Biology subjects

Garcia, B.

Publications and source records attributed to Garcia, B..

46 records · Page 3Linked to original sources

bioRxiv2021

A rare human centenarian variant of SIRT6 enhances genome stability and interaction with Lamin A

Sirtuin 6 (SIRT6) is a deacylase and mono-ADP ribosyl transferase (mADPr) enzyme involved in multiple cellular pathways implicated in the regulation of aging and metabolism. Targeted sequencing identified a SIRT6 allele containing two linked substitutions (N308K/A313S) as enriched in Ashkenazi Jewish (AJ) centenarians as compared to AJ control individuals. Characterization of this SIRT6 (centSIRT6) allele demonstrated it to be a stronger suppressor of LINE1 retrotransposons, confer enhanced stimulation of DNA double strand break repair, and more robust cancer cell killing compared to the wild type. Surprisingly, centSIRT6 displayed weaker deacetylase activity, but stronger mADPr activity, over a range of NAD+ concentrations and substrates. Additionally, centSIRT6 displayed a stronger interaction with Lamin A/C (LMNA), which correlated with enhanced ribosylation of LMNA. Our results suggest that enhanced SIRT6 function contributes to human longevity by improving genome maintenance via increased mADPr activity and enhanced interaction with LMNA.

molecular biology↗

bioRxiv2021

Poly (acetyl arginyl) glucosamine attenuates Pseudomonas aeruginosa in a rat lung infection model

Pseudomonas aeruginosa is a common opportunistic pathogen that can cause chronic infections in multiple disease states, including respiratory infections in patients with cystic fibrosis (CF) and non-CF bronchiectasis. Like many opportunists, P. aeruginosa forms multicellular biofilm communities that are widely thought to be an important determinant of bacterial persistence and resistance to antimicrobials and host immune effectors during chronic/recurrent infections. Poly (acetyl, arginyl) glucosamine (PAAG) is a glycopolymer which has antimicrobial activity against a broad range of bacterial species, and also has mucolytic activity which can normalize rheologic properties of cystic fibrosis mucus. In this study, we sought to evaluate the effect of PAAG on P. aeruginosa bacteria within biofilms in vitro, and in the context of experimental pulmonary infection in a rodent infection model. PAAG treatment caused significant bactericidal activity against P. aeruginosa biofilms, and a reduction in the total biomass of preformed P. aeruginosa biofilms on abiotic surfaces, as well as on the surface of immortalized cystic fibrosis human bronchial epithelial cells. Studies of membrane integrity indicated that PAAG causes changes to P. aeruginosa cell morphology and dysregulates membrane polarity. PAAG treatment reduced infection and consequent tissue inflammation in experimental P. aeruginosa rat infections. Based on these findings we conclude that PAAG represents a novel means to combat P. aeruginosa infection, which may warrant further evaluation as a therapeutic.

microbiology↗

bioRxiv2021

A dynamic and combinatorial histone code drives malaria parasite asexual and sexual development

A histone code defines system-level crosstalk between histone post-translational modifications (PTMs) to induce specific biological outcomes. Proteome-scale information of co-existing PTM across the entire chromatin landscape of the malaria parasite, Plasmodium falciparum, was lacking. Here, we used advanced quantitative middle-down proteomics to identify combinations of PTMs in both the proliferative, asexual stages and transmissible, sexual gametocyte stages of P. falciparum. We provide an updated, high-resolution compendium of 72 PTMs on H3 and H3.3, of which 30 are novel to the parasite. Co-existing PTMs with unique stage distinction was identified, indicating a dynamic and complex histone code with increased connectivity of novel PTMs seen in gametocytes. Chromatin proteomics of a gametocyte-specific combination, H3R17me2K18acK23ac, identified a SAGA-like effector complex (including the transcription factor AP2-G2) tied to this combination to regulate gene expression in mature gametocytes. Ultimately, this study unveils previously undiscovered histone PTMs and their functional relationship with co-existing partners. These results highlight that investigating chromatin regulation in the parasite using single histone PTM assays might overlook higher order gene regulation for distinct proliferation and differentiation processes.

biochemistry↗

bioRxiv2021

Structural and mechanistic insight into CRISPR-Cas9 inhibition by anti-CRISPR protein AcrIIC4

Phages, plasmids, and other mobile genetic elements express inhibitors of CRISPR-Cas immune systems, known as anti-CRISPR proteins, to protect themselves from targeted destruction. These anti-CRISPRs have been shown to function through very diverse mechanisms. In this work we investigate the activity of an anti-CRISPR isolated from a prophage in Haemophilus parainfluenzae that blocks CRISPR-Cas9 DNA cleavage activity. We determine the three-dimensional crystal struture of AcrIIC4 and show that it binds to the Cas9 Recognition Domain. This binding does not prevent the Cas9-anti-CRISPR complex from interacting with target DNA but does inhibit DNA cleavage. AcrIIC4 likely acts by blocking the conformational changes that allow the HNH and RuvC endonuclease domains to contact the DNA sites to be nicked.

biochemistry↗

bioRxiv2021

Maf/ham1-like pyrophosphatases, host-specific partners of viral RNA-dependent RNA polymerases

Cassava brown streak disease (CBSD), dubbed the "Ebola of plants", is a serious threat to food security in Africa caused by two viruses of the family Potyviridae: cassava brown streak virus (CBSV) and Ugandan (U)CBSV. Intriguingly, U/CBSV, along with another member of this family and one secoviridae, are the only known RNA viruses encoding a protein of the Maf/ham1-like family, a group of widespread pyrophosphatase of non-canonical nucleotides (ITPase) expressed by all living organisms. Despite the socio-economic impact of CDSD, the relevance and role of this atypical viral factor has not been yet established. Here, using an infectious cDNA clone and reverse genetics, we demonstrate that UCBSV requires the ITPase activity for infectivity in cassava, but not in the model plant Nicotiana benthamiana. HPLC MS/MS experiments showed that, quite likely, this host-specific constraint is due to an unexpected high concentration of non-canonical nucleotides in cassava. Finally, protein analyses and experimental evolution of mutant viruses indicated that keeping a fraction of the yielded UCBSV ITPase covalently bound to the viral RNA-dependent RNA polymerase (RdRP) optimizes viral fitness, and this seems to be a feature shared by the other members of the Potyviridae family expressing Maf/ham1-like proteins. All in all, our work (i) reveals that the over-accumulation of non-canonical nucleotides in the host might have a key role in antiviral defense, and (ii) provides the first example of an RdRP-ITPase partnership, reinforcing the idea that RNA viruses are incredibly versatile at adaptation to different host setups.

molecular biology↗

bioRxiv2019

Listeria phages induce Cas9 degradation to protect lysogenic genomes

Bacterial CRISPR-Cas systems employ RNA-guided nucleases to destroy foreign DNA. Bacteriophages, in turn, have evolved diverse \"anti-CRISPR\" proteins (Acrs) to counteract acquired immunity. In Listeria monocytogenes, prophages encode 2-3 distinct anti-Cas9 proteins, with acrIIA1 always present; however, its mechanism is unknown. Here, we report that AcrIIA1 binds with high affinity to Cas9 via the catalytic HNH domain and, in Listeria, triggers Cas9 degradation. AcrIIA1 displays broad-spectrum inhibition of Type II-A and II-C Cas9s, including an additional highly-diverged Listeria Cas9. During lytic infection, AcrIIA1 is insufficient for rapid Cas9 inactivation, thus phages require an additional \"partner\" Acr that rapidly blocks Cas9-DNA-binding. The AcrIIA1 N-terminal domain (AcrIIA1NTD) is dispensable for anti-CRISPR activity; instead it is required for optimal phage replication through direct transcriptional repression of the anti-CRISPR locus. AcrIIA1NTD is widespread amongst Firmicutes, can repress anti-CRISPR deployment by other phages, and has been co-opted by hosts potentially as an \"anti-anti-CRISPR.\" In summary, Listeria phages utilize narrow-spectrum inhibitors of DNA binding to rapidly inactivate Cas9 in lytic growth and the broad-spectrum AcrIIA1 to stimulate Cas9 degradation for protection of the Listeria genome in lysogeny.

microbiology↗

bioRxiv2019

Histone methyltransferase MMSET/NSD2 is essential for generation of B1 cell compartment in mice

Humoral immunity in mice and man relies on the function of two developmentally and functionally distinct B cell subsets - B1 and B2 cells. While B2 cells are responsible for most of the adaptive response to environmental antigens, B1 cells, which are comprised of phenotypically distinct B1a and B1b cells, are carriers of the innate humoral immunity that relies on production of poly-reactive and low affinity antibodies. The molecular mechanism of B cell specification into different subsets is not well established. Here we identified lysine methyltransferase MMSET/NSD2 as a critical regulator of the B1 cell population. We show that NSD2 deficiency in B cell precursors prevents generation of the B1 cell compartment, while having a minor impact on B2 cells. Our data revealed MMSET/NSD2, which catalyzes histone H3 lysine 36 di-methylation, as the first in class epigenetic master regulator of a major B cell lineage in mice.

immunology↗

bioRxiv2019

Myeloid Lineage Enhancers Drive Oncogene Synergy in CEBPA/CSF3R Mutant Acute Myeloid Leukemia

Acute Myeloid Leukemia (AML) develops due to the acquisition of mutations from multiple functional classes. Here, we demonstrate that activating mutations in the granulocyte colony stimulating factor receptor (CSF3R), cooperate with loss of function mutations in the transcription factor CEBPA to promote acute leukemia development. This finding of mutation-synergy is broadly applicable other mutations that activate the JAK/STAT pathway or disrupt CEBPA function (i.e. activating mutations in JAK3 and Core Binding Factor translocations). The interaction between these distinct classes of mutations occurs at the level of myeloid lineage enhancers where mutant CEBPA prevents activation of subset of differentiation associated enhancers. To confirm this enhancer-dependent mechanism, we demonstrate that CEBPA mutations must occur as the initial event in AML initiation, confirming predictions from clinical sequencing data. This improved mechanistic understanding will facilitate therapeutic development targeting the intersection of oncogene cooperativity.

cancer biology↗

bioRxiv2018

Potent Cas9 inhibition in bacterial and human cells by new anti-CRISPR protein families

CRISPR-Cas systems are widely used for genome engineering technologies, and in their natural setting, they play crucial roles in bacterial and archaeal adaptive immunity, protecting against phages and other mobile genetic elements. Previously we discovered bacteriophage-encoded Cas9-specific anti-CRISPR (Acr) proteins that serve as countermeasures against host bacterial immunity by inactivating their CRISPR-Cas systems1. We hypothesized that the evolutionary advantages conferred by anti-CRISPRs would drive the widespread occurrence of these proteins in nature2-4. We have identified new anti-CRISPRs using the bioinformatic approach that successfully identified previous Acr proteins1 against Neisseria meningitidis Cas9 (NmeCas9). In this work we report two novel anti-CRISPR families in strains of Haemophilus parainfluenzae and Simonsiella muelleri, both of which harbor type II-C CRISPR-Cas systems5. We characterize the type II-C Cas9 orthologs from H. parainfluenzae and S. muelleri, show that the newly identified Acrs are able to inhibit these systems, and define important features of their inhibitory mechanisms. The S. muelleri Acr is the most potent NmeCas9 inhibitor identified to date. Although inhibition of NmeCas9 by anti-CRISPRs from H. parainfluenzae and S. muelleri reveals cross-species inhibitory activity, more distantly related type II-C Cas9s are not inhibited by these proteins. The specificities of anti-CRISPRs and divergent Cas9s appear to reflect co-evolution of their strategies to combat or evade each other. Finally, we validate these new anti-CRISPR proteins as potent off-switches for Cas9 genome engineering applications.

molecular biology↗

bioRxiv2018

The Virtual Metabolic Human database: integrating human and gut microbiome metabolism with nutrition and disease

A multitude of factors contribute to complex diseases and can be measured with \"omics\" methods. Databases facilitate data interpretation for underlying mechanisms. Here, we describe the Virtual Metabolic Human (VMH, http://vmh.life) database encapsulating current knowledge of human metabolism within five interlinked resources \"Human metabolism\", \"Gut microbiome\", \"Disease\", \"Nutrition\", and \"ReconMaps\". The VMH captures 5,180 unique metabolites, 17,730 unique reactions, 3,288 human genes, 255 Mendelian diseases, 818 microbes, 632,685 microbial genes, and 8,790 food items. The VMHs unique features are i) the hosting the metabolic reconstructions of human and gut microbes amenable for metabolic modeling; ii) seven human metabolic maps for data visualization; iii) a nutrition designer; iv) a user-friendly webpage and application-programming interface to access its content; and v) user feedback option for community engagement. We demonstrate with four examples the VMHs utility. The VMH represents a novel, interdisciplinary database for data interpretation and hypothesis generation to the biomedical community.

systems biology↗