iNSpheroids mounted a mild and transient response to rAAV9, mediated by the activation of inflammatory pathways (e.g., TNF-via NF-{kappa}B activation) in glial cell populations. This model offers a valuable tool to dissect neuroinflammatory mechanisms, accelerating gene therapy development. TeaserImmune-competent 3D human CNS model recapitulates glial responses to rAAVs, enabling reliable preclinical gene therapy screening.

bioengineering↗

bioRxiv2024

Context specific ubiquitin modification of ribosomes regulate translation under oxidative stress

Cellular exposure to stress is known to activate several translational control pathways through ribosome ubiquitination. However, how unique patterns of ribosome ubiquitination act at the site-specific level to drive distinct modes of translation regulation remains unclear. To further understand the complexity of these ubiquitin signals, we developed a new targeted proteomics approach to quantify site-specific ubiquitin modification across the ribosome. This method increased the sensitivity and throughput of current approaches and allowed us to systematically measure the ubiquitin status of 78 ribosome peptides and ubiquitin linkages in response to stress. Using this method, we were able to detect the ubiquitination of several ribosome sites even in steady-state conditions, and to show that their modification increases non-stoichiometrically in a dynamic range of >4 orders of magnitude in response to hydrogen peroxide. Besides demonstrating new patterns of global ribosome ubiquitination, our study also revealed an unexpected increase of ubiquitination of ribosomal protein uS10/Rps20 and uS3/Rps3 independent of the canonical E3 ubiquitin ligase Hel2. Furthermore, we show that unique and mixed patterns of ribosome ubiquitination occur in a stress specific manner, depending on the nature of stressor and the enzymes involved. Finally, we showed that while deletion of HEL2 further induces the integrated stress response in response to the nucleotide alkylating agent 4-NQO, deletion of the E2 conjugase RAD6 leads to sustained translation only in response to H2O2. Our findings contribute to deciphering the complexity of the stress response at the translational level, revealing the induction of dynamic and selective ubiquitin codes, which shed light on the integration of important quality control pathways during cellular response to stress.

molecular biology↗

bioRxiv2024

Redox control of the deubiquitinating enzyme Ubp2 regulates translation during stress

Protein ubiquitination is essential to govern cells ability to cope with harmful environments by regulating many aspects of protein dynamics from synthesis to degradation. As important as the ubiquitination process, the reversal of ubiquitin chains mediated by deubiquitinating enzymes (DUBs) is critical for proper recovery from stress and re-establishment of proteostasis. Although it is known that ribosomes are decorated with K63-linked polyubiquitin (K63-ub) chains that control protein synthesis under stress, the mechanisms by which these ubiquitin chains are reversed and regulate proteostasis during stress recovery are still illusive. Here, we showed in budding yeast that the DUB Ubp2 is redox regulated during oxidative stress in a reversible manner, which determines the levels of K63-ub chains present on ribosomes. We also demonstrate that Ubp2 is a processive enzyme whose activity is modulated by a series of repeated domains and the formation of important disulfide bonds. By combining, cellular, biochemical, and proteomics analyses, we showed that Ubp2 is crucial for restoring translation after stress cessation, indicating an important role in determining cellular response to oxidative stress. Our work demonstrates a novel role for Ubp2, revealing that a range of signaling pathways can be controlled by redox regulation of DUB activity in eukaryotes, which in turn will define cellular states of health and diseases.

biochemistry↗

bioRxiv2023

Nonviral PDMAEMA Vectors Efficiently Express PDE6beta in a Mouse Model of Retinitis Pigmentosa

Retinitis Pigmentosa (RP) is one of main causes of inherited blindness, with about 6% of cases caused by a single mutation in the PDE6{beta} gene, making it an ideal candidate for a gene therapy intervention. Gene therapy has been shown to restore normal retinal and visual function in other monogenic diseases, such as Lebers Congenital Amaurosis and choroideremia, and RP could benefit from a similar therapeutic approach. Our goal was to combine efficient nonviral vectors and gene expression systems to express the PDE6{beta} gene in the retina of a mouse model of Retinitis Pigmentosa. We have firstly validated the potential of PDMAEMA polyplexes as a nonviral vector by subretinal injection, which were shown to efficiently promote gene expression in the RPE layer of the adult mouse retina. We have then produced polyplexes of PDMAEMA with a replicating plasmid expressing the PDE6{beta} gene and administered the polyplexes via subretinal injection in the rd10 mouse. We have observed that PDMAEMA polyplexes were able to efficiently enter the target retinal pigment epithelium cells and rapidly express the PDE6{beta} gene in the mouse retina, thus confirming their potential as nonviral vectors for retinal gene therapy.

bioengineering↗

bioRxiv2022

The miR156-targeted SlSBP15 represses tomato shoot branching via modulating auxin transport and interacting with GOBLET and BRANCHED1b

The microRNA156 (miR156)/SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL/SBP) regulatory hub is highly conserved among phylogenetically distinct species, but how it interconnects multiple pathways to converge to common integrators controlling shoot architecture is still unclear. Here, we demonstrated that the miR156/SlSBP15 hub modulates tomato shoot branching (SB) by connecting phytohormones with important genetic pathways regulating both axillary bud (AB) development and outgrowth. We verified that plants overexpressing the miR156 (156-OE plants) display high SB, whereas plants overexpressing a miR156-resistant SlSBP15 alelle (rSBP15 plants) display arrested SB and are able to partially restore the wild-type (WT) phenotype in156-OE background. Although rSBP15 plants showed ABs smaller than MT, its activation is dependent on shoot apex-derived auxin transport inhibition. Additionally, hormonal measurements reveal that IAA and ABA concentrations were lower in 156-OE and higher in rSBP15-OE plants. SlSBP15 regulates AB development and outgrowth by inhibiting auxin transport and the activity of GOBLET (GOB), and by interacting with BRANCHED1b (SlBRC1b) at the protein level to control abscisic acid (ABA) levels within ABs. Our data provide a new mechanism by which the miR156/SPL/SBP hub regulates SB, and suggest that SlSBP15 has potential applications in improving tomato architecture.

developmental biology↗

bioRxiv2022

The ubiquitin conjugase Rad6 mediates ribosome pausing during oxidative stress

Oxidative stress causes K63-linked ubiquitination of ribosomes by the E2 ubiquitin conjugase, Rad6. How Rad6-mediated ubiquitination of ribosomes affects global translation, however, is unclear. We therefore performed Ribo-seq and Disome-seq in Saccharomyces cerevisiae, and found that oxidative stress caused ribosome pausing at specific amino acid motifs, and this also led to ribosome collisions. However, these redox pausing signatures were lost in the absence of Rad6 but did not depend on the ribosome-associated quality control (RQC) pathway. We also found that Rad6 is needed to inhibit overall translation in response to oxidative stress and its deletion leads to increased expression of antioxidant genes. Finally, we observed that the lack of Rad6 leads to changes during translation initiation that affect activation of the integrated stress response (ISR) pathway. Our results provide a high-resolution picture of the gene expression changes during oxidative stress and unravel an additional stress response pathway affecting translation elongation. HIGHLIGHTSO_LIRad6 is required for sequence-specific ribosome pausing under oxidative stress. C_LIO_LIRad6 affects translation independently of the RQC pathway. C_LIO_LICells lacking Rad6 show dysregulated translational repression upon oxidative stress. C_LIO_LILoss of Rad6 leads to altered activation of the ISR pathway. C_LI

molecular biology↗

bioRxiv2022

Targeting C5aR1 signaling reduced neutrophil extracellular traps and ameliorates COVID-19 pathology

Patients with severe COVID-19 develop acute respiratory distress syndrome (ARDS) that may progress to cytokine storm syndrome, organ dysfunction, and death. Considering that complement component 5a (C5a), through its cellular receptor C5aR1, has potent proinflammatory actions, and plays immunopathological roles in inflammatory diseases, we investigated whether C5a/C5aR1 pathway could be involved in COVID-19 pathophysiology. C5a/C5aR1 signaling increased locally in the lung, especially in neutrophils of critically ill COVID-19 patients compared to patients with influenza infection, as well as in the lung tissue of K18-hACE2 Tg mice (Tg mice) infected with SARS-CoV-2. Genetic and pharmacological inhibition of C5aR1 signaling ameliorated lung immunopathology in Tg-infected mice. Mechanistically, we found that C5aR1 signaling drives neutrophil extracellular trap (NET)s-dependent immunopathology. These data confirm the immunopathological role of C5a/C5aR1 signaling in COVID-19 and indicate that antagonist of C5aR1 could be useful for COVID-19 treatment.

immunology↗

bioRxiv2022

Evaluation of PRKACA as a Therapeutic Target for Fibrolamellar Carcinoma

Background & AimsFibrolamellar carcinoma (FLC) is a rare, difficult-to-treat liver cancer primarily affecting pediatric and adolescent patients, and for which precision medicine approaches have historically not been possible. The DNAJB1-PRKACA gene fusion was identified as a driver of FLC pathogenesis. We aimed to assess whether FLC tumors maintain dependency on this gene fusion and determine if PRKACA is a viable therapeutic target. MethodsFLC patient-derived xenograft (PDX) shRNA cell lines were implanted subcutaneously into female NOD-SCID mice and tumors were allowed to develop prior to randomization to doxycycline (to induce knockdown) or control groups. Tumor development was assessed every 2 days. To assess the effect of treatment with novel selective PRKACA small molecule kinase inhibitors, BLU0588 and BLU2864, FLC PDX tumor cells were implanted subcutaneously into NOD-SCID mice and tumors allowed to develop. Mice were randomized to treatment (BLU0588 and BLU2864, orally, once daily) or control groups and tumor size determined as above. ResultsKnockdown of DNAJB1-PRKACA reversed a FLC-specific gene signature and reduced PDX tumor growth in mice compared to the control group. Furthermore, FLC PDX tumor growth was significantly reduced with BLU0588 and BLU2864 treatment versus control (P = 0.003 and P = 0.0005, respectively). ConclusionsWe demonstrated, using an inducible knockdown and small molecule approaches, that FLC PDX tumors were dependent upon DNAJB1-PRKACA fusion activity. In addition, this study serves as a proof-of-concept that PRKACA is a viable therapeutic target for FLC and warrants further investigation.

cancer biology↗

bioRxiv2021

Commercial vaccines do not confer protection against two genetic strains of Piscirickettsia salmonis, LF-89-like and EM-90-like, in Atlantic salmon.

In Atlantic salmon, vaccines have failed to control and prevent Piscirickettsiosis, for reasons that remain elusive. In this study, we report the efficacy of a commercial vaccine developed with the Piscirickettsia salmonis isolate AL100005 against other two isolates which are considered highly and ubiquitously prevalent in Chile: LF-89-like and EM-90-like. Two cohabitation trials were performed to mimic real-life conditions and vaccine performance: 1) post smolt fish were challenged with a single infection of LF-89-like, 2) adults were coinfected with EM-90-like and a low coinfection of sea lice. In the first trial, the vaccine delayed smolt mortalities by two days; however, unvaccinated and vaccinated fish did not show significant differences in survival (unvaccinated: 60.3%, vaccinated: 56.7%; p = 0.28). In the second trial, mortality started three days later for vaccinated fish than unvaccinated fish. However, unvaccinated and vaccinated fish did not show significant differences in survival (unvaccinated: 64.6%, vaccinated: 60.2%, p= 0.58). Thus, we found no evidence that the evaluated vaccines confer effective protection against of LF-89-like or EM-90-like with estimated relative survival proportions (RPSs) of -9% and -12%, respectively. More studies are necessary to evaluate whether pathogen heterogeneity is a key determinant of the vaccine efficacy against P. salmonis.

immunology↗

bioRxiv2017

Integrative meta-analysis reveals that most yeast proteins are very stable

Measurements for protein half-lives in yeast Saccharomyces cerevisiae reported large discrepancies, with median values between minutes to several hours. We present a unifying analysis that provides a consistent half-life estimate, based on our re-analysis of three published and one new dataset of cells grown under similar conditions. We found that degradation of many proteins can be approximated by exponential decay. Protein disappearance was primarily driven by dilution due to cell division, with cell doubling times ranging from [~]2 to 3.5 hours across the four experiments. After adjusting for doubling time, protein half-lives increased to median values between [~]7.5 to [~]40 hours. Half-lives correlated with cell doubling time even after adjustment, implying that slow growth also slows protein degradation. All estimates were validated by multiple means and were robust to different analysis methods. Overall, protein stability correlated with abundance and showed weak enrichment for degradation signals such as degrons and disordered regions. Long-lived proteins often functioned in oxidation-reduction and amino acid synthesis. Short-lived proteins often functioned in ribosome biogenesis. Despite some overall differences in behavior, all methods were able to resolve subtle difference in half-lives of ribosomal proteins, e.g. the short lifespan of RPL10. Finally, our results help the design of future experiments: time series measurements need to cover at least two to three cell doubling times for accurate estimates, exponential decay provides a reasonable proxy for protein stability, and it can be sufficiently estimated with four measurement points.

systems biology↗