Maturation and substrate processing topography of the Plasmodium falciparum invasion/egress protease plasmepsin X

During the intravascular stage of infection, the malaria parasite Plasmodium invades a host erythrocyte, multiplies within a parasitophorous vacuole (PV) and exits upon rupture of the PV and erythrocyte membranes in a process known as egress. Both egress and invasion are controlled by effector proteins discharged from specialized secretory organelles. The aspartic protease plasmepsin X (PM X) regulates activity for many of these effectors, but it is unclear how PM X accesses its diverse substrates that reside in different organelles. PM X also processes itself to generate different isoforms that remain present in terminal schizonts. The function of these different forms is not understood. We have mapped the autoprocessing cleavage sites and constructed parasites with cleavage site mutations. Surprisingly, all the cleavage mutant forms of PM X, including a quadruple mutant that remained full-length, retained in vitro activity, were trafficked normally in the parasites, and supported parasite growth and normal egress and invasion. Further analysis showed that the N-terminal half of the prodomain stays bound to the catalytic domain even after processing and is required for proper folding and intracellular trafficking of PM X. We find that this enzyme cleaves microneme and exoneme substrates before discharge, possibly in a common precursor organelle, while the rhoptry substrates that are dependent on PM X activity are cleaved after exoneme discharge into the PV. The data give insight into the temporal, spatial and biochemical control of this unusual but important aspartic protease.

The Association between Foot Surgery Type and Subsequent Healing in Veterans with Moderate-to-Severe Diabetic Foot Infections

Background Diabetic foot infections are a common precursor to lower extremity amputations. The treatment of diabetic foot infections involves both medical and surgical management, of which limb-sparing surgeries are increasingly preferred over amputations at or above the ankle to preserve mobility and quality of life. The outcomes following these limb-sparing surgeries are not well-described. Methods This was a single-center, retrospective cohort study of 90 Veterans with moderate-to-severe diabetic foot infections between 2017 and 2019 from the VA Maryland Health Care System. The exposure was foot surgery with bone resection (i.e., toe amputation, metatarsal resection, transmetatarsal amputation) versus debridement alone. The outcome was healing within 1 year. We used log-binomial regression to assess the association between foot surgery type and healing, stratify by infection location, and evaluate potential confounding variables. Results The cumulative incidence of healing after foot surgery with bone resection was greater than that following debridement (risk ratio 1.80, 95% confidence interval [1.17, 2.77]). This association was modified by infection location and greater for toe infections (4.52 [1.30, 15.7]) than other foot infections (1.19 [0.69, 2.02]). We found no evidence of confounding by comorbidities or infection severity. Conclusions For patients with toe infections, foot surgery with bone resection was associated with better healing than debridement alone. The multiple specialties caring for patients with diabetic foot infections need a stronger common knowledge base—from studies like this and future studies—to better counsel patients about their treatment and prognosis.

Engineering of Saccharomyces cerevisiae for 24-Methylene-Cholesterol Production

24-Methylene-cholesterol is a necessary substrate for the biosynthesis of physalin and withanolide, which show promising anticancer activities. It is difficult and costly to prepare 24-methylene-cholesterol via total chemical synthesis. In this study, we engineered the biosynthesis of 24-methylene-cholesterol in Saccharomyces cerevisiae by disrupting the two enzymes (i.e., ERG4 and ERG5) in the yeast’s native ergosterol pathway, with ERG5 being replaced with the DHCR7 (7-dehydrocholesterol reductase) enzyme. Three versions of DHCR7 originating from different organisms—including the DHCR7 from Physalis angulata (PhDHCR7) newly discovered in this study, as well as the previously reported OsDHCR7 from Oryza sativa and XlDHCR7 from Xenopus laevis—were assessed for their ability to produce 24-methylene-cholesterol. XlDHCR7 showed the best performance, producing 178 mg/L of 24-methylene-cholesterol via flask-shake cultivation. The yield could be increased up to 225 mg/L, when one additional copy of the XlDHCR7 expression cassette was integrated into the yeast genome. The 24-methylene-cholesterol-producing strain obtained in this study could serve as a platform for characterizing the downstream enzymes involved in the biosynthesis of physalin or withanolide, given that 24-methylene-cholesterol is a common precursor of these chemicals.

Biochemical and Structural Characteristics, Gene Regulation, Physiological, Pathological and Clinical Features of Lipocalin-Type Prostaglandin D2 Synthase as a Multifunctional Lipocalin

Lipocalin-type prostaglandin (PG) D2 synthase (L-PGDS) catalyzes the isomerization of PGH2, a common precursor of the two series of PGs, to produce PGD2. PGD2 stimulates three distinct types of G protein-coupled receptors: (1) D type of prostanoid (DP) receptors involved in the regulation of sleep, pain, food intake, and others; (2) chemoattractant receptor-homologous molecule expressed on T helper type 2 cells (CRTH2) receptors, in myelination of peripheral nervous system, adipocyte differentiation, inhibition of hair follicle neogenesis, and others; and (3) F type of prostanoid (FP) receptors, in dexamethasone-induced cardioprotection. L-PGDS is the same protein as β-trace, a major protein in human cerebrospinal fluid (CSF). L-PGDS exists in the central nervous system and male genital organs of various mammals, and human heart; and is secreted into the CSF, seminal plasma, and plasma, respectively. L-PGDS binds retinoic acids and retinal with high affinities (Kd < 100 nM) and diverse small lipophilic substances, such as thyroids, gangliosides, bilirubin and biliverdin, heme, NAD(P)H, and PGD2, acting as an extracellular carrier of these substances. L-PGDS also binds amyloid β peptides, prevents their fibril formation, and disaggregates amyloid β fibrils, acting as a major amyloid β chaperone in human CSF. Here, I summarize the recent progress of the research on PGD2 and L-PGDS, in terms of its “molecular properties,” “cell culture studies,” “animal experiments,” and “clinical studies,” all of which should help to understand the pathophysiological role of L-PGDS and inspire the future research of this multifunctional lipocalin.

Computer-aided engineering of a branching sucrase for the glucodiversification of a tetrasaccharide precursor of S. flexneri antigenic oligosaccharides

Enzyme engineering approaches have allowed to extend the collection of enzymatic tools available for synthetic purposes. However, controlling the regioselectivity of the reaction remains challenging, in particular when dealing with carbohydrates bearing numerous reactive hydroxyl groups as substrates. Here, we used a computer-aided design framework to engineer the active site of a sucrose-active $$\mathrm{\alpha }$$ α -transglucosylase for the 1,2-cis-glucosylation of a lightly protected chemically synthesized tetrasaccharide, a common precursor for the synthesis of serotype-specific S. flexneri O-antigen fragments. By targeting 27 amino acid positions of the acceptor binding subsites of a GH70 branching sucrase, we used a RosettaDesign-based approach to propose 49 mutants containing up to 15 mutations scattered over the active site. Upon experimental evaluation, these mutants were found to produce up to six distinct pentasaccharides, whereas only two were synthesized by the parental enzyme. Interestingly, we showed that by introducing specific mutations in the active site of a same enzyme scaffold, it is possible to control the regiospecificity of the 1,2-cis glucosylation of the tetrasaccharide acceptor and produce a unique diversity of pentasaccharide bricks. This work offers novel opportunities for the development of highly convergent chemo-enzymatic routes toward S. flexneri haptens.

miR-27b antagonizes BMP signaling in early differentiation of human induced pluripotent stem cells

Human induced pluripotent stem (hiPS) cells are feasible materials for studying the biological mechanisms underlying human embryogenesis. In early embryogenesis, definitive endoderm and mesoderm are differentiated from their common precursor, mesendoderm. Bone morphogenetic protein (BMP) signaling is responsible for regulating mesendoderm and mesoderm formation. Micro RNAs (miRNAs), short non-coding RNAs, broadly regulate biological processes via post-transcriptional repression. The expression of miR-27b, which is enriched in somatic cells, has been reported to increase through definitive endoderm and hepatic differentiation, but little is known about how miR-27b acts during early differentiation. Here, we used miR-27b-inducible hiPS cells to investigate the roles of miR-27b in the undifferentiated and early-differentiated stages. In undifferentiated hiPS cells, miR-27b suppressed the expression of pluripotency markers [alkaline phosphatase (AP) and nanog homeobox (NANOG)] and cell proliferation. Once differentiation began, miR-27b expression repressed phosphorylated SMAD1/5, the mediators of the BMP signaling, throughout definitive endoderm differentiation. Consistent with the above findings, miR-27b overexpression downregulated BMP-induced mesendodermal marker genes [Brachyury, mix paired-like homeobox 1 (MIXL1) and eomesodermin (EOMES)], suggesting that miR-27b had an inhibitory effect on early differentiation. Collectively, our findings revealed a novel antagonistic role of miR-27b in the BMP signaling pathway in the early differentiation of hiPS cells.

Enhancement of Phytosterol and Triterpenoid Production in Plant Hairy Root Cultures—Simultaneous Stimulation or Competition?

Plant in vitro cultures, including hairy roots, can be applied for controlled production of valuable natural products, such as triterpenoids and sterols. These compounds originate from the common precursor squalene. Sterols and triterpenoids distinctly differ in their functions, and the 2,3-oxidosqualene cyclization step is often regarded as a branch point between primary and secondary (more aptly: general and specialized) metabolism. Considering the crucial role of phytosterols as membrane constituents, it has been postulated that unconstrained biosynthesis of triterpenoids can occur when sterol formation is already satisfied, and these compounds are no longer needed for cell growth and division. This hypothesis seems to follow directly the growth-defense trade-off plant dilemma. In this review, we present some examples illustrating the specific interplay between the two divergent pathways for sterol and triterpenoid biosynthesis appearing in root cultures. These studies were significant for revealing the steps of the biosynthetic pathway, understanding the role of particular enzymes, and discovering the possibility of gene regulation. Currently, hairy roots of many plant species can be considered not only as an efficient tool for production of phytochemicals, but also as suitable experimental models for investigations on regulatory mechanisms of plant metabolism.

Orexin receptors in GtoPdb v.2021.3

Orexin receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Orexin receptors [42]) are activated by the endogenous polypeptides orexin-A and orexin-B (also known as hypocretin-1 and -2; 33 and 28 aa) derived from a common precursor, preproorexin or orexin precursor, by proteolytic cleavage and some typical peptide modifications [109]. Currently the only orexin receptor ligands in clinical use are suvorexant and lemborexant, which are used as hypnotics. Orexin receptor crystal structures have been solved [134, 133, 54, 117, 46].

Glucagon receptor family in GtoPdb v.2021.3

The glucagon family of receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on the Glucagon receptor family [162]) are activated by the endogenous peptide (27-44 aa) hormones glucagon, glucagon-like peptide 1, glucagon-like peptide 2, glucose-dependent insulinotropic polypeptide (also known as gastric inhibitory polypeptide), GHRH and secretin. One common precursor (GCG) generates glucagon, glucagon-like peptide 1 and glucagon-like peptide 2 peptides [119]. For a recent review on the current understanding of the structures of GLP-1 and GLP-1R, the molecular basis of their interaction, and the associated signaling events see de Graaf et al., 2016 [89].