D-Amino Acid Probes for Penicillin Binding Protein-based Bacterial Surface Labeling [Enzymology]

October 23rd, 2015 by Fura, J. M., Kearns, D., Pires, M. M.

Peptidoglycan is an essential and highly conserved mesh structure that surrounds bacterial cells. It plays a critical role in retaining a defined cell shape and, in the case of pathogenic Gram-positive bacteria, it lies at the interface between bacterial cells and the host organisms. Intriguingly, bacteria can metabolically incorporate unnatural D-amino acids into the peptidoglycan stem peptide directly from the surrounding media, a process mediated by penicillin binding proteins (PBPs). Metabolic peptidoglycan remodeling via unnatural D-amino acids has provided unique insight into peptidoglycan biosynthesis of live bacteria and has also served as the basis of a synthetic immunology strategy with potential therapeutic implications. A striking feature of this process is the vast promiscuity displayed by PBPs in tolerating entirely unnatural sidechains. Yet, the chemical space and physical features of this sidechain promiscuity has not been systematically determined. In this report, we designed and synthesized a library of variants displaying diverse sidechains to comprehensively establish the tolerability of unnatural D-amino acids by PBPs in both Gram-positive and Gram-negative organisms. In addition, nine Bacillus subtilis PBP-null mutants were evaluated with the goal of identifying a potential primary PBP responsible for unnatural D-amino acid incorporation and gaining insight into temporal control of PBP activity. Together, we have empirically established the scope of physical parameters that govern metabolic incorporation of unnatural D-amino acids into bacterial peptidoglycan.

Utilization of Dioxygen by Carotenoid Cleavage Oxygenases [Protein Structure and Folding]

October 23rd, 2015 by

Carotenoid cleavage oxygenases (CCOs) are non-heme, Fe(II)-dependent enzymes that participate in biologically important metabolic pathways involving carotenoids, apocarotenoids including retinoids, stilbenes and related compounds. CCOs typically catalyze the cleavage of non-aromatic double bonds by dioxygen (O2) to form aldehyde or ketone products. Expressed only in vertebrates, the RPE65 sub-group of CCOs catalyzes a non-canonical reaction consisting of concerted ester cleavage and trans-cis isomerization of all-trans-retinyl esters. It remains unclear whether the former group of CCOs function as mono- or di-oxygenases. Additionally, a potential role for O2 in catalysis by the RPE65 group of CCOs has not been evaluated to date. Here, we investigated the pattern of oxygen incorporation into apocarotenoid products of Synechocystis apocarotenoid oxygenase (ACO). Reactions performed in the presence of 18O-labeled water and 18O2 revealed an unambiguous dioxygenase pattern of O2 incorporation into the reaction products. Substitution of Ala for Thr at position 136 of ACO, a site predicted to govern the mono- vs. dioxygenase tendency of CCOs, greatly reduced enzymatic activity without altering the dioxygenase labeling pattern. Reevaluation of the oxygen-labeling pattern of the resveratrol-cleaving CCO, NOV2, previously reported to be a monooxygenase, using a purified enzyme sample revealed that it too is a dioxygenase. We also demonstrated that bovine RPE65 is not dependent on O2 for its cleavage/isomerase activity. In conjunction with prior research, the results of this study resolve key issues regarding the utilization of O2 by CCOs and indicate that dioxygenase activity is a feature common amongst double bond-cleaving CCOs.

The Blockade of NF-{kappa}B Activation by a Specific Inhibitory Peptide has a Strong Neuroprotective Role in a Sprague-Dawley Rat Kernicterus Model [Neurobiology]

October 23rd, 2015 by Li, M., Song, S., Li, S., Feng, J., Hua, Z.

Kernicterus, the permanent nerve damage occurs as a result of bilirubin precipitation, still occurs worldwide and may lead to death or permanent neurological impairments. However, the underlying mechanisms remain unclear and effective therapeutic strategies are lacking. The present study aims to investigate the activation of nuclear factor kappa B (NF-κB) and to identify the effect of NF-κB inhibition on the newborn rat Kernicterus model. The NF-κB essential modifier-binding domain peptide (NBD), coupled with the HIV trans-activator of transcription peptide (TAT) was used to inhibit NF-κB. NF-κB was significantly activated in the cerebrum at 1 and 3 h (P < 0.05) after the model was established, as measured by the electrophoretic mobility shift assay (EMSA). NF-κB activation was inhibited by intraperitoneal administration of TAT-NBD. The general conditions of the TAT-NBD-treated rats were improved, meanwhile, these rats performed much better on the neurological evaluation, the rotarod test and the Morris water maze test (P<0.05) than the vehicle-treated rats at 28 days. Furthermore, the morphology of the nerve cells was better preserved in the TAT-NBD group, and displayed less neurodegeneration and astrocytosis. Simultaneously, apoptosis in the brain was attenuated, and the levels of the TNF-α and IL-1β proteins were decreased (P < 0.01). These results suggested that NF-κB was activated and inhibition of NF-κB activation by TAT-NBD not only attenuates the acute neurotoxicity, apoptosis and inflammation, but also improved the long-term neurobehavioral impairments in the Kernicterus model rats in vivo. Thus, inhibiting NF-κB activation might be a potential therapeutic approach for Kernicterus.
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The Protein Interaction of RNA Helicase B (RhlB) and Polynucleotide Phosphorylase (PNPase) Contributes to the Homeostatic Control of Cysteine in Escherichia coli [Microbiology]

October 22nd, 2015 by Tseng, Y.-T., Chiou, N.-T., Gogiraju, R., Lin-Chao, S.

PNPase, one of the major enzymes with 3'-to-5' single-stranded RNA (ssRNA) degradation and processing activities, can interact with the RNA helicase RhlB independently of RNA degradosome formation in E. coli. Here, we report that loss of interaction between RhlB and PNPase impacts cysteine homeostasis in E. coli. By random mutagenesis, we identified a mutant RhlBP238L that loses 75% of its ability to interact with PNPase, but retains normal interaction with RNase E and RNA, in addition to exhibiting normal helicase activity. Applying microarray analyses to an E. coli strain with impaired RNA degradosome formation, we investigated the biological consequences of a weakened interaction between RhlB and PNPase. We found significant increases in 11 out of 14 genes involved in cysteine biosynthesis. Subsequent Northern blot analyses showed that the upregulated transcripts were the result of stabilization of the cysB transcript encoding a transcriptional activator for the cys operons. Furthermore, Northern blots of PNPase or RhlB mutants showed that RhlB-PNPase plays both a catalytic and structural role in regulating cysB degradation. Cells expressing the RhlBP238L mutant exhibited an increase in intracellular cysteine and an enhanced anti-oxidative response. Collectively, this study suggests a mechanism by which bacteria use the PNPase-RhlB exosome-like complex to combat oxidative stress by modulating cysB mRNA degradation.
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Enhancing autophagy with drugs or lung-directed gene therapy reverses the pathological effects of respiratory epithelial cell proteinopathy [Protein Synthesis and Degradation]

October 22nd, 2015 by

Recent studies have shown that autophagy mitigates the pathological effects of proteinopathies in the liver, heart and skeletal muscle but this has not been investigated for proteinopathies that affect the lung. This may be due at least in part to the lack of an animal model robust enough for spontaneous pathological effects from proteinopathies even though several rare proteinopathies, surfactant protein A and C deficiencies, cause severe pulmonary fibrosis. In this report we show that the PiZ mouse, transgenic for the common misfolded variant α1-antitrypsin Z (ATZ), is a model of respiratory epithelial cell proteinopathy with spontaneous pulmonary fibrosis. Intracellular accumulation of misfolded ATZ in respiratory epithelial cells of the PiZ model resulted in activation of autophagy, leukocyte infiltration and spontaneous pulmonary fibrosis severe enough to elicit functional restrictive deficits. Treatment with autophagy enhancer drugs or lung-directed gene transfer of TFEB, a master transcriptional activator of the autophagolysosomal system, reversed these proteotoxic consequences. We conclude that this mouse is an excellent model of respiratory epithelial proteinopathy with spontaneous pulmonary fibrosis, that autophagy is an important endogenous proteostasis mechanism and an attractive target for therapy.
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Store-Operated Ca2+ Entry Mediated by Orai1 and TRPC1 participates to Insulin Secretion in Rat Beta-cells [Cell Biology]

October 22nd, 2015 by

Store-Operated Ca2+ Channels (SOCs) are voltage-independent Ca2+ channels activated upon depletion of the endoplasmic reticulum (ER) Ca2+ stores. Early studies suggest the contribution of such channels to Ca2+ homeostasis in insulin-secreting pancreatic β-cells. However, their composition and contribution to glucose-stimulated insulin secretion (GSIS) remains unclear. In this study, ER Ca2+ depletion triggered by acetylcholine (ACh) or thapsigargin (Tg) stimulated the formation of a ternary complex composed of Orai1, TRPC1 and STIM1, the key proteins involved in the formation of SOCs. Ca2+ imaging further revealed that Orai1 and TRPC1 are required to form functional SOCs and that these channels are activated by STIM1 in response to Tg or ACh. Pharmacological SOCs inhibition or dominant-negative blockade of Orai1 or TRPC1 using the specific pore mutants Orai1-E106D or TRPC1-F562A impaired GSIS in rat β-cells and fully blocked the potentiating effect of ACh on secretion. In contrast, pharmacological or dominant-negative blockade of TRPC3 had no effect on extracellular Ca2+ entry and GSIS. Finally, we observed that prolonged exposure to supraphysiological glucose concentration impaired SOCs function without altering the expression levels of STIM1, Orai1 and TRPC1. We conclude that Orai1 and TRPC1, which form SOCs regulated by STIM1, play a key role in the effect of ACh on GSIS, a process which may be impaired in type 2 diabetes.

Lecithin:Cholesterol Acyltransferase (LCAT) Deficiency Promotes Differentiation of Satellite Cells to Brown Adipocytes in a Cholesterol-Dependent Manner [Lipids]

October 22nd, 2015 by Nesan, D., Tavallaee, G., Koh, D., Bashiri, A., Abdin, R., Ng, D. S.

Our lab previously reported that lecithin:cholesterol acyltransferase (LCAT) and LDL receptor double knockout mice (Ldlr-/-xLcat-/- or DKO) spontaneously develop functioning ectopic brown adipose tissue (BAT) in skeletal muscle, putatively contributing to the protection from diet-induced obesity phenotype. Here we further investigated their developmental origin and the mechanistic role of LCAT deficiency. Gene profiling of skeletal muscle in DKO newborns and adults revealed a classical lineage. Primary quiescent satellite cells (SC) from chow-fed DKO mice, not in Ldlr-/-xLcat+/+ single-knockout (SKO) or C57BL/6 wild type, were found to (i) express exclusively classical BAT-selective genes, (ii) be primed to express key functional BAT genes and (iii) exhibit markedly increased ex vivo adipogenic differentiation into brown adipocytes. This gene priming effect was abrogated upon feeding the mice a 2% high cholesterol diet (HCD) in association with accumulation of excess intracellular cholesterol. Ex vivo cholesterol loading of chow-fed DKO SC recapitulated the effect, indicating that cellular cholesterol is a key regulator of SC-to-BAT differentiation. Comparing adipogenicity of Ldlr+/+xLcat-/- (LCAT-KO) SC to those of DKO mice identified a specific role for LCAT deficiency in priming of SC to express BAT genes, and that cellular cholesterol depletion is important for adipogenic differentiation, as evidenced by comparable induction of adipogenesis by depleting cholesterol in SC from both LCAT-KO and SKO mice. Taken together, we conclude that ectopic BAT in DKO cells are classical in origin and their development begins in utero. We further showed complementary roles of LCAT deficiency and cellular cholesterol reduction in the SC-to-BAT adipogenesis.
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Structural and Functional highlights of Vacuolar Soluble Protein 1 from pathogen T. brucei. brucei [Microbiology]

October 22nd, 2015 by

Trypanosoma brucei (T. brucei) is responsible for the fatal human disease called African trypanosomiasis or sleeping sickness. The causative parasite Trypanosoma encodes soluble versions of inorganic pyrophosphatases (PPase), also called vacuolar soluble proteins (VSPs), which are localized to its acidocalcisomes. Latter are acidic membrane enclosed organelles rich in polyphosphate chains and divalent cations whose significance in these parasites remains unclear. We here report the crystal structure of T. brucei brucei acidocalcisomal PPases in a ternary complex with Mg2+ and imidodiphosphate. The crystal structure reveals a novel structural architecture distinct from known class I PPases in its tetrameric oligomeric state in which a fused EF-hand domain arranges around the catalytic PPase domain. This unprecedented assembly evident from TbbVSP1 crystal structure is further confirmed by small angle X-ray scattering (SAXS) and electron microscopy data. Solution scattering data suggests structural flexibility in EF-hand domains indicative of conformational plasticity within TbbVSP1.

The {beta}5-loop and lid-domain contribute to the substrate specificity of pancreatic lipase related protein 2 (PNLIPRP2) [Enzymology]

October 21st, 2015 by Xiao, X., Lowe, M. E.

Pancreatic triglyceride lipase (PNLIP) is essential for dietary fat digestion in children and adults whereas a homologue, pancreatic lipase related protein 2 (PNLIPRP2), is critical in newborns. The two lipases are structurally similar yet they have different substrate specificities. PNLIP only cleaves neutral fats. PNLIP cleaves neutral and polar fats. To test the hypothesis that the differences in activity between PNLIP and PNLIPRP2 are governed by surface loops around the active site, we created multiple chimeras of both lipases by exchanging the surface loops singly or in combination. The chimeras were expressed, purified and tested for activity against various substrates. The structural determinants of PNLIPRP2 galactolipase activity were contained in the N-terminal domain. Of the lid-domain, β5- and β9-loops, the lid-domain and the β5-loop influenced activity against triglycerides and galactolipids. Any chimera on PNLIP with the PNLIPRP2 lid-domain or β5-loop had decreased triglyceride lipase activity similar to that of PNLIPRP2. The corresponding chimeras of PNLIPRP2 did not increase activity against neutral lipids. Galactolipase activity was abolished by the PNLIP β5-loop and decreased by the PNLIP lid-domain. The source of the β9-loop had minimal effect on activity. We conclude that the lid-domain and β5-loop contribute to substrate specificity but do not completely account for the differing activities of PNLIP and PNLIPRP2. Other regions in the N-terminal domain must contribute to the galactolipase activity of PNLIPRP2 through direct interactions with the substrate or by altering the conformation of the residues surrounding the hydrophilic cavity in PNLIPRP2.

Evaluating the use of antibody variable region (Fv) charge as a risk assessment tool for predicting typical cynomolgus monkey pharmacokinetics [Protein Structure and Folding]

October 21st, 2015 by

The pharmacokinetic (PK) behavior of monoclonal antibodies in cynomolgus monkeys (cynos) is generally translatable to that in humans. Unfortunately, about 39% of the antibodies evaluated for PK in cynos have fast non-specific (or non-target mediated) clearance (in-house data). An empirical model relating variable region (Fv) charge and hydrophobicity to cyno non-specific clearance was developed to gauge the risk an antibody would have for fast non-specific clearance in the monkey. The purpose of this study was to evaluate the predictability of this empirical model on cyno non-specific clearance with antibodies specifically engineered to have either high or low Fv charge. These amino acid changes were made in the Fv region of two test antibodies, humAb4D5-8 and anti-LTα. HumAb4D5-8 has a typical non-specific clearance in cynos and by making it more positively charged, the antibody acquires fast non-specific clearance, while making it less positively charged did not impact its clearance. Anti-LTα has fast non-specific clearance in cynos and making it more positively charged caused it to clear even faster, while making it less positively charged caused it to clear slower and within the typical range. These trends in clearance were also observed in two other preclinical species, mice and rats. The effect of modifying Fv charge on subcutaneous bioavailability was also examined and in general bioavailability was inversely related to the direction of Fv charge change. Thus, modifying Fv charge appears to impact antibody PK and the changes tended to correlate with those predicted by the empirical model.
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