Biology subjects

Schenkman, S.

Publications and source records attributed to Schenkman, S..

3 recordsLinked to original sources

bioRxiv2023

Trypanosoma cruzi VDU deubiquitinase mediates surface protein trafficking and infectivity

Ubiquitylation is a post-translational modification promoting protein degradation. Within the endomembrane system ubiquitylation marks proteins for lysosomal processing. Deubiquitinases (DUBs) cleave ubiquitin from modified proteins and, in the case of surface proteins, prevent lysosomal targeting and thus play a major role in controlling turnover. In Trypanosoma cruzi, the etiological agent of Chagas disease, acquisition of nutrients in parasites proliferating within the blood meal of insect vectors occurs via the cytostome, a unique structure connected to a long tubular cytopharynx. Contents are delivered to late endosomes and accumulate in reservosomes, equivalent to lysosomes. When starved, T. cruzi differentiates into mammalian infective trypomastigotes, which are cell cycle arrested. Here we asked what roles ubiquitylation plays in this unique endocytic process by interrogation of the T. cruzi ortholog of VDU (von Hippel-Lindau-interacting deubiquitylating enzyme)/USP33 (ubiquitin-specific protease). We found that TcVDU expression level inversely correlated with transferrin endocytosis, and that overexpression led to a longer retention of the endocytic cargo near the cytostome. TcVDU itself was found enriched in the anterior region of the parasite, in proximity to endocytic cargo. Most importantly, TcVDU overexpression reduced parasite invasion capacity and led to increased release of trans-sialidase. These alterations in the abundance of multiple surface proteins in TcVDU mutants indicate a key role of TcVDU in modulating the T. cruzi surface by affecting the endosomal traffic and consequently the host-parasite interface. Author summaryTrypanosoma cruzi is the cause of Chagas disease that affects large populations of Central and South America. Disease is spreading to other continents due to poor control of blood transfusion from donors with chronic and undiagnosed infection, migration and drug treatment with limited performance and undesired side effects. Proliferating forms of T. cruzi acquire nutrients through the cytostome, a cell surface opening connected to a long tubular cytopharynx. Internalized material is endocytosed in the cytopharynx. This is distinct from other members of the Trypanosomatids, which endocytose material exclusively through the flagellar pocket. By using CRISPR gene editing and overexpression we demonstrated that a conserved deubiquitinase (VDU) is a key control element for traffic from the cytopharynx to endosomal compartments in T. cruzi. Changed levels of VDU modify endosomal traffic and largely impact the parasite surface, causing changes in infectivity.

microbiology↗

bioRxiv2023

Development and characterization of a multimeric recombinant protein based on the spike protein receptor binding domain of SARS-CoV-2 that can neutralize virus infection

BackgroundThe SARS-CoV-2 virus, responsible for the COVID-19 pandemic, has four structural proteins and sixteen non-structural proteins. The S-protein is one of the structural proteins exposed on the surface of the virus and is the main target for producing neutralizing antibodies and vaccines. The S-protein forms a trimer that can bind the angiotensin-converting enzyme 2 (ACE2) through its receptor binding domain (RBD) for cell entry. MethodsWe stably expressed in a constitutive manner in HEK293 cells a new recombinant protein containing a signal sequence of immunoglobulin to produce an extended C-terminal portion of the RBD followed by a region responsible for the trimerization inducer of the bacteriophage T4, and a sequence of 6 histidines. The protein was produced and released in the culture supernatant of cells and was purified by Ni-agarose column and exclusion chromatography. It was then characterized by SDS-polyacrylamide gel and used as antigen to generate protective antibodies to inhibit ACE2 receptor interaction and virus entry into Vero cells. ResultsThe purified protein displayed a molecular mass of 135 kDa and with a secondary structure like the monomeric RBD. Electrophoresis analysis in SDS-polyacrylamide gel with and without reducing agents, and in the presence of crosslinkers indicated that it forms a multimeric structure composed of trimers and hexamers. The purified protein was able to bind the ACE2 receptor and generated high antibody titers in mice (1:10000), capable of inhibiting the binding of biotin labeled ACE2 to the virus S1 subunit, and to neutralize the entry of the SARS-CoV-2 Wuhan strain into cells. ConclusionOur results characterize a new multimeric protein based on S1 subunit to combat COVID-19, as a possible immunogen or antigen for diagnosis.

microbiology↗

bioRxiv2021

Mitochondrial sirtuin TcSir2rp3 affects TcSODA activity and oxidative stress response in Trypanosoma cruzi

Trypanosoma cruzi, the etiological agent of Chagas disease, faces a variety of environmental scenarios during its life cycle in both invertebrate and vertebrate hosts, which include changes in the redox environment that requires a fine regulation of a complex antioxidant arsenal of enzymes. Reversible post-translational modifications, as lysine acetylation, are a fast and economical way for cells to react to environmental conditions. Acetylation neutralizes the lysine positive charge conferring novel properties to the modified proteins, from changes in enzymatic activity to subcellular localization. Recently, we found that the main antioxidant enzymes, including the mitochondrial superoxide dismutase A (TcSODA) are acetylated in T. cruzi, suggesting that protein acetylation could participate in the oxidative stress response in T. cruzi. Therefore, we investigated whether mitochondrial lysine deacetylase sirtuin 3 (TcSir2rp3) was involved in the activity control of TcSODA. We observed an increased resistance to hydrogen peroxide and menadione two oxidant compounds in parasites overexpressing TcSir2rp3. Increased resistance was also found for benznidazole and nifurtimox, the two drugs available for treatment of Chagas disease, known to induce reactive oxidative and nitrosactive species in the parasite. In parallel, TcSir2rp3 overexpressing parasites showed parasites showed a reduction in the ROS levels after treatment with benznidazole and nifurtimox, suggesting a role of TcSir2rp3 in the oxidative stress response. To better understand the way TcSir2rp3 could contributes to oxidative stress response, we analyzed the expression of a key antioxidant enzyme, TcSODA, in the TcSir2rp3 overexpressing parasites and did not detect any increase in protein levels of this enzyme. However, we found that parasites overexpressing TcSir2rp3 presented higher levels of superoxide dismutase activity, and also that TcSir2rp3 and TcSODA interacts in vivo. Knowing that TcSODA is acetylated at lysine residues K44 and K97, and that K97 is located at similar region in the protein structure as K68 in human manganese superoxide dismutase (MnSOD), responsible to regulates MnSOD activity, we generated mutated versions of TcSODA at K44 and K97 and found that replacing K97 by glutamine, which mimics an acetylated lysine, negatively affects the enzyme activity in vitro. By using molecular dynamics approaches we revealed that acetylation of K97 induces specific conformational changes in TcSODA with respect of hydrogen bonding pattern to neighbor residues, specifically D94 and E96, suggesting a key participation of this residue to modulate the affinity to O2- by changing the charge availability on the surface of the enzyme. Taken together, our results showed for the first time the involvement of lysine acetylation in the maintenance of homeostatic redox state in trypanosomatids, contributing to the understanding of mechanisms used by T. cruzi to progress during the infection and opening the opportunity to explore protein acetylation as potential drug target in this parasite.

microbiology↗