Biology subjects

Lucero, R. M.

Publications and source records attributed to Lucero, R. M..

4 recordsLinked to original sources

bioRxiv2025

Aedes aegypti midgut chymotrypsin has no significant role in blood meal protein digestion or fecundity

The Aedes aegypti mosquito is a vector of dengue, Zika, and chikungunya. The mosquitos reliance on blood facilitates the transmission of these viral pathogens to humans. Digestion of blood proteins depends on the biphasic expression of serine proteases, with trypsin-like activity contributing to most of the activity in the midgut. Other proteases found (serine collagenase- and chymotrypsin-like) are thought to contribute to digestion, but their roles are largely understudied. Thus, elucidating the activity and specific roles of all midgut proteases will help understand the complexity of the digestion process and help validate them as potential targets for the development of a new vector control strategy. Herein, we focused on characterizing the activity profile and role of Ae. aegypti chymotrypsin (AaCHYMO). Knockdown studies resulted in elimination and significant reduction of chymotrypsin-like activity in blood fed midgut extracts, while in vitro fluorescent and blood protein digestion assays revealed important substrate specificity differences. Interestingly, knockdown of AaCHYMO did not impact fecundity, indicating the presence of an intricate network of proteases working collectively to degrade blood proteins. Further, knockdown of the ecdysone receptor (EcR) led to a decrease in overall AaCHYMO expression and activity in the mosquito, which may play an important regulatory role.

biochemistry↗

bioRxiv2024

Comprehensive proteolytic profiling of Aedes aegypti mosquito midgut extracts: Unraveling the blood meal protein digestion system

To sustain the gonotrophic cycle, the Aedes aegypti mosquito must acquire a blood meal from a human or other vertebrate host. However, in the process of blood feeding, the mosquito may facilitate the transmission of several bloodborne viral pathogens (e.g., dengue, Zika, and chikungunya). The blood meal is essential as it contains proteins that are digested into polypeptides and amino acid nutrients that are eventually used for egg production. These proteins are digested by several midgut proteolytic enzymes. As such, the female mosquitos reliance on blood may serve as a potential target for vector and viral transmission control. However, this strategy may prove to be challenging since midgut proteolytic activity is a complex process dependent on several exo- and endo-proteases. Therefore, to understand the complexity of Ae. aegypti blood meal digestion, we used Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS) to generate global proteolytic profiles of sugar- and blood-fed midgut tissue extracts, along with substrate profiles of recombinantly expressed midgut proteases. Our results reveal a shift from high exoproteolytic activity in sugar-fed mosquitoes to an expressive increase in endoproteolytic activity in blood-fed mosquitoes. This approach allowed for the identification of 146 cleaved peptide bonds (by the combined 6 h and 24 h blood-fed samples) in the MSP-MS substrate library, and of these 146, 99 (68%) were cleaved by the five recombinant proteases evaluated. These reveal the individual contribution of each recombinant midgut protease to the overall blood meal digestion process of the Ae. aegypti mosquito. Further, our molecular docking simulations support the substrate specificity of each recombinant protease. Therefore, the present study provides key information of midgut proteases and the blood meal digestion process in mosquitoes, which may be exploited for the development of potential inhibitor targets for vector and viral transmission control strategies. Author SummaryThe Aedes aegypti mosquito is a vector of viral pathogens that can be transmitted directly to humans. For instance, the transmission of dengue, Zika, or chikungunya viruses may happen during the Ae. aegypti acquisition of an infected blood meal. This blood meal is important for the anautogenous mosquito because without the digestion of blood proteins the mosquito will not obtain the necessary nutrients needed for egg production. After imbibing a blood meal, midgut digestive enzymes (proteases) are expressed and secreted into the lumen. To fully understand their roles in blood meal digestion, we used a special technique called Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS). This method allows us to generate global proteolytic activity profiles of Ae. aegypti midgut tissue extracts that were fed with sugar or blood. In addition, we generated substrate cleavage profiles of recombinantly expressed midgut proteases allowing us to understand the enzyme preferences for blood proteins. Therefore, utilizing this approach, we found the contribution of each individual recombinant protease tested relative to the global activity profile of blood-fed midgut tissue extracts. This may be a starting point for the validation of midgut protease inhibition and the development of a new potential vector control strategy.

biochemistry↗

bioRxiv2024

Peripheral mutations underlie promiscuous transport of quaternary ammonium antiseptics by Small Multidrug Resistance transporters

The mechanistic basis of transport promiscuity in multidrug exporters is not well understood. We examine this question using the Small Multidrug Resistance (SMR) transporters. We engineer a selective SMR protein to promiscuously export quaternary ammonium antiseptics, similar to multidrug exporters in this family. Using combinatorial mutagenesis and deep sequencing, we identify the necessary and sufficient molecular determinants of this new activity. Using x-ray crystallography, electrophysiology, and a novel proteoliposome-based antiseptic transport assay, we tease apart the mechanistic roles that these residues play in transport polyspecificity. We find that substrate preference changes not through modification of the residues that directly interact with the substrate, but through mutations peripheral to the binding pocket. Our new molecular insights into substrate promiscuity among the SMRs can be applied to understand multidrug export and the evolution of novel transport functions more generally.

biochemistry↗

bioRxiv2023

Transport of metformin metabolites by guanidinium exporters of the Small Multidrug Resistance family

Proteins from the Small Multidrug Resistance (SMR) family are frequently associated with horizontally transferred multidrug resistance gene arrays found in bacteria from wastewater and the human-adjacent biosphere. Recent studies suggest that a subset of SMR transporters might participate in metabolism of the common pharmaceutical metformin by bacterial consortia. Here, we show that both genomic and plasmid-associated transporters of the SMRGdx functional subtype export byproducts of microbial metformin metabolism, with particularly high export efficiency for guanylurea. We use solid supported membrane electrophysiology to evaluate the transport kinetics for guanylurea and native substrate guanidinium by four representative SMRGdx homologues. Using an internal reference to normalize independent electrophysiology experiments, we show that transport rates are comparable for genomic and plasmid-associated SMRGdx homologues, and using a proteoliposome-based transport assay, we show that 2 proton:1 substrate transport stoichiometry is maintained. Additional characterization of guanidinium and guanylurea export properties focuses on the structurally characterized homologue, Gdx-Clo, for which we examined the pH dependence and thermodynamics of substrate binding and solved an x-ray crystal structure with guanylurea bound. Together, these experiments contribute in two main ways. By providing the first detailed kinetic examination of the structurally characterized SMRGdx homologue Gdx-Clo, they provide a functional framework that will inform future mechanistic studies of this model transport protein. Second, this study casts light on a potential role for SMRGdx transporters in microbial handling of metformin and its microbial metabolic byproducts, providing insight into how native transport physiologies are co-opted to contend with new selective pressures. SummaryUsing solid supported membrane electrophysiology, structural biology, and binding assays, we characterize binding and transport of metformin metabolites by bacterial SMR transporters, including proteins associated with horizontal gene transfer in wastewater bacteria that degrade metformin.

biophysics↗