Variation on a theme of SDR. dTDP-6-deoxy-L- lyxo-4-hexulose reductase (RmlD) shows a new Mg2+-dependent dimerization mode

dTDP-6-deoxy-L-lyxo-4-hexulose reductase (RmlD) catalyzes the final step in the conversion of dTDP-D-glucose to dTDP-L-rhamnose in an NAD(P)H- and Mg2+-dependent reaction. L-rhamnose biosynthesis is an antibacterial target. The structure of RmlD from Salmonella enterica serovar Typhimurium has been determined, and complexes with NADH, NADPH, and dTDP-L-rhamnose are reported. RmlD differs from other short chain dehydrogenases in that it has a novel dimer interface that contains Mg2+. Enzyme catalysis involves hydride transfer from the nicotinamide ring of the cofactor to the C4'-carbonyl group of the substrate. The substrate is activated through protonation by a conserved tyrosine. NAD(P)H is bound in a solvent-exposed cleft, allowing facile replacement. We suggest a novel role for the conserved serine/threonine residue of the catalytic triad of SDR enzymes.     

Glucose sensing by gut endocrine cells and activation of the vagal afferent pathway is impaired in a rodent model of type 2 diabetes mellitus

Glucose in the gut lumen activates gut endocrine cells to release 5-HT, glucagon-like peptide 1/2 (GLP-1/2), and glucose-dependent insulinotropic polypeptide (GIP), which act to change gastrointestinal function and regulate postprandial plasma glucose. There is evidence that both release and action of incretin hormones is reduced in type 2 diabetes (T2D). We measured cellular activation of enteroendocrine and enterochromaffin cells, enteric neurons, and vagal afferent neurons in response to intestinal glucose in a model of type 2 diabetes mellitus, the UCD-T2DM rat. Prediabetic (PD), recent-diabetic (RD, 2 wk postonset), and 3-mo diabetic (3MD) fasted UCD-T2DM rats were given an orogastric gavage of vehicle (water, 0.5 ml /100 g body wt) or glucose (330 μmol/100 g body wt); after 6 min tissue was removed and cellular activation was determined by immunohistochemistry for phosphorylated calcium calmodulin-dependent kinase II (pCaMKII). In PD rats, pCaMKII immunoreactivity was increased in duodenal 5-HT (P < 0.001), K (P < 0.01) and L (P < 0.01) cells in response to glucose; glucose-induced activation of all three cell types was significantly reduced in RD and 3MD compared with PD rats. Immunoreactivity for GLP-1, but not GIP, was significantly reduced in RD and 3MD compared with PD rats (P < 0.01). Administration of glucose significantly increased pCaMKII in enteric and vagal afferent neurons in PD rats; glucose-induced pCaMKII immunoreactivity was attenuated in enteric and vagal afferent neurons (P < 0.01, P < 0.001, respectively) in RD and 3MD. These data suggest that glucose sensing in enteroendocrine and enterochromaffin cells and activation of neural pathways is markedly impaired in UCD-T2DM rats.     

Dopamine- and cAMP-regulated Phosphoprotein of 32-kDa (DARPP-32)-dependent Activation of Extracellular Signal-regulated Kinase (ERK) and Mammalian Target of Rapamycin Complex 1 (mTORC1) Signaling in Experimental Parkinsonism

Dyskinesia, a motor complication caused by prolonged administration of the antiparkinsonian drug l-3,4-dihydroxyphenylalanine (l-DOPA), is accompanied by activation of cAMP signaling and hyperphosphorylation of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32). Here, we show that the abnormal phosphorylation of DARPP-32 occurs specifically in medium spiny neurons (MSNs) expressing dopamine D1 receptors (D1R). Using mice in which DARPP-32 is selectively deleted in D1R-expressing MSNs, we demonstrate that this protein is required for l-DOPA-induced activation of the extracellular signal-regulated protein kinases 1 and 2 and the mammalian target of rapamycin complex 1 (mTORC1) pathways, which are implicated in dyskinesia. We also show that mutation of the phosphorylation site for cAMP-dependent protein kinase on DARPP-32 attenuates l-DOPA-induced dyskinesia and reduces the concomitant activations of ERK and mTORC1 signaling. These studies demonstrate that, in D1R-expressing MSNs, l-DOPA-induced activation of ERK and mTORC1 requires DARPP-32 and indicates the importance of the cAMP/DARPP-32 signaling cascade in dyskinesia.     

The Prion Protein Preference of Sporadic Creutzfeldt-Jakob Disease Subtypes

Sporadic Creutzfeldt-Jakob disease (CJD) is the most prevalent manifestation of the transmissible spongiform encephalopathies or prion diseases affecting humans. The disease encompasses a spectrum of clinical phenotypes that have been correlated with molecular subtypes that are characterized by the molecular mass of the protease-resistant fragment of the disease-related conformation of the prion protein and a polymorphism at codon 129 of the gene encoding the prion protein. A cell-free assay of prion protein misfolding was used to investigate the ability of these sporadic CJD molecular subtypes to propagate using brain-derived sources of the cellular prion protein (PrP^C). This study confirmed the presence of three distinct sporadic CJD molecular subtypes with PrP^C substrate requirements that reflected their codon 129 associations in vivo. However, the ability of a sporadic CJD molecular subtype to use a specific PrP^C substrate was not determined solely by codon 129 as the efficiency of prion propagation was also influenced by the composition of the brain tissue from which the PrP^C substrate was sourced, thus indicating that nuances in PrP^C or additional factors may determine sporadic CJD subtype. The results of this study will aid in the design of diagnostic assays that can detect prion disease across the diversity of sporadic CJD subtypes.     

Diverse energetic effects of charge reversal mutations of poxvirus topoisomerase IB

A key aspect of the reaction mechanism of type IB topoisomerases is the controlled unwinding of DNA supercoils while the enzyme is transiently bound to one strand of the DNA duplex via a phosphotyrosyl linkage. In this complex, the mobile segment of the bound DNA downstream from the site of cleavage must rotate around the helical axis, requiring that interactions with the enzyme must break and re-form multiple times during the course of removing supercoils. A crystal structure of variola virus type IB topoisomerase (vTopo) bound to DNA shows several positively charged side chains that interact with the downstream mobile and upstream rigid segments, suggesting that these groups may play a role in catalysis, including the processive unwinding of supercoils. We have mutated three such residues, R67, K35, and K271, to Ala and Glu and determined the energetic effects of these mutations at each point along the reaction coordinate of vTopo. R67 interacts with a phosphate group in the rigid DNA segment across from the site of DNA strand cleavage. The ~30-fold damaging effects of the R67A and R67E mutations were primarily on the phosphoryl transfer step, with little effect on enzyme-DNA binding, or the processivity of supercoil unwinding. Removal of the K35 interaction shows mutational effects similar to those of R67, even though this residue interacts with the mobile segment 3 bp from the cleavage site. The two mutations of K271, which interacts with the mobile region even further from the site of covalent linkage, show significant effects not only on phosphoryl transfer but also on downstream DNA strand positioning. Moreover, supercoil unwinding measurements indicate that the K271A and K271E mutations increase the average number of supercoils that are removed during the lifetime of the covalent complex, enhancing the processivity of supercoil unwinding. These measurements support the proposal that the processivity of supercoil unwinding can be regulated by electrostatic interactions between the enzyme and the mobile DNA phosphate backbone.     

Toward Fast Determination of Protein Stability Maps: Experimental and Theoretical Analysis of Mutants of a Nocardiopsis prasina Serine Protease

The stability of serine proteases is of major importance for their application in industrial processes. Here we study the determinants of the stability of a Nocardiopsis prasina serine protease using fast residual activity assays, a feature classification algorithm, and structure-based energy calculation algorithms for 121 micropurified mutant enzyme clones containing multiple point mutations. Using a multivariate regression analysis, we deconvolute the data for the mutant clones and find that mutations of residues Asn47 and Pro124 are deleterious to the stability of the enzyme. Both of these residues are situated in loops that are known to be important for the stability of the highly homologous α-lytic protease. Structure-based energy calculations with PEATSA give a good general agreement with the trend of experimentally measured values but also identify a number of clones that the algorithm fails to predict correctly. We discuss the significance of the results in relation to the structure and function of closely related proteases, comment on the optimal experimental design when performing high-throughput experiments for characterizing the determinants of protein stability, and discuss the performance of structure-based energy calculations with complex data sets such as the one presented here.     

A role for the vesicle-associated tubulin binding protein ARL6 (BBS3) in flagellum extension in Trypanosoma brucei

The small GTPase Arl6 is implicated in the ciliopathic human genetic disorder Bardet-Biedl syndrome, acting at primary cilia in recruitment of the octomeric BBSome complex, which is required for specific trafficking events to and from the cilium in eukaryotes. Here we describe functional characterisation of Arl6 in the flagellated model eukaryote Trypanosoma brucei, which requires motility for viability. Unlike human Arl6 which has a ciliary localisation, TbARL6 is associated with electron-dense vesicles throughout the cell body following co-translational modification by N-myristoylation. Similar to the related protein ARL-3A in T. brucei, modulation of expression of ARL6 by RNA interference does not prevent motility but causes a significant reduction in flagellum length. Tubulin is identified as an ARL6 interacting partner, suggesting that ARL6 may act as an anchor between vesicles and cytoplasmic microtubules. We provide evidence that the interaction between ARL6 and the BBSome is conserved in unicellular eukaryotes. Overexpression of BBS1 leads to translocation of endogenous ARL6 to the site of exogenous BBS1 at the flagellar pocket. Furthermore, a combination of BBS1 overexpression and ARL6 RNAi has a synergistic inhibitory effect on cell growth. Our findings indicate that ARL6 in trypanosomes contributes to flagellum biogenesis, most likely through an interaction with the BBSome.