Biology subjects

Nigro, G.

Publications and source records attributed to Nigro, G..

11 recordsLinked to original sources

bioRxiv2026

The replication checkpoint couples fork progression to the release of transcribed genes from nuclear pores

The nuclear envelope contributes to chromatin compartmentalization in eukaryotes by anchoring heterochromatin to the lamina and active transcription sites to nuclear pore complexes (NPCs). During DNA replication, chromatin association with the nuclear envelope is thought to act as a topological barrier to DNA polymerases, potentially exacerbating transcription-replication conflicts (TRCs). While limited evidence suggests that the DNA replication checkpoint kinase Rad53/CHK1 promotes the release of genes from the NPC during replicative stress, the global scale and precise regulation of this process remain unknown. Through a candidate-based screen, we demonstrate that gene release from NPCs is a general feature of the cellular response to replication stress, tightly coordinated with replication fork progression. We show that the checkpoint kinases Mec1/ATR and Rad53/CHK1 are both essential for managing gene association with NPCs, but operate in a distinct, stepwise manner: Mec1/ATR triggers the initial dissociation by promoting the removal of RNAPII from the DNA, while Rad53/CHK1 prevents the premature reassociation of transcribed loci by phosphorylating the nucleoporin Mlp1. Finally, we demonstrate that maintaining this control over genes-NPC association is critical for efficient DNA replication under stress. Collectively, our findings reveal that the DNA replication checkpoint integrates genome duplication with chromosome spatial positioning to maintain genome integrity during replication stress.

genetics↗

bioRxiv2026

ILC3s are Required for Enterocyte Homeostasis to Food Intake

Food provides nutrients that are selectively absorbed by the intestine, but, at the same time, may contain elements that challenge the intestinal barrier and induce post-prandial inflammation (PPI). How PPI is controlled in order to avoid pathological perturbation of homeostasis remains unclear. Here, we report that during fasting, enterocytes increase their absorptive potential and oxidative metabolism, a program that is largely reversed upon food intake of lipids that perturb the intestinal barrier and induce PPI. Such perturbation is countered by ILC3s, in the absence of which PPI increases, program reversal does not occur, and enterocytes engage into excessive oxidative metabolism. This enterocyte state leads to critical hypoglycemia as a consequence of decreased glucose absorption and increased insulinemia, recapitulating the pathological situation found in patients suffering from intestinal damage and sepsis. We hereby uncover a critical function for ILC3s in maintaining enterocyte homeostasis upon challenging food intake.

immunology↗

bioRxiv2026

A crypt-operating DNA repair checkpoint for uncoupling regeneration and tumorigenesis

The rapid turnover of the intestinal epithelium increases its vulnerability to genomic instability and environmental insults such as irradiation. Defects in DNA damage resolution can compromise epithelial regeneration, promote chronic tissue injury, and predispose to colorectal tumorigenesis. However, the intrinsic mechanisms that coordinate DNA repair with epithelial regeneration at the crypt level remain poorly defined. Here, we identify the Nod-like receptor protein 6 (Nlrp6) as a key epithelial regulator of genome surveillance and regenerative control in intestinal crypts. Nlrp6 is strategically expressed in crypt base columnar cells, where it preserves crypt homeostasis by restraining proliferation under genotoxic stress conditions. Loss of Nlrp6 in crypt base columnar cells results in uncontrolled oncogenic stress, defective epithelial regeneration, and accumulation of unrepaired DNA damage, features associated with poor prognosis in colorectal cancer. Conversely, aberrant Nlrp6 overexpression induces cytoplasmic retention of Csnk2 catalytic subunits, limiting their nuclear availability when DNA repair is required. These findings position Nlrp6 as a non-canonical, cell-intrinsic surveillance mechanism that links DNA damage responses to epithelial regeneration through Csnk2-dependent signaling. Collectively, our study reveals a crypt-intrinsic DNA repair pathway that governs epithelial regeneration and disease outcomes, providing new insight into how genome instability and regenerative failure contribute to colorectal cancer progression.

cell biology↗

bioRxiv2025

The E. coli CNF1 toxin induces fetal reprogramming of intestinal stem cells and a serrated colorectal cancer transcriptional signature in organoids

Mechanistic insights are essential to understand how gut microbiota pathobionts contribute to tumorigenesis, given the mounting evidence linking them to colorectal cancer. We report a higher prevalence of the Rho GTPases-targeting cnf1 toxin gene from Escherichia coli in the microbiota of early-stage, proximal colorectal cancer. Comparative gene set enrichment analysis reveals a concordant serrated pathway signature between colorectal cancer tissue of patients colonized with cnf1+ bacteria and CNF1-treated mouse intestinal organoids. RNA sequencing of organoids shows that CNF1 induces a fetal-like transcriptional reprogramming. Using integrated approaches, we demonstrate that CNF1 reprograms Lgr5 stem cells into a Ly6a/Sca-1 fetal-like state, that exhibits enhanced stemness potential. This reprogramming is preceded by a Yap/Taz-driven early transcriptional program and nuclear translocation of Yap. Functional analyses identify a RhoA/Rock-Yap/Taz-dependent transition to Ly6a/Sca-1 stem cells, highlighting a mechanistic link between bacterial effectors and stem cell plasticity in colorectal tumorigenesis.

cancer biology↗

bioRxiv2024

CD300e as a Driver of Immunosuppressive Tumor Microenvironment in Colorectal Cancer

Colorectal cancer (CRC) progression is shaped by the tumor microenvironment, particularly tumor-associated macrophages (TAMs), which often adopt immunosuppressive functions. CD300e, a myeloid receptor involved in immune regulation, has an uncharacterized role in CRC. Here, we show that CD300e is selectively upregulated in tumor-infiltrating monocytes and macrophages, driving a suppressive phenotype marked by impaired antigen presentation. In vitro cocultures of patient-derived tumor organoids and human monocytes revealed that tumor-derived signals induce CD300e expression and promote a protumorigenic macrophage profile. Using CD300e knockout mice in AOM/DSS and MC38 CRC models, we found that CD300e loss reduced tumor burden, enhanced MHC expression on TAMs, and improved T-cell responses. Transcriptomic and functional analyses demonstrated that CD300e-deficient macrophages exhibit increased phagocytosis, upregulated antigen presentation, and greater support for T-cell proliferation and cytotoxicity. Adoptive transfer confirmed that macrophage-intrinsic CD300e expression is sufficient to suppress T-cell function and promote tumor growth. Our findings identify CD300e as a critical regulator of macrophage-mediated immune suppression in CRC and a potential target for reprogramming TAMs to enhance immunotherapy.

cancer biology↗

bioRxiv2023

The oncogenic role of Streptococcus gallolyticus subsp. gallolyticus is linked to activation of multiple cancer-related signaling pathways

Streptococcus gallolyticus subsp. gallolyticus (SGG), an opportunistic gram-positive pathogen responsible for septicemia and endocarditis in the elderly, is often associated with colon cancer (CRC). In this work, we investigated the oncogenic role of SGG strain UCN34 using the azoxymethane (AOM)-induced CRC model in vivo, organoid formation ex vivo and proteomic and phosphoproteomic analyses from murine colons. We showed that SGG UCN34 accelerates colon tumor development in the murine CRC model. Full proteome and phosphoproteome analysis of murine colons chronically colonized by SGG UCN34 or the closely related non-pathogenic S. gallolyticus subsp. macedonicus (SGM) revealed that 164 proteins and 725 phosphorylation sites were differentially regulated following colonization by SGG UCN34. Ingenuity Pathway Analysis (IPA) indicates a pro-tumoral shift specifically induced following colonization by SGG UCN34, as most proteins and phosphoproteins identified were associated with digestive cancer. Comprehensive analysis of the altered phosphoproteins using ROMA software revealed significantly elevated activities in several cancer hallmark pathways affecting tumoral cells and their microenvironment, i.e. MAPK (ERK, JNK and p38), mTOR and integrin/ILK/actin signaling, in SGG UCN34 colonized colon. Importantly, analysis of protein arrays of human colon tumors colonized with SGG showed up-regulation of PI3K/Akt/mTOR and MAPK pathways, providing clinical relevance to our findings. To test SGGs capacity to induce pre-cancerous transformation of the murine colonic epithelium, we grew ex vivo organoids which revealed unusual structures with compact morphology following exposure to SGG. Taken together, our results reveal that the oncogenic role of SGG UCN34 is associated with activation of multiple cancer-related signaling pathways. Author SummaryColorectal cancer is the third most common cause of cancer mortality worldwide. The colon is a very singular organ, colonized by a vast and complex community of microorganisms, known as the gut microbiota. Strong evidence supports a role of the microbiota in colon cancer development. Streptococcus gallolyticus subsp. gallolyticus (SGG), a gut commensal, was one of the first bacteria to be associated with colorectal cancer. A better understanding of the role of SGG in colon cancer development is critical to developing novel strategies to improve clinical diagnosis and treatment of this disease. Here, using a global proteomic analysis of mouse colonic tissue colonized by SGG, we show that over 90% of the proteins with altered levels are involved in cancer. SGG colonization promotes autocrine and paracrine pro-tumor signals contributing to transformation of the colonic epithelium but also modifying the stromal microenvironment, which in turn sustains tumor development. Importantly, two tumor hallmark pathways (PI3K/Akt/mTOR and MAPK) identified in our mouse model were also found in human colon tumor biopsies colonized by SGG, strengthening the clinical relevance to our study.

microbiology↗

bioRxiv2022

Redesigning methionyl-tRNA synthetase for β-methionine activity with adaptive landscape flattening and experiments

Amino acids (AAs) with a noncanonical backbone would be a valuable tool for protein engineering, enabling new structural motifs and building blocks. To incorporate them into an expanded genetic code, the first, key step is to obtain an appropriate aminoacyl-tRNA synthetase (aaRS). Currently, directed evolution is not available to optimize such AAs, since an appropriate selective pressure is not available. Computational protein design (CPD) is an alternative. We used a new CPD method to redesign MetRS and increase its activity towards {beta}-Met, which has an extra backbone methylene. The new method considered a few active site positions for design and used a Monte Carlo exploration of the corresponding sequence space. During the exploration, a bias energy was adaptively learned, such that the free energy landscape of the apo enzyme was flattened. Enzyme variants could then be sampled, in the presence of the ligand and the bias energy, according to their {beta}-Met binding affinities. Eleven predicted variants were chosen for experimental testing; all exhibited detectable activity for {beta}-Met adenylation. Top predicted hits were characterized experimentally in detail. Dissociation constants, catalytic rates, and Michaelis constants for both -Met and {beta}-Met were measured. The best mutant retained a preference for -Met over {beta}-Met; however, the preference was reduced, compared to the wildtype, by a factor of 29. For this mutant, high resolution crystal structures were obtained in complex with both -Met and {beta}-Met, indicating that the predicted, active conformation of {beta}-Met in the active site was retained. Author summaryAmino acids (AAs) with a noncanonical backbone would be valuable for protein engineering, enabling new structural motifs. To incorporate them into an expanded genetic code, the key step is to obtain an appropriate aminoacyl-tRNA synthetase (aaRS). Currently, directed evolution is not available to optimize such AAs. Computational protein design is an alternative. We used a new method to redesign MetRS and increase its activity towards {beta}-Met, which has an extra backbone methylene. The method considered a few active site positions for design and used a Monte Carlo exploration of sequence space, during which a bias energy was adaptively learned, such that the free energy landscape of the apo enzyme was flattened. Enzyme variants could then be sampled, in the presence of the ligand and the bias energy, according to their {beta}-Met binding affinities. Eleven predicted variants were chosen for experimental testing; all exhibited detectable {beta}-Met adenylation activity. Top hits were characterized experimentally in detail. The best mutant had its preference for -Met over {beta}-Met reduced by a factor of 29. Crystal structures indicated that the predicted, active conformation of {beta}-Met in the active site was retained.

biochemistry↗

bioRxiv2022

Parvimonas micra, an oral pathobiont associated with colorectal cancer, epigenetically reprograms human primary intestinal epithelial cells

Recently, an intestinal dysbiotic microbiota with enrichment in oral cavity bacteria has been described in colorectal cancer (CRC) patients. Here we characterized and investigated one of these oral pathobionts, the Gram-positive anaerobic coccus Parvimonas micra. We identified two phylotypes (A and B) exhibiting different phenotypes and adhesion capabilities. We observed a strong association of phylotype A with CRC, with its higher abundance in feces and in tumoral tissue compared with the normal homologous colonic mucosa, which was associated with a distinct methylation status of patients. By developing an in vitro hypoxic co-culture system of human primary colonic cells with anaerobic bacteria, we showed that P. micra phylotype A alters the DNA methylation profile promoters of key tumor-suppressor genes, oncogenes, and genes involved in epithelial-mesenchymal transition. In colonic mucosa of CRC patients carrying P. micra phylotype A, we found similar DNA methylations alterations, together with significant enrichment of differentially expressed genes in pathways involved in inflammation, cell adhesion, and regulation of actin cytoskeleton, providing evidence of P. micra possible role in the carcinogenic process.

microbiology↗

bioRxiv2021

A virus-encoded microRNA contributes to evade innate immune response during SARS-CoV-2 infection

SARS-CoV-2 infection results in impaired interferon response in severe COVID-19 patients. However, how SARS-CoV-2 interferes with host immune response is incompletely understood. Here, we sequenced small RNAs from SARS-CoV-2-infected human cells and identified a micro-RNA (miRNA) encoded in a recently evolved region of the viral genome. We show that the virus-encoded miRNA produces two miRNA isoforms in infected cells by the enzyme Dicer and they are loaded into Argonaute proteins. Moreover, the predominant miRNA isoform targets the 3UTR of interferon-stimulated genes and represses their expression in a miRNA-like fashion. Finally, the two viral miRNA isoforms were detected in nasopharyngeal swabs from COVID-19 patients. We propose that SARS-CoV-2 employs a virus-encoded miRNA to hijack the host miRNA machinery and evade the interferon-mediated immune response.

molecular biology↗

bioRxiv2019

Adaptive landscape flattening allows the design of both enzyme:substrate binding and catalytic power

Designed enzymes are of fundamental and technological interest. Experimental directed evolution still has significant limitations, and computational approaches are complementary. A designed enzyme should satisfy multiple criteria: stability, substrate binding, transition state binding. Such multi-objective design is computationally challenging. Two recent studies used adaptive importance sampling Monte Carlo to redesign proteins for ligand binding. By first flattening the energy landscape of the apo protein, they obtained positive design for the bound state and negative design for the unbound. We extend the method to the design of an enzyme for specific transition state binding, i.e., for catalytic power. We consider methionyl-tRNA synthetase (MetRS), which attaches methionine (Met) to its cognate tRNA, establishing codon identity. MetRS and other synthetases have been extensively redesigned by experimental directed evolution to accept noncanonical amino acids as substrates, leading to genetic code expansion. We redesigned MetRS computationally to bind several ligands: the Met analog azidonorleucine, methionyl-adenylate (MetAMP), and the activated ligands that form the transition state for MetAMP production. Enzyme mutants known to have azidonorleucine activity were recovered, and mutants predicted to bind MetAMP were characterized experimentally and found to be active. Mutants predicted to have low activation free energies for MetAMP production were found to be active and the predicted reaction rates agreed well with the experimental values. We expect the present method will become the paradigm for computational enzyme design.

biochemistry↗

bioRxiv2019

Innate immune recognition of a bacterial MAMP leads to conditional activation of pro- or anti-inflammatory responses

Microbe-associated molecular patterns (MAMPs) are recognized by pattern recognition receptors (PRRs) of the innate immune system. Flagellin, the primary component of bacterial flagella, is recognized by membrane TLR5 and cytoplasmic NLRC4 receptors, which promote a vigorous pro-inflammatory response typically associated with bacterial infection. However, herein, we report that the nature of the flagellin-induced response is highly dependent on the physiological state of the tissue. Specifically, in the steady state, epithelial cell detection of flagellin orchestrates an anti-inflammatory response mediated by IL-33-dependent type 2 regulatory T cells while, in the context of injury, it induces a pro-inflammatory response mediated by myeloid cells, IL-18 and Th17 cells. Likewise, in the absence of infection, bacterial symbionts expressing high levels of flagellin induce a type 2 response. These data demonstrate that, depending on the inflammatory state of the milieu, MAMPs can function both as immunogens or tolerogens.

immunology↗