Structure of putative tumor suppressor ALDH1L1

Putative tumor suppressor ALDH1L1, the product of natural fusion of three unrelated genes, regulates folate metabolism by catalyzing NADP+-dependent conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2. Cryo-EM structures of tetrameric rat ALDH1L1 revealed the architecture and functional domain interactions of this complex enzyme. Highly mobile N-terminal domains, which remove formyl from 10-formyltetrahydrofolate, undergo multiple transient inter-domain interactions. The C-terminal aldehyde dehydrogenase domains, which convert formyl to CO2, form unusually large interfaces with the intermediate domains, homologs of acyl/peptidyl carrier proteins (A/PCPs), which transfer the formyl group between the catalytic domains. The 4′-phosphopantetheine arm of the intermediate domain is fully extended and reaches deep into the catalytic pocket of the C-terminal domain. Remarkably, the tetrameric state of ALDH1L1 is indispensable for catalysis because the intermediate domain transfers formyl between the catalytic domains of different protomers. These findings emphasize the versatility of A/PCPs in complex, highly dynamic enzymatic systems.

Engineered chimeras unveil swappable modular features of fatty acid and polyketide synthase acyl carrier proteins

The strategic redesign of microbial biosynthetic pathways is a compelling route to access molecules of diverse structure and function in a potentially environmentally sustainable fashion. The promise of this approach hinges on an improved understanding of acyl carrier proteins (ACPs), which serve as central hubs in biosynthetic pathways. These small, flexible proteins mediate the transport of molecular building blocks and intermediates to enzymatic partners that extend and tailor the growing natural products. Past combinatorial biosynthesis efforts have failed due to incompatible ACP-enzyme pairings. Herein we report the design of chimeric ACPs with features of the actinorhodin polyketide synthase ACP (ACT) and of the E. coli fatty acid synthase (FAS) ACP (AcpP). We evaluate the ability of the chimeric ACPs to interact with the E. coli FAS ketosynthase FabF, which represents an interaction essential to building the carbon backbone of the synthase molecular output. Given that AcpP interacts with FabF but ACT does not, we sought to exchange modular features of ACT with AcpP to confer functionality with FabF. The interactions of chimeric ACPs with FabF were interrogated using sedimentation velocity experiments, surface plasmon resonance analyses, mechanism-based crosslinking assays, and molecular dynamics simulations. Results suggest that the residues guiding AcpP-FabF compatibility and ACT-FabF incompatibility may reside in the loop I, α-helix II region. These findings can inform the development of strategic secondary element swaps that expand the enzyme compatibility of ACPs across systems and therefore represent a critical step towards the strategic engineering of unnatural natural products.

Human Glucose Transporters in Renal Glucose Homeostasis

The kidney plays an important role in glucose homeostasis by releasing glucose into the blood stream to prevent hypoglycemia. It is also responsible for the filtration and subsequent reabsorption or excretion of glucose. As glucose is hydrophilic and soluble in water, it is unable to pass through the lipid bilayer on its own; therefore, transport takes place using carrier proteins localized to the plasma membrane. Both sodium-independent glucose transporters (GLUT proteins) and sodium-dependent glucose transporters (SGLT proteins) are expressed in kidney tissue, and mutations of the genes coding for these glucose transporters lead to renal disorders and diseases, including renal cancers. In addition, several diseases may disturb the expression and/or function of renal glucose transporters. The aim of this review is to describe the role of the kidney in glucose homeostasis and the contribution of glucose transporters in renal physiology and renal diseases.

Effect of diabetes mellitus on levels of insulin-like growth factors and their carrier proteins in Guerin’s carcinoma and it’s perifocal tissue in rats

Purpose of the study. Diabetes mellitus (DM) is considered an independent risk factor for higher cancer incidence and death rates. The system of insulin-like growth factors and their carrier proteins (IGF and IGFBP) and hyperglycemia create favorable conditions for the proliferation and metastasis of cancer cells.Materials and methods. Outbred male and female rats were divided into groups (n = 8 each): controls - with Guerin's carcinoma; main group - Guerin's carcinoma growing in presence of DM. Experimental DM was reproduces in animals by the single intraperitoneal alloxan injection (150 mg/kg body weight). After 10 days of the carcinoma growth, levels of IGF and IGFBP in the tumor and in it's perifocal area were measured using ELISA.Results. DM in females upregulated levels of glucose both in the tumor and in perifocal tissues by 1.8 (p < 0.05) and 8.1 times, respectively, but caused opposite changes in IGF-I - it's increase by 6.3 times in the tumor and decrease by 3.2 times in the perifocal area; as a result, such tumors with small primary nodes were more "aggressive" and actively metastasized. In males, induced DM downregulated levels of glucose, IGF-II and IGFBP2 in the carcinoma by 8.4, 3.1 and 1.7 (p < 0.05) times, respectively, and increased levels of IGF-I and IGFBP2 by 1.4 and 1.3 times (p < 0.05) in the perifocal area without changing glucose levels; as a result, tumor volumes exceeded the values in the standard growth, without metastasizing into visceral organs.Conclusion. We revealed gender differences in changing levels of glucose and IGF both in the tumor and in it's perifocal tissue in rats with Guerin's carcinoma growing in presence of DM; these differences could determine different tumor growth dynamics in male and female rats.

Combined effects of glycan chain length and linkage type on the immunogenicity of glycoconjugate vaccines

The development and use of antibacterial glycoconjugate vaccines have significantly reduced the occurrence of potentially fatal childhood and adult diseases such as bacteremia, bacterial meningitis, and pneumonia. In these vaccines, the covalent linkage of bacterial glycans to carrier proteins augments the immunogenicity of saccharide antigens by triggering T cell-dependent B cell responses, leading to high-affinity antibodies and durable protection. Licensed glycoconjugate vaccines either contain long-chain bacterial polysaccharides, medium-sized oligosaccharides, or short synthetic glycans. Here, we discuss factors that affect the glycan chain length in vaccines and review the available literature discussing the impact of glycan chain length on vaccine efficacy. Furthermore, we evaluate the available clinical data on licensed glycoconjugate vaccine preparations with varying chain lengths against two bacterial pathogens, Haemophilus influenzae type b and Neisseria meningitidis group C, regarding a possible correlation of glycan chain length with their efficacy. We find that long-chain glycans cross-linked to carrier proteins and medium-sized oligosaccharides end-linked to carriers both achieve high immunogenicity and efficacy. However, end-linked glycoconjugates that contain long untethered stretches of native glycan chains may induce hyporesponsiveness by T cell-independent activation of B cells, while cross-linked medium-sized oligosaccharides may suffer from suboptimal saccharide epitope accessibility.

Modification of Glyceraldehyde-3-Phosphate Dehydrogenase with Nitric Oxide: Role in Signal Transduction and Development of Apoptosis

This review focuses on the consequences of GAPDH S-nitrosylation at the catalytic cysteine residue. The widespread hypothesis according to which S-nitrosylation causes a change in GAPDH structure and its subsequent binding to the Siah1 protein is considered in detail. It is assumed that the GAPDH complex with Siah1 is transported to the nucleus by carrier proteins, interacts with nuclear proteins, and induces apoptosis. However, there are several conflicting and unproven elements in this hypothesis. In particular, there is no direct confirmation of the interaction between the tetrameric GAPDH and Siah1 caused by S-nitrosylation of GAPDH. The question remains as to whether the translocation of GAPDH into the nucleus is caused by S-nitrosylation or by some other modification of the catalytic cysteine residue. The hypothesis of the induction of apoptosis by oxidation of GAPDH is considered. This oxidation leads to a release of the coenzyme NAD+ from the active center of GAPDH, followed by the dissociation of the tetramer into subunits, which move to the nucleus due to passive transport and induce apoptosis. In conclusion, the main tasks are summarized, the solutions to which will make it possible to more definitively establish the role of nitric oxide in the induction of apoptosis.

Restoration of mRNA Expression of Solute Carrier Proteins in Liver of Diet-Induced Obese Mice by Metformin

Metformin (MET), the most common medicine for type 2 diabetes (T2DM), improves insulin sensitivity by targeting the liver, intestine and other organs. Its impact on expression of the solute carrier (Slc) transporter genes have not been reported in the mechanism of insulin sensitization. In this study, we examined Slc gene expression in the liver and colon of diet-induced obese (DIO) mice treated with MET by transcriptomic analysis. There were 939 differentially expressed genes (DEGs) in the liver of DIO mice vs lean mice, which included 34 Slc genes. MET altered 489 DEGs in the liver of DIO mice, in which 23 were Slc genes. Expression of 20 MET-responsive Slc DEGs was confirmed by qRT-PCR, in which 15 Slc genes were altered in DIO mice and their expressions were restored by MET, including Slc2a10, Slc2a13, Slc5a9, Slc6a14, Slc7a9, Slc9a2, Slc9a3, Slc13a2, Slc15a2, Slc26a3, Slc34a2, Slc37a1, Slc44a4, Slc51b and Slc52a3. While, there were only 97 DEGs in the colon of DIO mice with 5 Slc genes, whose expression was not restored by MET. The data suggest that more genes were altered in the liver over the colon by the high fat diet (HFD). There were 20 Slc genes with alteration confirmed in the liver of DIO mice and 15 of them were restored by MET, which was associated with improvement of insulin sensitivity and obesity. The restoration may improve the uptake of glucose, amino acids, mannose, fructose, 1,5-anhydro-D-glucitol and bumetanide in hepatocytes of the liver of DIO mice. The study provides new insight into the mechanism of metformin action in insulin sensitization and obesity.

Metabolite trafficking enables programmed terpene secretion by yeast

Metabolites are often unable to permeate cell membranes and are thus accumulated inside cells 1. We investigated whether engineered microbes could exclusively secrete intracellular metabolites because sustainable metabolite secretion holds a great potential for mass-production of high-value chemicals in an efficient and continuous manner 2,3. In this study, we demonstrated a synthetic pathway for a metabolite trafficking system that enables lipophilic terpene secretion by yeast cells. When metabolite-binding proteins are tagged with signal peptides, metabolite trafficking becomes programmable; loaded metabolites can be precisely delivered to a desired location within or outside the cell. As a proof of concept, we systematically coupled a terpene-specific protein with an export signal peptide and subsequently demonstrated exceptionally efficient, yet selective terpene secretion by yeast (~225 mg/L for squalene and ~1.6 mg/L for β-carotene). Other carrier proteins could also be readily fused with desired signal peptides, thereby tailoring different metabolite trafficking pathways in different microbes. To the best of our knowledge, this is the first and most efficient cognate pathway for metabolite secretion by microorganisms.

The Hitchhiker's guide to glycoproteomics

Protein glycosylation is one of the most common post-translational modifications that are essential for cell function across all domains of life. Changes in glycosylation are considered a hallmark of many diseases, thus making glycoproteins important diagnostic and prognostic biomarker candidates and therapeutic targets. Glycoproteomics, the study of glycans and their carrier proteins in a system-wide context, is becoming a powerful tool in glycobiology that enables the functional analysis of protein glycosylation. This ‘Hitchhiker's guide to glycoproteomics’ is intended as a starting point for anyone who wants to explore the emerging world of glycoproteomics. The review moves from the techniques that have been developed for the characterisation of single glycoproteins to technologies that may be used for a successful complex glycoproteome characterisation. Examples of the variety of approaches, methodologies, and technologies currently used in the field are given. This review introduces the common strategies to capture glycoprotein-specific and system-wide glycoproteome data from tissues, body fluids, or cells, and a perspective on how integration into a multi-omics workflow enables a deep identification and characterisation of glycoproteins — a class of biomolecules essential in regulating cell function.