Biology subjects

Pereira, P. J. B.

Publications and source records attributed to Pereira, P. J. B..

4 recordsLinked to original sources

bioRxiv2026

Chemical Synthesis of Thrombostasin: An Anticoagulant Protein Produced by the Horn Fly Haematobia irritans

Thrombostasin is a cysteine-free anticoagulant protein produced by the horn fly Haematobia irritans, featuring an unusual stretch of seven consecutive aspartate residues within the polypeptide sequence. Here, we report the first chemical synthesis of native thrombostasin and a hepta-glutamate analogue, together with assessment of their thrombin inhibitory and anticoagulant activity.

molecular biology↗

bioRxiv2023

Structural and functional characterization of the NF-κB-targeting toxin AIP56 from Photobacterium damselae subsp. piscicida reveals a novel mechanism for membrane interaction and translocation

Bacterial AB toxins are secreted virulence factors that are internalized by target cells through receptor-mediated endocytosis, translocating their enzymatic domain to the cytosol from endosomes (short-trip) or the endoplasmic reticulum (long-trip). To accomplish this, they evolved a multidomain structure organized into either a single polypeptide chain or non-covalently associated polypeptide chains. The prototypical short-trip single-chain toxin is characterized by a receptor-binding domain that confers cellular specificity and a translocation domain responsible for pore formation whereby the catalytic domain translocates to the cytosol in an endosomal acidification-dependent way. In this work, the resolution of the three-dimensional structure of AIP56 showed that instead of a two-domain organization, as previously predicted, AIP56 has a three-domain organization, with a NleC-like catalytic domain associated with a small middle domain that contains the linker-peptide, followed by the receptor-binding domain. In contrast to prototypical single-chain AB toxins, AIP56 does not comprise a typical structurally complex translocation domain; instead, the elements involved in translocation are scattered across its domains. Thus, the catalytic domain contains a helical hairpin that serves as a molecular switch for triggering the conformational changes necessary for membrane insertion only with endosomal acidification, whereas the middle and receptor-binding domains are required for pore formation. Considering the increasing number of putative AIP56 homologous toxins in databases, the data presented here have implications far beyond the AIP56 intoxication process and the virulence of Phdp.

microbiology↗

bioRxiv2022

Pathogen-specific structural features of two key players in Candida albicans morphogenetic switch

Ras-like protein 1 (CaRas1) is a key regulator of the switch between the yeast and hyphal forms of Candida albicans, a feature associated with pathogenesis. CaRas1 is activated by the guanine nucleotide exchange factor (GEF) CaCdc25, triggering hyphal growth-related signaling pathways through its highly conserved GTP-binding domain (G-domain). An important function in hyphal growth has also been proposed for the long hypervariable region downstream of the G-domain of CaRas1, whose unusual content of polyQ stretches and Q/N repeats make CaRas1 unique within Ras-family proteins. Despite its biological importance, both the structure of CaRas1 and the molecular basis of its activation by CaCdc25 remain unexplored. Here, we show that CaRas1 displays an elongated shape and that its hypervariable region contains helical structural elements with intramolecular coiled-coil propensity and limited conformational flexibility. Functional assays revealed that CaRas1 activation by CaCdc25 is highly efficient, with 5-to 2000-fold higher activity levels than reported for human GEFs. In addition, the threedimensional structure of the catalytic region of CaCdc25, together with the structural characterization of CaRas1/CaCdc25 complexes, unveiled a specific region located in the -helical hairpin of CaCdc25, critical for CaRas1 activation, where negatively charged substitutions reduce its activity. The unique structural features of the low complexity region of CaRas1 and the distinctive properties of CaRas1 activation by CaCdc25, common in the homologous proteins from CTG-clade species, uncover novel strategies to target key virulence factors in human-infecting fungal pathogens.

biochemistry↗

bioRxiv2019

A simple linearization method unveils hidden enzymatic assay interferences

Enzymes are among the most important drug targets in the pharmaceutical industry. The bioassays used to screen enzyme modulators can be affected by unaccounted interferences such as time-dependent inactivation and inhibition effects. Using procaspase-3, caspase-3, and -thrombin as model enzymes, we show that some of these effects are not eliminated by merely ignoring the reaction phases that follow initial-rate measurements. We thus propose a linearization method (LM) for detecting spurious changes of enzymatic activity based on the representation of progress curves in modified coordinates. This method is highly sensitive to signal readout distortions, thereby allowing rigorous selection of valid kinetic data. The method allows the detection of assay interferences even when their occurrence is not suspected a priori. By knowing the assets and liabilities of the bioassay, enzymology results can be reported with enhanced reproducibility and accuracy. Critical analysis of full progress curves is expected to help discriminating experimental artifacts from true mechanisms of enzymatic inhibition.

biophysics↗