Biology subjects

Julio, A.

Publications and source records attributed to Julio, A..

3 recordsLinked to original sources

bioRxiv2024

An activation-based high throughput screen identifies caspase-10 inhibitors

Caspases are a family of highly homologous cysteine proteases that play critical roles in inflammation and apoptosis. Small molecule inhibitors are useful tools for studying caspase biology, complementary to genetic approaches. However, achieving inhibitor selectivity for individual members of this highly homologous enzyme family remains a major challenge in developing such tool compounds. Prior studies have revealed that one strategy to tackle this selectivity gap is to target the precursor or zymogen forms of individual caspases, which share reduced structural homology when compared to active proteases. To establish a screening assay that favors the discovery of zymogen-directed caspase-10 selective inhibitors, we engineered a low-background and high-activity tobacco etch virus (TEV)--activated caspase-10 protein. We then subjected this turn-on protease to a high-throughput screen of approximately 100,000 compounds, with an average Z value of 0.58 across all plates analyzed. Counter screening, including against TEV protease, delineated bona fide procaspase-10 inhibitors. Confirmatory studies identified a class of low-pH activatable caspase-10 inhibitors. In parallel, mode-of-action studies revealed that pifithrin-{micro} (PFT{micro}), a reported TP53 inhibitor, also functions as a promiscuous caspase inhibitor. Both inhibitor classes showed preferential zymogen inhibition. Given the generalized utility of activation assays, we expect our screening platform to have widespread applications in identifying state-specific protease inhibitors.

biochemistry↗

bioRxiv2023

Gene editing in the Chagas disease vector Rhodnius prolixus by Cas9-mediated ReMOT Control

Rhodnius prolixus is currently the model vector of choice for studying Chagas disease transmission, a debilitating disease caused by Trypanosoma cruzi parasites. Despite the broad use of double-stranded RNA interference for the knockdown of gene function in R. prolixus, transgenesis and gene editing protocols are still lacking. Here we tested Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) and direct parental injection of CRISPR (DIPA-CRISPR) for the maternal delivery of CRISPR/Cas9 elements to developing R. prolixus oocytes, and strategies for the identification of insertions/deletions (indels) in target loci of resulting gene-edited G0 nymphs. We demonstrate successful ReMOT Control-mediated gene editing of the eye color markers Rp-scarlet and Rp-white, and the cuticle color marker Rp-yellow, with highest effectiveness obtained using the ovary-targeting BtKV ligand. These results provide proof-of-concepts for generating somatic mutations in R. prolixus and potentially for generating germline edited lines in triatomines. Our studies also suggest that optimal strategies for recovery of mutations include performing multiple gRNA injections and the use of visible phenotypes such as those displayed in the Rp-scarlet, Rp-white and Rp-yellow loci for future Co-CRISPR experiments. These results will lay the foundation for gene editing protocols for triatomines and could lead to the development of novel control strategies for vectors of Chagas disease. Author SummaryRhodnius prolixus is an insect vector of the protozoan Trypanossoma cruzi, causative agent of debilitating Chagas disease. To fight the spread of the disease, it has been suggested that biological control of the insect should be attempted. Gene editing by the novel CRISPR methodology holds great promise in this sense, as it enables to target almost any gene in the genome for mutagenesis, thus allowing the control of insect physiology and reproduction. Here we have tested protocols for the delivery of CRISPR reagents as an attempt to enable genome editing of the vector. Our results show that maternal delivery of CRISPR by the ReMOT Control method is efficient for mutating eye and cuticle color genes in the resulting nymphs, generating edited animals with red eyes, white eyes or a yellow cuticle. This is the first report of gene editing in a vector of Chagas disease and should lay the basis to produce modified animals either unable to carry the T. cruzi parasite or to reproduce.

molecular biology↗

bioRxiv2020

Pigmentation loci as markers for genome editing in the Chagas disease vector Rhodnius prolixus

The kissing bug Rhodnius prolixus is a major vector for Chagas disease in the Americas, and also considered as the primary model for functional studies. Prospective transgenic approaches and genome editing strategies hold great promise for controlling insect populations as well as disease propagation. In this context, identifying visible genetic markers for transgenic methodologies is of paramount importance to advance the field. Here we have identified and analyzed the function of putative cuticle and eye color genes by investigating the effect of gene knockdown on fertility, viability, and the generation of visible phenotypes. Synthesis of the dark, yellow and tan pigments present in the cuticle of most insects depends on the function of key genes encoding enzymes in the tyrosine pathway. Knockdown of the R. prolixus yellow and aaNAT/pro orthologs produces striking alterations in cuticle color. Surprisingly, knockdown of ebony does not generate visible phenotypes. Since loss of ebony function results in a dark cuticle in several insect orders, we conclude that R. prolixus evolved alternative strategies for cuticle coloration, possibly including the loss of a pigmentation function for an entire branch of the tyrosine pathway. Knockdown of the scarlet and brown genes - encoding ABC transporters - alters cuticle and eye pigmentation, implying that the transport of pigment into proper organelles is an important process both for cuticle and eye coloration in this species. Therefore, this analysis identifies for the first time potential visible markers for transgenesis in a hemipteran vector for a debilitating human disease. Author SummaryThe hemipteran Rhodnius prolixus - also known as a kissing bug - is a main vector transmitting the parasite Trypanosoma cruzi, the causative agent of Chagas disease, a debilitating infection estimated to affect more than 6 million people in Central and South America. In order to limit disease spread, an important measure is insect vector control. However, kissing bugs - like other insects - develop resistance to insecticides. Alternative strategies based on transgenesis and the recently developed CRISPR- based genome edition hold great promise to control vector population or generate parasite-resistant insects. For these approaches to be feasible in R. prolixus, it is critical to identify visible phenotypic markers. Here we identify and describe several genes controlling cuticle and eye pigmentation that are well-suited putative landing sites for transformation strategies. Among these, loss-of-function mutations in the ABC transporter encoding scarlet and the tyrosine pathway enzyme encoding aaNAT/pro generate striking and easily visible phenotypes. Importantly, the knockdown of these genes does not affect insect viability and fertility under laboratory conditions. Our results suggest that R. prolixus has developed alternative strategies for cuticle coloration involving the loss of an entire branch of tanning loci, while the other branch producing cuticle patterns by generating non-pigmented areas has gained critical importance.

genetics↗