Biology subjects

Pereira, C.

Publications and source records attributed to Pereira, C..

23 records · Page 2Linked to original sources

bioRxiv2020

A secreted NlpC/P60 endopeptidase from Photobacterium damselae subsp. piscicida cleaves the peptidoglycan of potentially competing bacteria

Peptidoglycan (PG) is a major component of the bacterial cell wall, forming a mesh-like structure enwrapping the bacteria that is essential for maintaining structural integrity and providing support for anchoring other components of the cell envelope. PG biogenesis is highly dynamic and requires multiple enzymes, including several hydrolases that cleave glycosidic or amide bonds in the PG. Here, it is described the structural and functional characterization of an NlpC/P60-containing peptidase from Photobacterium damselae subsp. piscicida (Phdp), a Gram-negative bacterium that causes high mortality of warm-water marine fish with great impact for the aquaculture industry. PnpA (Photobacterium NlpC-like Protein A) has a four-domain structure with a hydrophobic and narrow access to the catalytic center and specificity for the {gamma}-D-glutamyl-meso-diaminopimelic acid bond. However, PnpA does not cleave the PG of Phdp and neither PG of several Gram-negative and Gram-positive bacterial species. Interestingly, it is secreted by the Phdp type II secretion system and degrades the PG of Vibrio anguillarum and V. vulnificus. This suggests that PnpA is used by Phdp to gain an advantage over bacteria that compete for the same resources or to obtain nutrients in nutrient-scarce environments. Comparison of the muropeptide composition of PG susceptible and resistant to the catalytic activity of PnpA, showed that the global content of muropeptides is similar, suggesting that susceptibility to PnpA is determined by the three-dimensional organization of the muropeptides in the PG. IMPORTANCEPeptidoglycan (PG) is a major component of the bacterial cell wall formed by long chains of two alternating sugars interconnected by short peptides, originating a mesh-like structure that enwraps the bacterial cell. Although PG provides structural integrity and support for anchoring other components of the cell envelope, it is constantly being remodeled through the action of specific enzymes that cleave or joint its components. Here, it is shown that Photobacterium damselae subsp. piscicida, a bacterium that causes high mortality in warm-water marine fish, produces PnpA, an enzyme that is secreted into the environment and is able to cleave the PG of potentially competing bacteria, either for gaining competitive advantages and/or to get nutrients. The specificity of PnpA to the PG of some bacteria and its inability to cleave others may be explained by differences in the structure of the PG mesh and not by different muropeptide composition.

microbiology↗

bioRxiv2020

Reprogramming of Human Cells to Pluripotency Induces CENP-A Chromatin Depletion

Pluripotent stem cells (PSCs) are central to development as they are the precursors of all cell types in the embryo. Therefore, maintaining a stable karyotype is essential, both for their physiological role as well as for use in regenerative medicine. In culture, an estimated 10-30% of PSC lines present karyotypic abnormalities, but the underlying causes remain unknown. To gain insight into the mitotic capacity of human embryonic stem cells and induced pluripotent stem cells, we explore the structure of the centromere and kinetochore. Centromere function depends on CENP-A nucleosome-defined chromatin. We show that while PSCs maintain abundant pools of CENP-A, CENP-C and CENP-T, these essential centromere components are strongly reduced at stem cell centromeres. Outer kinetochore recruitment is also impaired to a lesser extent, indicating an overall weaker kinetochore. This impairment is specific for the kinetochore forming centromere complex while the inner centromere protein Aurora B remains unaffected. We further show that, similar to differentiated human cells, CENP-A chromatin assembly in PSCs requires transition into G1 phase. Finally, reprogramming experiments indicate that reduction of centromeric CENP-A levels is an early event during dedifferentiation, coinciding with global chromatin remodelling. Our characterisation of centromeres in human stem cells drives new hypotheses including a possible link between impaired centromere function and stem cell aneuploidies.

cell biology↗

bioRxiv2018

CD44v6 expression is a novel predictive marker of therapy response and poor prognosis in gastric cancer patients

Late diagnosis, modest treatment options and lack of predictive markers of therapy response dictate the poor overall survival (OS) of [~]1 year in most gastric cancer (GC) patients. We hypothesized that the level of CD44v6 expression in tumor cells could predict therapy response and prognosis in GC patients.\n\nWe analyzed a surgical tumor series of GC patients for the extension of CD44v6 membranous immuno-expression, clinical-pathological features, patient survival, and response to therapy. By integrating this information, we assessed the value of CD44v6 expression to predict benefit from current treatment regimens and prognosis in GC patients. We used GC cell lines and mouse xenografts to assess and/validate the biological impact of CD44v6 expression in GC cells behavior.\n\nWe demonstrated that GC patients whose tumors present higher levels of CD44v6 membranous expression benefit from adding chemotherapy to surgery as opposed to those without CD44v6 expression. Moreover, patients bearing CD44_high tumors presented worse OS than those bearing CD44_absent/low tumors, consolidating the role of CD44v6 expression as an independent factor of poor prognosis in this disease. Finally, our in vitro and patients data pinpoints the CD44v6+ cell population as the driver of tumor recurrence following conventional chemotherapy, in heterogeneous tumors composed by CD44v6- and CD44v6+ cells.\n\nOur study pioneers the identification of CD44v6 as a potential predictive marker of response to conventional chemotherapy, and consolidates CD44v6 as an independent marker of poor prognosis in GC. Overall, our data strongly supports selection of patients with high CD44v6 expressing tumors for conventional chemotherapy with or without surgery, regardless of the TNM stage.

cancer biology↗

bioRxiv2018

Self-assembly of the RZZ complex into filaments drives kinetochore expansion in the absence of microtubule attachment

The kinetochore is a dynamic multi-protein assembly that forms on each sister chromatid and interacts with microtubules of the mitotic spindle to drive chromosome segregation. In animals, kinetochores without attached microtubules expand their outermost layer into crescent and ring shapes to promote microtubule capture and spindle assembly checkpoint (SAC) signalling. Kinetochore expansion is an example of protein co-polymerization, but the mechanism is not understood. Here, we present evidence that kinetochore expansion is driven by oligomerization of the Rod-Zw10-Zwilch (RZZ) complex, an outer kinetochore component that recruits the motor dynein and the SAC proteins Mad1-Mad2. Depletion of ROD in human cells suppresses kinetochore expansion, as does depletion of Spindly, the adaptor that connects RZZ to dynein, while dynein itself is dispensable. Expansion is also suppressed by mutating ZWILCH residues implicated in Spindly binding. Conversely, supplying cells with excess ROD facilitates kinetochore expansion under otherwise prohibitive conditions. Using the C. elegans early embryo, we demonstrate that ROD-1 has a concentration-dependent propensity for oligomerizing into {micro}m-scale filaments, and we identify the ROD-1 {beta}-propeller as a key regulator of self-assembly. Finally, we show that a minimal ROD-1-Zw10 complex efficiently oligomerizes into filaments in vitro. Our results suggest that RZZs capacity for oligomerization is harnessed by kinetochores to assemble the expanded outermost domain, in which RZZ filaments serve as recruitment platforms for SAC components and microtubule-binding proteins. Thus, we propose that RZZ self-assembly into filaments underlies the adaptive change in kinetochore size that contributes to chromosome segregation fidelity.

cell biology↗

bioRxiv2017

SQANTI: extensive characterization of long read transcript sequences for quality control in full-length transcriptome identification and quantification

High-throughput sequencing of full-length transcripts using long reads has paved the way for the discovery of thousands of novel transcripts, even in very well annotated organisms as mice and humans. Nonetheless, there is a need for studies and tools that characterize these novel isoforms. Here we present SQANTI, an automated pipeline for the classification of long-read transcripts that computes 47 descriptors that can be used to assess the quality of the data and of the preprocessing pipelines. We applied SQANTI to a neuronal mouse transcriptome using PacBio long reads and illustrate how the tool is effective in readily describing the composition of and characterizing the full-length transcriptome. We perform extensive evaluation of ToFU PacBio transcripts by PCR to reveal that an important number of the novel transcripts are technical artifacts of the sequencing approach, and that SQANTI quality descriptors can be used to engineer a filtering strategy to remove them. Most novel transcripts in this curated transcriptome are novel combinations of existing splice sites, result more frequently in novel ORFs than novel UTRs and are enriched in both general metabolic and neural specific functions. We show that these new transcripts have a major impact in the correct quantification of transcript levels by state-of-the-art short-read based quantification algorithms. By comparing our iso-transcriptome with public proteomics databases we find that alternative isoforms are elusive to proteogenomics detection and are variable in protein changes with respect to the principal isoform of their genes. SQANTI allows the user to maximize the analytical outcome of long read technologies by providing the tools to deliver quality-evaluated and curated full-length transcriptomes. SQANTI is available at https://bitbucket.org/ConesaLab/sqanti.

bioinformatics↗