RUTBC1 protein, a Rab9A effector that activates GTP hydrolysis by Rab32 and Rab33B proteins

Rab GTPases regulate all steps of membrane trafficking. Their interconversion between active, GTP-bound states and inactive, GDP-bound states is regulated by guanine nucleotide exchange factors and GTPase-activating proteins. The substrates for most Rab GTPase-activating proteins (GAPs) are unknown. Rab9A and its effectors regulate transport of mannose 6-phosphate receptors from late endosomes to the trans-Golgi network. We show here that RUTBC1 is a Tre2/Bub2/Cdc16 domain-containing protein that binds to Rab9A-GTP both in vitro and in cultured cells, but is not a GTPase-activating protein for Rab9A. Biochemical screening of RUTBC1 Rab protein substrates revealed highest in vitro GTP hydrolysis-activating activity with Rab32 and Rab33B. Catalysis required Arg-803 of RUTBC1, and RUTBC1 could activate a catalytically inhibited Rab33B mutant (Q92A), in support of a dual finger mechanism for RUTBC1 action. Rab9A binding did not influence GAP activity of bead-bound RUTBC1 protein. In cells and cell extracts, RUTBC1 influenced the ability of Rab32 to bind its effector protein, Varp, consistent with a physiological role for RUTBC1 in regulating Rab32. In contrast, binding of Rab33B to its effector protein, Atg16L1, was not influenced by RUTBC1 in cells or extracts. The identification of a protein that binds Rab9A and inactivates Rab32 supports a model in which Rab9A and Rab32 act in adjacent pathways at the boundary between late endosomes and the biogenesis of lysosome-related organelles.     

Prosaposin sorting is mediated by oligomerization

The compartmental nature of eukaryotic cells requires sophisticated mechanisms of protein sorting. Prosaposin, the precursor of four sphingolipid activator proteins, is transported from the trans-Golgi network (TGN) to lysosomes as a partially glycosylated (65 kDa) protein with high-mannose/hybrid oligosaccharides. Prosaposin is also found in the extracellular space where it is secreted as a fully glycosylated (70 kDa) protein composed of complex glycans. Although the trafficking of prosaposin to lysosomes is known to be mediated by sortilin, the mechanism of secretion of this protein is still unknown. In this study, we report that prosaposin may covalently aggregate into oligomers. Our results demonstrate that while prosaposin oligomers are secreted into the extracellular space, monomeric prosaposin remains inside the cell bound to sortilin. We also found that deletion of the C-terminus of prosaposin, previously shown to block its lysosomal transport, did not abolish its oligomerization and secretion. On the other hand, elimination of the N-terminus and of each saposin domain inhibited its oligomerization and resulted in its retention as a fully glycosylated protein. In conclusion, we are reporting for the first time that oligomerization of prosaposin is crucial for its entry into the secretory pathway.     

CK2 phosphorylation of an acidic Ser/Thr di-isoleucine motif in the Na+/H+ exchanger NHE5 isoform promotes association with beta-arrestin2 and endocytosis

Internalization of the Na(+)/H(+) exchanger NHE5 into recycling endosomes is enhanced by the endocytic adaptor proteins β-arrestin1 and -2, best known for their preferential recognition of ligand-activated G protein-coupled receptors (GPCRs). However, the mechanism underlying their atypical association with non-GPCRs, such as NHE5, is unknown. In this study, we identified a highly acidic, serine/threonine-rich, di-isoleucine motif (amino acids 697-723) in the cytoplasmic C terminus of NHE5 that is recognized by β-arrestin2. Gross deletions of this site decreased the state of phosphorylation of NHE5 as well as its binding and responsiveness to β-arrestin2 in intact cells. More refined in vitro analyses showed that this site was robustly phosphorylated by the acidotropic protein kinase CK2, whereas other kinases, such as CK1 or the GPCR kinase GRK2, were considerably less potent. Simultaneous mutation of five Ser/Thr residues within 702-714 to Ala ((702)ST/AA(714)) abolished phosphorylation and binding of β-arrestin2. In transfected cells, the CK2 catalytic α subunit formed a complex with NHE5 and decreased wild-type but not (702)ST/AA(714) NHE5 activity, further supporting a regulatory role for this kinase. The rate of internalization of (702)ST/AA(714) was also diminished and relatively insensitive to overexpression of β-arrestin2. However, unlike in vitro, this mutant retained its ability to form a complex with β-arrestin2 despite its lack of responsiveness. Additional mutations of two di-isoleucine-based motifs (I697A/L698A and I722A/I723A) that immediately flank the acidic cluster, either separately or together, were required to disrupt their association. These data demonstrate that discrete elements of an elaborate sorting signal in NHE5 contribute to β-arrestin2 binding and trafficking along the recycling endosomal pathway.     

Structure of surface layer homology (SLH) domains from Bacillus anthracis surface array protein

Surface (S)-layers, para-crystalline arrays of protein, are deposited in the envelope of most bacterial species. These surface organelles are retained in the bacterial envelope through the non-covalent association of proteins with cell wall carbohydrates. Bacillus anthracis, a Gram-positive pathogen, produces S-layers of the protein Sap, which uses three consecutive repeats of the surface-layer homology (SLH) domain to engage secondary cell wall polysaccharides (SCWP). Using x-ray crystallography, we reveal here the structure of these SLH domains, which assume the shape of a three-prong spindle. Each SLH domain contributes to a three-helical bundle at the spindle base, whereas another α-helix and its connecting loops generate the three prongs. The inter-prong grooves contain conserved cationic and anionic residues, which are necessary for SLH domains to bind the B. anthracis SCWP. Modeling experiments suggest that the SLH domains of other S-layer proteins also fold into three-prong spindles and capture bacterial envelope carbohydrates by a similar mechanism.     

Computational analysis reveals abundance of potential glycoproteins in Archaea, Bacteria and Eukarya

Glycosylation is the most common type of post-translational modification (PTM) and is known to affect protein stability, folding and activity. Inactivity of enzymes mediating glycosylation can result in serious disorders including colon cancer and brain disorders. Out of five main types of glycosylation, N-linked glycosylation is most abundant and characterized by the addition of a sugar group to an Asparagine residue at the N-X-S/T motif. Enzyme mediating such transfer is known as oligosaccharyl transferase (OST). It has been hypothesized before that a significant number of proteins serve as glycoproteins. In this study, we used programming implementations of Python to statistically quantify the representation of glycoproteins by scanning all the available proteome sequence data at ExPASy server for the presence of glycoproteins and also the enzyme which plays critical role in glycosylation i.e. OST. Our results suggest that more than 50% of the proteins carry N-X-S/T motif i.e. they could be potential glycoproteins. Furthermore, approximately 28-36% (1/3) of proteins possesses signature motifs which are characteristic features of enzyme OST. Quantifying this bias individually reveals that both the number of proteins tagged with N-X-S/T motif and the average number of motifs per protein is significantly higher in case of eukaryotes when compared to prokaryotes. In the light of these results we conclude that there is a significant bias in the representation of glycoproteins in the proteomes of all species and is manifested substantially in eukaryotes and claim for glycosylation to be the most common and ubiquitous PTM in cells, especially in eukaryotes.     

Structural and functional analysis of two glutamate racemase isozymes from Bacillus anthracis and implications for inhibitor design

Glutamate racemase (RacE) is responsible for converting l-glutamate to d-glutamate, which is an essential component of peptidoglycan biosynthesis, and the primary constituent of the poly-gamma-d-glutamate capsule of the pathogen Bacillus anthracis. RacE enzymes are essential for bacterial growth and lack a human homolog, making them attractive targets for the design and development of antibacterial therapeutics. We have cloned, expressed and purified the two glutamate racemase isozymes, RacE1 and RacE2, from the B. anthracis genome. Through a series of steady-state kinetic studies, and based upon the ability of both RacE1 and RacE2 to catalyze the rapid formation of d-glutamate, we have determined that RacE1 and RacE2 are bona fide isozymes. The X-ray structures of B. anthracis RacE1 and RacE2, in complex with d-glutamate, were determined to resolutions of 1.75 A and 2.0 A. Both enzymes are dimers with monomers arranged in a "tail-to-tail" orientation, similar to the B. subtilis RacE structure, but differing substantially from the Aquifex pyrophilus RacE structure. The differences in quaternary structures produce differences in the active sites of racemases among the various species, which has important implications for structure-based, inhibitor design efforts within this class of enzymes. We found a Val to Ala variance at the entrance of the active site between RacE1 and RacE2, which results in the active site entrance being less sterically hindered for RacE1. Using a series of inhibitors, we show that this variance results in differences in the inhibitory activity against the two isozymes and suggest a strategy for structure-based inhibitor design to obtain broad-spectrum inhibitors for glutamate racemases.     

A computational approach to animal breeding

We propose a computational model of mating strategies for controlled animal breeding programs. A mating strategy in a controlled breeding program is a heuristic with some optimization criteria as a goal. Thus, it is appropriate to use the computational tools available for analysis of optimization heuristics. In this paper, we propose the first discrete model of the controlled animal breeding problem and analyse heuristics for two possible objectives: (1) breeding for maximum diversity and (2) breeding a target individual. These two goals are representative of conservation biology and agricultural livestock management, respectively. We evaluate several mating strategies and provide upper and lower bounds for the expected number of matings. While the population parameters may vary and can change the actual number of matings for a particular strategy, the order of magnitude of the number of expected matings and the relative competitiveness of the mating heuristics remains the same. Thus, our simple discrete model of the animal breeding problem provides a novel viable and robust approach to designing and comparing breeding strategies in captive populations.     

The neuronal dynamics underlying cognitive flexibility in set shifting tasks

The ability to switch attention from one aspect of an object to another or in other words to switch the “attentional set” as investigated in tasks like the “Wisconsin Card Sorting Test” is commonly referred to as cognitive flexibility. In this work we present a biophysically detailed neurodynamical model which illustrates the neuronal base of the processes related to this cognitive flexibility. For this purpose we conducted behavioral experiments which allow the combined evaluation of different aspects of set shifting tasks: uninstructed set shifts as investigated in Wisconsin-like tasks, effects of stimulus congruency as investigated in Stroop-like tasks and the contribution of working memory as investigated in “Delayed-Match-to-Sample” tasks. The work describes how general experimental findings are usable to design the architecture of a biophysical detailed though minimalistic model with a high orientation on neurobiological findings and how, in turn, the simulations support experimental investigations. The resulting model is able to account for experimental and individual response times and error rates and enables the switch of attention as a system inherent model feature: The switching process suggested by the model is based on the memorization of the visual stimuli and does not require any synaptic learning. The operation of the model thus demonstrates with at least a high probability the neuronal dynamics underlying a key component of human behavior: the ability to adapt behavior according to context requirements—cognitive flexibility. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Action Editor: Peter Dayan     

SNX11 Identified as an Essential Host Factor for SFTS Virus Infection by CRISPR Knockout Screening

Severe fever with thrombocytopenia syndrome virus(SFTSV) is a highly pathogenic tick-borne bunyavirus that causes lethal infectious disease and severe fever with thrombocytopenia syndrome(SFTS) in humans. The molecular mechanisms and host cellular factors required for SFTSV infection remain uncharacterized. Using a genome-wide CRISPR-based screening strategy, we identified a host cellular protein, sorting nexin 11(SNX11) which is involved in the intracellular endosomal trafficking pathway, as an essential cell factor for SFTSV infection. An SNX11-KO HeLa cell line was established, and SFTSV replication was significantly reduced. The glycoproteins of SFTSV were detected and remained in later endosomal compartments but were not detectable in the endoplasmic reticulum(ER) or Golgi apparatus. pH values in the endosomal compartments of the SNX11-KO cells increased compared with the pH of normal HeLa cells, and lysosomal-associated membrane protein 1(LAMP1) expression was significantly elevated in the SNX11-KO cells. Overall,these results indicated that penetration of SFTSV from the endolysosomes into the cytoplasm of host cells was blocked in the cells lacking SNX11. Our study for the first time provides insight into the important role of the SNX11 as an essential host factor in the intracellular trafficking and penetrating process of SFTSV infection via potential regulation of viral protein sorting, membrane fusion, and other endocytic machinery.     

Formation of Disulfide Bridges Drives Oligomerization,Membrane Pore Formation,and Translocation of Fibroblast Growth Factor 2 to Cell Surfaces

Fibroblast growth factor 2 (FGF2) is a key signaling molecule in tumor-induced angiogenesis. FGF2 is secreted by an unconventional secretory mechanism that involves phosphatidylinositol 4,5-bisphosphate-dependent insertion of FGF2 oligomers into the plasma membrane. This process is regulated by Tec kinase-mediated tyrosine phosphorylation of FGF2. Molecular interactions driving FGF2 monomers into membrane-inserted FGF2 oligomers are unknown. Here we identify two surface cysteines that are critical for efficient unconventional secretion of FGF2. They represent unique features of FGF2 as they are absent from all signal-peptide-containing members of the FGF protein family. We show that phosphatidylinositol 4,5-bisphosphate-dependent FGF2 oligomerization concomitant with the generation of membrane pores depends on FGF2 surface cysteines as either chemical alkylation or substitution with alanines impairs these processes. We further demonstrate that the FGF2 variant forms lacking the two surface cysteines are not secreted from cells. These findings were corroborated by experiments redirecting a signal-peptide-containing FGF family member from the endoplasmic reticulum/Golgi-dependent secretory pathway into the unconventional secretory pathway of FGF2. Cis elements known to be required for unconventional secretion of FGF2, including the two surface cysteines, were transplanted into a variant form of FGF4 without signal peptide. The resulting FGF4/2 hybrid protein was secreted by unconventional means. We propose that the formation of disulfide bridges drives membrane insertion of FGF2 oligomers as intermediates in unconventional secretion of FGF2.