Biology subjects

Diaz, R. E.

Publications and source records attributed to Diaz, R. E..

5 recordsLinked to original sources

bioRxiv2023

Structural characterization of ligand binding and pH-specific enzymatic activity of mouse Acidic Mammalian Chitinase

Chitin is an abundant biopolymer and pathogen-associated molecular pattern that stimulates a host innate immune response. Mammals express chitin-binding and chitin-degrading proteins to remove chitin from the body. One of these proteins, Acidic Mammalian Chitinase (AMCase), is an enzyme known for its ability to function under acidic conditions in the stomach but is also active in tissues with more neutral pHs, such as the lung. Here, we used a combination of biochemical, structural, and computational modeling approaches to examine how the mouse homolog (mAMCase) can act in both acidic and neutral environments. We measured kinetic properties of mAMCase activity across a broad pH range, quantifying its unusual dual activity optima at pH 2 and 7. We also solved high resolution crystal structures of mAMCase in complex with oligomeric GlcNAcn, the building block of chitin, where we identified extensive conformational ligand heterogeneity. Leveraging these data, we conducted molecular dynamics simulations that suggest how a key catalytic residue could be protonated via distinct mechanisms in each of the two environmental pH ranges. These results integrate structural, biochemical, and computational approaches to deliver a more complete understanding of the catalytic mechanism governing mAMCase activity at different pH. Engineering proteins with tunable pH optima may provide new opportunities to develop improved enzyme variants, including AMCase, for therapeutic purposes in chitin degradation.

biochemistry↗

bioRxiv2021

Type 2 innate immunity regulates hair follicle homeostasis to control Demodex pathosymbionts

Allergic skin diseases are common, but basal roles for type 2 immunity in cutaneous homeostasis are incompletely understood. Here, we show that skin group 2 innate lymphoid cells (ILC2s) are the predominant resident cells that secretes IL-13, which attenuates epithelial cell proliferation during anagen throughout the hair follicle (HF), including in HF stem cells. Although HF are normal in the absence of type 2 immunity, colonization with the commensal mite, Demodex musculi, results in epithelial proliferation and aberrant hair follicle morphology accompanied by loss of stable commensalism with massive infestation and expansion of inflammatory ILC2s and other immune cells. Topical anti-parasitic agents, but not broad-spectrum antibiotics, reversed the phenotype. Commensal Demodex colonization of mammalian hair follicles is ubiquitous, including in humans, revealing an unanticipated role for ILC2s and type 2 immunity for skin homeostasis in the normal environment. One Sentence SummaryDynamic regulation of skin physiology by Type 2 innate immunity regulates healthy commensalism by parasitic Demodex mites.

immunology↗

bioRxiv2020

Fragment binding to the Nsp3 macrodomain of SARS-CoV-2 identified through crystallographic screening and computational docking

The SARS-CoV-2 macrodomain (Mac1) within the non-structural protein 3 (Nsp3) counteracts host-mediated antiviral ADP-ribosylation signalling. This enzyme is a promising antiviral target because catalytic mutations render viruses non-pathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of diverse fragment libraries resulted in 214 unique macrodomain-binding fragments, out of 2,683 screened. An additional 60 molecules were selected from docking over 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several crystallographic and docking fragment hits were validated for solution binding using three biophysical techniques (DSF, HTRF, ITC). Overall, the 234 fragment structures presented explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.

biochemistry↗

bioRxiv2019

Dissecting the chitinolytic activity of mammalian chitinases

Chitin is an abundant polysaccharide used by a large range of organisms for structural rigidity and water repulsion. As such, the insoluble crystalline structure of chitin poses significant challenges for enzymatic degradation. Vertebrates do not produce chitin, but do express chitin degrading enzymes. Acidic mammalian chitinase, the primary enzyme involved in the degradation of environmental chitin in mammalian lungs, is a processive glycosyl hydrolase that may be able to make multiple hydrolysis events for each binding event. Mutations to acidic mammalian chitinase have been associated with asthma, and genetic deletion of the enzyme in mice results in significantly increased morbidity and mortality with age. We initially set out to reverse this phenotype by engineering hyperactive acidic mammalian chitinase variants. Using a directed evolution screening approach using commercial fluorogenic substrates, we identified mutations with consistent increases in activity. To determine whether the activity increases observed with oligomeric substrates were consistent with more biologically relevant chitin substrates, we developed new assays to quantify chitinase activity with colloidal crystalline chitin, and identified a high throughput fluorogenic assay that gives sufficient signal to noise advantages to quantify changes to activity due to the addition or removal of a chitin binding domain to the enzyme. We show that the activity increasing mutations derived from our directed evolution screen were lost when crystalline substrates were used. In contrast, naturally occurring gain-of-function mutations gave similar results with oligomeric and crystalline substrates. We also show that the activity differences between acidic mammalian chitinase and chitotriosidase are reduced in the context of crystalline substrate, suggesting that previously reported activity differences with oligomeric substrates may have been largely driven by differential substrate specificity for the oligomers. These results highlight the need for assays against more physiological substrates when engineering complex metabolic enzymes, and provide a new approach that may be broadly applicable to engineering glycosyl hydrolases.

biochemistry↗

bioRxiv2018

Robust and conserved stochastic self-assembly mechanism for dynamic ParB-parS partition complexes on bacterial chromosomes and plasmids

Chromosome and plasmid segregation in bacteria are mostly driven by ParABS systems. These DNA partitioning machineries rely on large nucleoprotein complexes assembled on centromere sites (parS). However, the mechanism of how a few parS-bound ParB proteins nucleate the formation of highly concentrated ParB clusters remains unclear despite several proposed physico-mathematical models. We discriminated between these different models by varying some key parameters in vivo using the plasmid F partition system. We found that Nucleation & caging is the only coherent model recapitulating in vivo data. We also showed that the stochastic self-assembly of partition complexes (i) does not directly involve ParA, (ii) results in a dynamic structure of discrete size independent of ParB concentration, and (iii) is not perturbed by active transcription but is by protein complexes. We refined the Nucleation & Caging model and successfully applied it to the chromosomally-encoded Par system of Vibrio cholerae, indicating that this stochastic self-assembly mechanism is widely conserved from plasmids to chromosomes.

systems biology↗