Biology subjects

Cieplak, P.

Publications and source records attributed to Cieplak, P..

5 recordsLinked to original sources

bioRxiv2021

Salt leverages polyethylene terephthalate hydrolase (PETase) enzymatic activity via the predicted conformational switch

Plastic pollution spawned a global challenge caused by the environmental accumulation of polyethylene terephthalate (PET) plastics. Ongoing remediation efforts using microbial and engineered PET hydrolyzing enzymes (PETases) are hindered by slow depolymerization activities. Here, we report the optimized reaction conditions that leveraged the PETase hydrolase activity 2 to 3.8-fold in the presence of high NaCl concentrations. Molecular dynamics simulations (MDS) were applied to model salt-dependent conformational changes of the PETase enzyme bound to a 3-unit PET polymer. MDS demonstrated that PETase interaction with flanking polymer units exhibited a striking structural disparity at low and high salt concentrations. At low salt concentrations, flanking polymer units displayed significant bending. In contrast, flanking units extended at high salt concentrations, thus residues D206, H237, and S160 of the catalytic triad positioned in close vicinity of the scissile ester bond of the polymer substrate. The resulting high salt-specific enzyme/substrate geometry can potentially facilitate hydrolysis. We theorized that a salt-dependent conformational switch could attenuate the enzyme to a broad range of natural and artificial polymers consumed as carbon sources. Altogether, new knowledge may advance the engineering of PET hydrolase enzymes and benefit bioconversion programs.

molecular biology↗

bioRxiv2020

Toxoplasma gondii serine hydrolases regulate parasite lipid mobilization during growth and replication within the host

The intracellular protozoan parasite Toxoplasma gondii must scavenge cholesterol and other lipids from the host to facilitate intracellular growth and replication. Enzymes responsible for neutral lipid synthesis have been identified but there is no evidence for enzymes that catalyze lipolysis of cholesterol esters and esterified lipids. Here we characterize several T. gondii serine hydrolases with esterase and thioesterase activities that were previously thought to be depalmitoylating enzymes. We find they do not cleave palmitoyl thiol esters but rather hydrolyze short chain lipid esters. Deletion of one of the hydrolases results in alterations in levels of multiple lipids species. We also identify small molecule inhibitors of these hydrolases and show that treatment of parasites results in phenotypic defects reminiscent of parasites exposed to excess cholesterol or oleic acid. Together, these data characterize enzymes necessary for processing lipids critical for infection and highlight the potential for targeting parasite hydrolases for therapeutic applications. HighlightsO_LIBioinformatic and biochemical characterization of T. gondii serine hydrolases reveals substrate preference between enzymes with similar catalytic fold C_LIO_LIT. gondii serine hydrolases previously thought to be depalmitoylases are lipid metabolizing enzymes C_LIO_LIT. gondii lipid metabolism pathways utilize enzymes that are viable therapeutic targets C_LI

microbiology↗

bioRxiv2020

Epitope-resolved profiling of the SARS-CoV-2 antibody response identifies cross-reactivity with an endemic human CoV

A high-resolution understanding of the antibody response to SARS-CoV-2 is important for the design of effective diagnostics, vaccines and therapeutics. However, SARS-CoV-2 antibody epitopes remain largely uncharacterized, and it is unknown whether and how the response may cross-react with related viruses. Here, we use a multiplexed peptide assay ( PepSeq) to generate an epitope-resolved view of reactivity across all human coronaviruses. PepSeq accurately detects SARS-CoV-2 exposure and resolves epitopes across the Spike and Nucleocapsid proteins. Two of these represent recurrent reactivities to conserved, functionally-important sites in the Spike S2 subunit, regions that we show are also targeted for the endemic coronaviruses in pre-pandemic controls. At one of these sites, we demonstrate that the SARS-CoV-2 response strongly and recurrently cross-reacts with the endemic virus hCoV-OC43. Our analyses reveal new diagnostic and therapeutic targets, including a site at which SARS-CoV-2 may recruit common pre-existing antibodies and with the potential for broadly-neutralizing responses.

immunology↗

bioRxiv2020

Quantitative profiling of protease specificity.

Proteases comprise an important class of enzymes, whose activity is central to many physiologic and pathologic processes. Detailed knowledge of protease specificity is key to understanding their function. Although many methodologies have been developed to profile specificities of proteases, few have the diversity and quantitative grasp necessary to fully define specificity of a protease, both in terms of substrate numbers and their catalytic efficiencies. We have developed a concept of “selectome”, which defines the set of substrates that uniquely represents specificity of a protease. We applied it to two closely related members of the Matrixin family – MMP-2 and MMP-9 by using substrate phage display coupled with Next Generation Sequencing and information theory-based data analysis. We have also derived a quantitative measure of substrate specificity, which accounts for both the numbers and relative catalytic efficiencies of substrates. Using these advances greatly facilitates uncovering selectivity between closely related members of protease families and provides insight into to the degree of contribution of catalytic cleft specificity to protein substrate recognition, thus providing basis to overcoming two of the major challenges in the field of proteolysis: 1) development of highly selective activity probes and inhibitors for studying proteases with overlapping specificities, and 2) distinguishing targeted proteolysis from bystander proteolytic events.View Full Text

biochemistry↗

bioRxiv2019

The antimalarial natural product salinipostin A identifies essential α/β serine hydrolases involved in lipid metabolism in P. falciparum parasites

Salinipostin A (Sal A) is a potent antimalarial marine natural product with an undefined mechanism of action. Using a Sal A-derived activity-based probe, we identify its targets in the Plasmodium falciparum parasite. All of the identified proteins contain /{beta} serine hydrolase domains, and several are essential for parasite growth. One of the essential targets displays high homology to human monoacylglycerol lipase (MAGL) and is able to process lipid esters including a MAGL acylglyceride substrate. This Sal A target is inhibited by the anti-obesity drug Orlistat, which disrupts lipid metabolism and produces disorganized and stalled schizonts similar to Sal A. Resistance selections yielded parasites that showed only minor reductions in sensitivity and that acquired mutations in a protein linked to drug resistance in Toxoplasma gondii. This inability to evolve efficient resistance mechanisms combined with the non-essentiality of human homologs makes the serine hydrolases identified here promising antimalarial targets.

cell biology↗