Biology subjects

Brambilla, L.

Publications and source records attributed to Brambilla, L..

5 recordsLinked to original sources

bioRxiv2023

A modular model integrating metabolism, growth, and cell cycle predicts that fermentation is required to modulate cell size in yeast populations

For unicellular organisms, the reproduction rate and growth are crucial determinants of fitness and, therefore, essential functional manifestations of the organism genotype. Using the budding yeast Saccharomyces cerevisiae as a model organism, we integrated metabolism, which provides energy and building blocks for growth, with cell mass growth and cell cycle progression into a low-granularity, multiscale (from cell to population) computational model. This model predicted that cells with constitutive respiration do not modulate cell size according to the growth conditions. We experimentally validated the model predictions using mutants with defects in the upper part of glycolysis or glucose transport. Plugging in molecular details of cellular subsystems allowed us to refine predictions from the cellular to the molecular level. Our hybrid multiscale modeling approach provides a framework for structuring molecular knowledge and predicting cell phenotypes under various genetic and environmental conditions.

systems biology↗

bioRxiv2023

Structure guided mutagenesis of Henipavirus Receptor Binding Proteins reveals molecular determinants of receptor usage and antibody binding epitopes

Nipah virus (NiV) is a highly lethal, zoonotic henipavirus (HNV) that causes respiratory and neurological signs and symptoms in humans. Similar to other paramyxoviruses, HNVs mediate entry into host cells through the concerted actions of two surface glycoproteins: a receptor binding protein (RBP) that mediates attachment and a fusion glycoprotein (F) that triggers fusion in an RBP-dependent manner. NiV uses ephrin-B2 (EFNB2) and ephrin-B3 (EFNB3) as entry receptors. Ghana virus (GhV), a novel HNV identified in a Ghanaian bat, use EFNB2 but not EFNB3. In this study, we employ a structure-informed approach to identify receptor interfacing residues and systematically introduce GhV-RBP residues into a NiV-RBP backbone to uncover the molecular determinants of EFNB3 usage. We reveal two regions that severely impair EFNB3 binding by NiV-RBP and EFNB3-mediated entry by NiV pseudotyped viral particles. Further analyses uncovered two point mutations (NiVN557SGhV and NiVY581TGhV) pivotal for this phenotype. Moreover, we identify NiV interaction with Y120 of EFNB3 as important for usage of this receptor. Beyond these EFNB3-related findings, we reveal two domains that restrict GhV binding of EFNB2, identify the HNV-head as an immunodominant target for polyclonal and monoclonal antibodies, and describe putative epitopes for GhV and NiV-specific monoclonal antibodies. Cumulatively, the work presented here generates useful reagents and tools that shed insight to residues important for NiV usage of EFNB3, reveals regions critical for GhV binding of EFNB2, and describes putative HNV antibody binding epitopes.

molecular biology↗

bioRxiv2023

An in vitro experimental pipeline to characterize the binding specificity of SARS-CoV-2 neutralizing antibodies

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has led to over 760 million cases and >6.8 million deaths worldwide. We developed a panel of human neutralizing monoclonal antibodies (mAbs) targeting the SARS-CoV-2 Spike protein using Harbour H2L2 transgenic mice immunized with Spike receptor binding domain (RBD) (1). Representative antibodies from genetically-distinct families were evaluated for inhibition of replication-competent VSV expressing SARS-CoV-2 Spike (rcVSV-S) in place of VSV-G. One mAb (denoted FG-10A3) inhibited infection of all rcVSV-S variants; its therapeutically-modified version, STI-9167, inhibited infection of all tested SARS-CoV-2 variants, including Omicron BA.1 and BA.2, and limited virus proliferation in vivo (1). To characterize the binding specificity and epitope of FG-10A3, we generated mAb-resistant rcVSV-S virions and performed structural analysis of the antibody/antigen complex using cryo-EM. FG-10A3/STI-9167 is a Class 1 antibody that prevents Spike-ACE2 binding by engaging a region within the Spike receptor binding motif (RBM). Sequencing of mAb-resistant rcVSV-S virions identified F486 as a critical residue for mAb neutralization, with structural analysis revealing that both the variable heavy and light chains of STI-9167 bound the disulfide-stabilized 470-490 loop at the Spike RBD tip. Interestingly, substitutions at position 486 were later observed in emerging variants of concern BA.2.75.2 and XBB. This work provides a predictive modeling strategy to define the neutralizing capacity and limitations of mAb therapeutics against emerging SARS-CoV-2 variants. ImportanceThe COVID-19 pandemic remains a significant public health concern for the global population; development and characterization of therapeutics, especially ones that are broadly effective, will continue to be essential as SARS-CoV-2 variants emerge. Neutralizing monoclonal antibodies remain an effective therapeutic strategy to prevent virus infection and spread with the caveat that they interact with the circulating variants. The epitope and binding specificity of a broadly neutralizing anti-SARS-CoV-2 Spike RBD antibody clone against many SARS-CoV-2 VOC was characterized by generating antibody-resistant virions coupled with cryo-EM structural analysis. This workflow can serve to predict the efficacy of antibody therapeutics against emerging variants and inform the design of therapeutics and vaccines.

microbiology↗

bioRxiv2023

PGM3 inhibition Shows cooperative Effects With Erastin inducing Pancreatic cancer cell death via activation of the Unfolded Protein Response

1Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with a poor patient prognosis. Remarkably, PDAC is one of the most aggressive and deadly tumor types and is notorious for its resistance to all types of treatment. PDAC resistance is frequently associated with a wide metabolic rewiring and in particular of the glycolytic branch named Hexosamine Biosynthetic Pathway (HBP). Here we show the effect of the combined treatment between an HBPs Phosphoglucomutase 3 (PGM3) enzyme inhibitor, named FR054, and erastin (ERA), a recognized ferroptosis inducer, on PDAC cell growth and survival. Noteworthy, the combined treatment applied to PDAC cell lines induces a significant decrease in cell proliferation and a concurrent enhancement of cell death. Furthermore, we show that this combined treatment induces Unfolded Protein Response (UPR), NFE2 Like BZIP Transcription Factor 2 (NRF2) activation, a change in cellular redox state, a greater sensitivity to oxidative stress, a major dependence on glutamine metabolism, and finally ferroptosis cell death. Our study discloses that HBP inhibition enhances, through UPR activation, the ERA effect and therefore might be a novel anticancer mechanism to be exploited as PDAC therapy.

cancer biology↗

bioRxiv2021

Tonic interferon restricts pathogenic IL-17-driven inflammatory disease via balancing the microbiome

Maintenance of immune homeostasis involves a synergistic relationship between the host and the microbiome. Canonical interferon (IFN) signaling controls responses to acute microbial infection, through engagement of the STAT1 transcription factor. However, the contribution of tonic levels of IFN to immune homeostasis in absence of acute infection remains largely unexplored. We report that STAT1 KO mice spontaneously developed an inflammatory disease marked by myeloid hyperplasia and splenic accumulation of hematopoietic stem cells. Moreover, these animals developed inflammatory bowel disease. Profiling gut bacteria revealed a profound dysbiosis in absence of tonic IFN signaling, which triggered expansion of TH17 cells and loss of splenic Treg cells. Resolution of dysbiosis by antibiotic treatment averted the TH17 bias, and blocking IL17 signaling prevented myeloid expansion and splenic stem cell accumulation. Thus, tonic IFNs regulate gut microbial ecology, which is crucial for maintaining physiologic immune homeostasis and preventing inflammation.

immunology↗