Role of ureides in source-to-sink transport of photoassimilates in non-fixing soybean

Nitrogen (N)-fixing soybean plants use the ureides allantoin and allantoic acid as major long-distance transport forms of N, but in non-fixing, non-nodulated plants amino acids mainly serve in source-to-sink N allocation. However, some ureides are still synthesized in roots of non-fixing soybean, and our study addresses the role of ureide transport processes in those plants. In previous work, legume ureide permeases (UPSs) were identified that are involved in cellular import of allantoin and allantoic acid. Here, UPS1 from common bean was expressed in the soybean phloem, which resulted in enhanced source-to-sink transport of ureides in the transgenic plants. This was accompanied by increased ureide synthesis and elevated allantoin and allantoic acid root-to-sink transport. Interestingly, amino acid assimilation, xylem transport, and phloem partitioning to sinks were also strongly up-regulated. In addition, photosynthesis and sucrose phloem transport were improved in the transgenic plants. These combined changes in source physiology and assimilate partitioning resulted in increased vegetative growth and improved seed numbers. Overall, the results support that ureide transport processes in non-fixing plants affect source N and carbon acquisition and assimilation as well as source-to-sink translocation of N and carbon assimilates with consequences for plant growth and seed development.

Impact of the Monocarboxylate Transporter-1 (MCT1)-Mediated Cellular Import of Lactate on Stemness Properties of Human Pancreatic Adenocarcinoma Cells

Metabolite exchange between stromal and tumor cells or among tumor cells themselves accompanies metabolic reprogramming in cancer including pancreatic adenocarcinoma (PDAC). Some tumor cells import and utilize lactate for oxidative energy production (reverse Warburg-metabolism) and the presence of these “reverse Warburg“ cells associates with a more aggressive phenotype and worse prognosis, though the underlying mechanisms are poorly understood. We now show that PDAC cells (BxPc3, A818-6, T3M4) expressing the lactate-importer monocarboxylate transporter-1 (MCT1) are protected by lactate against gemcitabine-induced apoptosis in a MCT1-dependent fashion, contrary to MCT1-negative PDAC cells (Panc1, Capan2). Moreover, lactate administration under glucose starvation, resembling reverse Warburg co a phenotype of BxPc3 and T3M4 cells that confers greater potential of clonal growth upon re-exposure to glucose, along with drug resistance and elevated expression of the stemness marker Nestin and reprogramming factors (Oct4, KLF4, Nanog). These lactate dependent effects on stemness properties are abrogated by the MCT1/lactate-uptake inhibitor 7ACC2 or MCT1 knock-down. Furthermore, the clinical relevance of these observations was supported by detecting co-expression of MCT1 and reprogramming factors in human PDAC tissues. In conclusion, the MCT1-dependent import of lactate supplies “reverse Warburg “PDAC cells with an efficient driver of metabostemness. This condition may essentially contribute to malignant traits including therapy resistance.

Current Progress on Equilibrative Nucleoside Transporter Function and Inhibitor Design

Physiological nucleosides are used for the synthesis of DNA, RNA, and ATP in the cell and serve as universal mammalian signaling molecules that regulate physiological processes such as vasodilation and platelet aggregation by engaging with cell surface receptors. The same pathways that allow uptake of physiological nucleosides mediate the cellular import of synthetic nucleoside analogs used against cancer, HIV, and other viral diseases. Physiological nucleosides and nucleoside drugs are imported by two families of nucleoside transporters: the SLC28 concentrative nucleoside transporters (CNTs) and SLC29 equilibrative nucleoside transporters (ENTs). The four human ENT paralogs are expressed in distinct tissues, localize to different subcellular sites, and transport a variety of different molecules. Here we provide an overview of the known structure–function relationships of the ENT family with a focus on ligand binding and transport in the context of a new hENT1 homology model. We provide a generic residue numbering system for the different ENTs to facilitate the interpretation of mutational data produced using different ENT homologs. The discovery of paralog-selective small-molecule modulators is highly relevant for the design of new therapies and for uncovering the functions of poorly characterized ENT family members. Here, we discuss recent developments in the discovery of new paralog-selective small-molecule ENT inhibitors, including new natural product-inspired compounds. Recent progress in the ability to heterologously produce functional ENTs will allow us to gain insight into the structure and functions of different ENT family members as well as the rational discovery of highly selective inhibitors.

Nutritional Immunity and Antibiotic Drug Treatments Influence Microbial Composition but Fail to Eliminate Urethral Catheter Biofilms in Recurrently Catheterized Patients

Polymicrobial biofilms that form on indwelling urethral catheters used by neurogenic bladder patients are known to recur following catheter replacements. Uropathogens dominate in catheter biofilms (CBs), grow and disperse as multi-cellular aggregates. Their microbial complexity, the characteristics of host immune responses and the molecular crosstalk in this ecosystem are incompletely understood. By surveying eight patients over up to six months with meta-omics analysis methods, we shed new light on the longitudinal microbial dynamics in CBs and the microbial-host crosstalk. There was evidence of chronic innate immune responses in all patients. Pathogens dominated the microbial contents.Proteus mirabilisoften out-competed other species in cases of salt encrustation of catheters. The examination of proteomes in CBs and associated urinary pellets revealed many abundant bacterial systems for transition metal ion (TMI) acquisition. TMIs are sequestered by effector proteins released by activated neutrophils and urothelial cells, such as lactotransferrin and calgranulins, which were abundant in the host proteomes. We identified positive quantitative correlations among systems responsible for siderophore biosynthesis, TMI/siderophore uptake and TMI cellular import in bacterial species, suggesting competition for TMIs to support their metabolism and growth in CBs.Enterococcus faecaliswas prevalent as a cohabitant of CBs and expressed three lipoproteins with apparent TMI acquisition functions. Fastidious anaerobic bacteria such asVeillonella,Actinobaculum, andBifidobacteriumgrew in CB communities that appeared to be oxygen starved. Finally, antibiotic drug treatments were shown to influence microbial composition of CBs but failed to prevent re-colonization of urethral catheters with persisting and/or drug-resistant newly emerging pathogens.

Metal cofactors trafficking and assembly in the cell: a molecular view

Metal ions are essential cofactors required by the proteome of organisms from any kingdom of life to correctly exert their functions. Dedicated cellular import, transport and homeostasis systems assure that the needed metal ion is correctly delivered and inserted into the target proteins and avoid the presence of free metal ions in the cell, preventing oxidative damaging. Among metal ions, in eukaryotic organisms copper and iron are required by proteins involved in absolutely essential functions, such as respiration, oxidative stress protection, catalysis, gene expression regulation. Copper and iron binding proteins are localized in essentially all cellular compartments. Copper is physiologically present mainly as individual metal ion. Iron can be present both as individual metal ion or as part of cofactors, such as hemes and iron-sulfur (Fe-S) clusters. Both metal ions are characterized by the ability to cycle between different oxidation states, which enable them to catalyze redox reactions and to participate in electron transfer processes. Here we describe in detail the main processes responsible for the trafficking of copper and iron sulfur clusters, with particular interest for the structural aspects of the maturation of copper and iron-sulfur-binding proteins.

Trends in the Binding of Cell Penetrating Peptides to siRNA: A Molecular Docking Study

The use of gene therapeutics, including short interfering RNA (siRNA), is limited by the lack of efficient delivery systems. An appealing approach to deliver gene therapeutics involves noncovalent complexation with cell penetrating peptides (CPPs) which are able to penetrate the cell membranes of mammals. Although a number of CPPs have been discovered, our understanding of their complexation and translocation of siRNA is as yet insufficient. Here, we report on computational studies comparing the binding affinities of CPPs with siRNA, considering a variety of CPPs. Specifically, seventeen CPPs from three different categories, cationic, amphipathic, and hydrophobic CPPs, were studied. Molecular mechanics were used to minimize structures, while molecular docking calculations were used to predict the orientation and favorability of sequentially binding multiple peptides to siRNA. Binding scores from docking calculations were highest for amphipathic peptides over cationic and hydrophobic peptides. Results indicate that initial complexation of peptides will likely occur along the major groove of the siRNA, driven by electrostatic interactions. Subsequent binding of CPPs is likely to occur in the minor groove and later on bind randomly, to siRNA or previously bound CPPs, through hydrophobic interactions. However, hydrophobic CPPs do not show this binding pattern. Ultimately binding yields a positively charged nanoparticle capable of noninvasive cellular import of therapeutic molecules.

Differential Requirements for Clathrin-dependent Endocytosis at Sites of Cell–Substrate Adhesion

Clathrin-dependent endocytosis is a major route for the cellular import of macromolecules and occurs at the interface between the cell and its surroundings. However, little is known about the influences of cell–substrate attachment in clathrin-coated vesicle formation. Using biochemical and imaging-based methods, we find that cell–substrate adhesion reduces the rate of endocytosis. Clathrin-coated pits (CCPs) in proximity to substrate contacts exhibit slower dynamics in comparison to CCPs found more distant from adhesions. Direct manipulation of the extracellular matrix (ECM) to modulate adhesion demonstrates that tight adhesion dramatically reduces clathrin-dependent endocytosis and extends the lifetimes of clathrin structures. This reduction is in part mediated by integrin-matrix engagement. In addition, we demonstrate that actin cytoskeletal dynamics are differentially required for efficient endocytosis, with a stronger requirement for actin polymerization in areas of adhesion. Together, these results reveal that cell–substrate adhesion regulates clathrin-dependent endocytosis and suggests that actin assembly facilitates vesicle formation at sites of adhesion.

Identification of multidrug resistance protein 1 (MRP1/ABCC1) as a molecular gate for cellular export of cobalamin

Cobalamin (Cbl, vitamin B12) deficiency in humans is a cause of hematologic and neurologic disorders. We show here that the cellular export of Cbl, in contrast to the carrier- and receptor-dependent cellular import of Cbl, occurs by transmembrane transport of “free” Cbl. Screening of candidate transporters by cellular gene silencing showed a role in cellular Cbl efflux of the ATP-binding cassette (ABC)–drug transporter, ABCC1, alias multidrug resistance protein 1 (MRP1), which is present in the basolateral membrane of intestinal epithelium and in other cells. The ability of MRP1 to mediate ATP-dependent Cbl transport was confirmed by vesicular transport experiments, and a physiologic role of MRP1 in mammalian Cbl homeostasis is indicated by the phenotype of knockout mice with targeted disruption of MRP1. These animals have a reduced concentration of Cbl in plasma and in the storage organs liver and kidney. In contrast, Cbl accumulates in the terminal part of the intestine of these mice, suggesting a functional malabsorption because of a lower epithelial basolateral Cbl efflux. The identification of this Cbl export mechanism now allows the delineation of a coherent pathway for Cbl trafficking from food to the body cells.