Structure and Mechanistic Insights into Novel Iron-mediated Moonlighting Functions of Human J-protein Cochaperone, Dph4

J-proteins are obligate cochaperones of Hsp70s and stimulate their ATPase activity via the J-domain. Although the functions of J-proteins have been well understood in the context of Hsp70s, their additional co-evolved ``physiological functions'' are still elusive. We report here the solution structure and mechanism of novel iron-mediated functional roles of human Dph4, a type III J-protein playing a vital role in diphthamide biosynthesis and normal development. The NMR structure of Dph4 reveals two domains: a conserved J-domain and a CSL-domain connected via a flexible linker-helix. The linker-helix modulates the conformational flexibility between the two domains, regulating thereby the protein function. Dph4 exhibits a unique ability to bind iron in tetrahedral coordination geometry through cysteines of its CSL-domain. The oxidized Fe-Dph4 shows characteristic UV-visible and electron paramagnetic resonance spectral properties similar to rubredoxins. Iron-bound Dph4 (Fe-Dph4) also undergoes oligomerization, thus potentially functioning as a transient ``iron storage protein,'' thereby regulating the intracellular iron homeostasis. Remarkably, Fe-Dph4 exhibits vital redox and electron carrier activity, which is critical for important metabolic reactions, including diphthamide biosynthesis. Further, we observed that Fe-Dph4 is conformationally better poised to perform Hsp70-dependent functions, thus underlining the significance of iron binding in Dph4. Yeast Jjj3, a functional ortholog of human Dph4 also shows a similar iron-binding property, indicating the conserved nature of iron sequestration across species. Taken together, our findings provide invaluable evidence in favor of additional co-evolved specialized functions of J-proteins, previously not well appreciated.

Autophosphorylation of Ser(428) of EhC2PK Plays a Critical Role in Regulating Erythrophagocytosis in the Parasite Entamoeba histolytica

The protozoan parasite Entamoeba histolytica can invade both intestinal and extra intestinal tissues resulting in amoebiasis. During the process of invasion E. histolytica ingests red blood and host cells using phagocytic processes. Though phagocytosis is considered to be a key virulence determinant, the mechanism is not very well understood in E. histolytica. We have recently demonstrated that a novel C2 domain-containing protein kinase, EhC2PK is involved in the initiation of erythrophagocytosis. Because cells overexpressing the kinase-dead mutant of EhC2PK displayed a reduction in erythrophagocytosis, it appears that kinase activity is necessary for initiation. Biochemical analysis showed that EhC2PK is an unusual Mn2+-dependent serine kinase. It has a trans-autophosphorylated site at Ser(428) as revealed by mass spectrometric and biochemical analysis. The autophosphorylation defective mutants (S428A, KD Delta C) showed a reduction in auto and substrate phosphorylation. Time kinetics of in vitro kinase activity suggested two phases, an initial short slow phase followed by a rapid phase for wild type protein, whereas mutations in the autophosphorylation sites that cause defect (S428A) or conferred phosphomimetic property (S428E) displayed no distinct phases, suggesting that autophosphorylation may be controlling kinase activity through an autocatalytic mechanism. A reduction and delay in erythrophagocytosis was observed in E. histolytica cells overexpressing S428A and KD Delta C proteins. These results indicate that enrichment of EhC2PK at the site of phagocytosis enhances the rate of trans-autophosphorylation, thereby increasing kinase activity and regulating the initiation of erythrophagocytosis in E. histolytica.

Cyclic AMP-induced Conformational Changes in Mycobacterial Protein Acetyltransferases

The activities of a number of proteins are regulated by the binding of cAMP and cGMP to cyclic nucleotide binding (CNB) domains that are found associated with one or more effector domains with diverse functions. Although the conserved architecture of CNB domains has been extensively studied by x-ray crystallography, the key to unraveling the mechanisms of cAMP action has been protein dynamics analyses. Recently, we have identified a novel cAMP-binding protein from mycobacteria, where cAMP regulates the activity of an associated protein acetyltransferase domain. In the current study, we have monitored the conformational changes that occur upon cAMP binding to the CNB domain in these proteins, using a combination of bioluminescence resonance energy transfer and amide hydrogen/deuterium exchange mass spectrometry. Coupled with mutational analyses, our studies reveal the critical role of the linker region (positioned between the CNB domain and the acetyltransferase domain) in allosteric coupling of cAMP binding to activation of acetyltransferase catalysis. Importantly, major differences in conformational change upon cAMP binding were accompanied by stabilization of the CNB and linker domain alone. This is in contrast to other cAMP-binding proteins, where cyclic nucleotide binding has been shown to involve intricate and parallel allosteric relays. Finally, this powerful convergence of results from bioluminescence resonance energy transfer and hydrogen/deuterium exchange mass spectrometry reaffirms the power of solution biophysical tools in unraveling mechanistic bases of regulation of proteins in the absence of high resolution structural information.

A full-length bifunctional protein involved in c-di-GMP turnover is required for long-term survival under nutrient starvation in Mycobacterium smegmatis

The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) plays an important role in a variety of cellular functions, including biofilm formation, alterations in the cell surface, host colonization and regulation of bacterial flagellar motility, which enable bacteria to survive changing environmental conditions. The cellular level of c-di-GMP is regulated by a balance between opposing activities of diguanylate cyclases (DGCs) and cognate phosphodiesterases (PDE-As). Here, we report the presence and importance of a protein, MSDGC-1 (an orthologue of Rv1354c in Mycobacterium tuberculosis), involved in c-di-GMP turnover in Mycobacterium smegmatis. MSDGC-1 is a multidomain protein, having GAF, GGDEF and EAL domains arranged in tandem, and exhibits both c-di-GMP synthesis and degradation activities. Most other proteins containing GGDEF and EAL domains have been demonstrated to have either DGC or PDE-A activity. Unlike other bacteria, which harbour several copies of the protein involved in c-di-GMP turnover, M. smegmatis has a single genomic copy, deletion of which severely affects long-term survival under conditions of nutrient starvation. Overexpression of MSDGC-1 alters the colony morphology and growth profile of M. smegmatis. In order to gain insights into the regulation of the c-di-GMP level, we cloned individual domains and tested their activities. We observed a loss of activity in the separated domains, indicating the importance of full-length MSDGC-1 for controlling bifunctionality.

Crystal Structure of a Monomeric Thiolase-Like Protein Type 1 (TLP1) from Mycobacterium smegmatis

An analysis of the Mycobacterium smegmatis genome suggests that it codes for several thiolases and thiolase-like proteins. Thiolases are an important family of enzymes that are involved in fatty acid metabolism. They occur as either dimers or tetramers. Thiolases catalyze the Claisen condensation of two acetyl-Coenzyme A molecules in the synthetic direction and the thiolytic cleavage of 3-ketoacyl-Coenzyme A molecules in the degradative direction. Some of the M. smegmatis genes have been annotated as thiolases of the poorly characterized SCP2-thiolase subfamily. The mammalian SCP2-thiolase consists of an N-terminal thiolase domain followed by an additional C-terminal domain called sterol carrier protein-2 or SCP2. The M. smegmatis protein selected in the present study, referred to here as the thiolase-like protein type 1 (MsTLP1), has been biochemically and structurally characterized. Unlike classical thiolases, MsTLP1 is a monomer in solution. Its structure has been determined at 2.7 angstrom resolution by the single wavelength anomalous dispersion method. The structure of the protomer confirms that the N-terminal domain has the thiolase fold. An extra C-terminal domain is indeed observed. Interestingly, it consists of six beta-strands forming an anti-parallel beta-barrel which is completely different from the expected SCP2-fold. Detailed sequence and structural comparisons with thiolases show that the residues known to be essential for catalysis are not conserved in MsTLP1. Consistent with this observation, activity measurements show that MsTLP1 does not catalyze the thiolase reaction. This is the first structural report of a monomeric thiolase-like protein from any organism. These studies show that MsTLP1 belongs to a new group of thiolase related proteins of unknown function.

Lipopeptides from the Banyan Endophyte, Bacillus subtilis K1: Mass Spectrometric Characterization of a Library of Fengycins

Mass spectrometric analysis of a banyan endophyte, Bacillus subtilis K1, extract showing broad spectrum antifungal activity revealed a complex mixture of lipopeptides, iturins, surfactins, and fengycins. Fractionation by reversed-phase high performance liquid chromatography (HPLC) facilitated a detailed analysis of fengycin microheterogeneity. Matrix assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) mass spectrometric studies permitted the identification of several new fengycin variants. Four major sites of heterogeneity are identified: (1) N-terminus beta-hydroxy fatty acid moiety, where chain length variation and the presence of unsaturation occur, (2) position 6 (Ala/Val/Ile/Leu), (3) position 10 (Val/Ile) within the macrocyclic ring, and (4) Gln to Glu replacement at position 8, resulting in fengycin variants that differ in mass by 1 Da. Diagnostic fragment ions provide a quick method for localizing the sites of variation in the macrocycle or the linear segment. Subsequent establishment of the sequences is achieved by MS/MS analysis of linear fengycin species produced by hydrolysis of the macrocyclic lactone. Unsaturation in the fatty acid chain and the presence of linear precursors in the B. subtilis K1 extract are also established by mass spectrometry. The anomalous distribution of intensities within isotopic multiplets is a diagnostic for Gln/Glu replacements. High resolution mass spectrometry facilitates the identification of fengycin species differing by 1 Da by localizing the variable position (Gln(8)/Glu(8)) in the fengycin variants.

Novel ATPase activity of the polyprotein intermediate, Viral Protein genome-linked-Nuclear Inclusion-a protease, of Pepper vein banding potyvirus

Potyviruses temporally regulate their protein function by polyprotein processing. Previous studies have shown that VPg (Viral Protein genome-linked) of Pepper vein banding virus interacts with the NIa-Pro (Nuclear Inclusion-a protease) domain, and modulates the kinetics of the protease. In the present study, we report for the first time that VPg harbors the Walker motifs A and B, and the presence of NIa-Pro, especially in cis (cleavage site (E191A) VPg-Pro mutant), is essential for manifestation of the ATPase activity. Mutation of Lys47 (Walker motif A) and Asp88:Glu89 (Walker motif B) to alanine in E191A VPg-Pro lead to reduced ATPase activity, confirming that this activity was inherent to VPg. We propose that potyviral VPg, established as an intrinsically disordered domain, undergoes plausible structural alterations upon interaction with globular NIa-Pro which induces the ATPase activity. (C) 2012 Elsevier Inc. All rights reserved.

Mycobacterium tuberculosis RsdA provides a conformational rationale for selective regulation of sigma-factor activity by proteolysis

The relative levels of different sigma factors dictate the expression profile of a bacterium. Extracytoplasmic function sigma factors synchronize the transcriptional profile with environmental conditions. The cellular concentration of free extracytoplasmic function sigma factors is regulated by the localization of this protein in a sigma/anti-sigma complex. Anti-sigma factors are multi-domain proteins with a receptor to sense environmental stimuli and a conserved anti-sigma domain (ASD) that binds a sigma factor. Here we describe the structure of Mycobacterium tuberculosis anti-sigma(D) (RsdA) in complex with the -35 promoter binding domain of sigma(D) (sigma(D)(4)). We note distinct conformational features that enable the release of sigma(D) by the selective proteolysis of the ASD in RsdA. The structural and biochemical features of the sigma(D)/RsdA complex provide a basis to reconcile diverse regulatory mechanisms that govern sigma/anti-sigma interactions despite high overall structural similarity. Multiple regulatory mechanisms embedded in an ASD scaffold thus provide an elegant route to rapidly re-engineer the expression profile of a bacterium in response to an environmental stimulus.

Cyclic AMP-dependent protein lysine acylation in mycobacteria regulates fatty acid and propionate metabolism

Acetylation of lysine residues is a posttranslational modification that is used by both eukaryotes and prokaryotes to regulate a variety of biological processes. Here we identify multiple substrates for the cAMP-dependent protein lysine acetyltransferase from Mycobacterium tuberculosis (KATmt). We demonstrate that a catalytically important lysine residue in a number of FadD (fatty acyl CoA synthetase) enzymes is acetylated by KATmt in a cAMP-dependent manner and that acetylation inhibits the activity of FadD enzymes. A sirtuin-like enzyme can deacetylate multiple FadDs, thus completing the regulatory cycle. Using a strain deleted for the KATmt ortholog in Mycobacterium bovis Bacillus Calmette-Guerin (BCG), we show for the first time that acetylation is dependent on intracellular cAMP levels. KATmt can utilize propionyl CoA as a substrate and, therefore, plays a critical role in alleviating propionyl CoA toxicity in mycobacteria by inactivating acyl CoA synthetase (ACS). The precision by which mycobacteria can regulate the metabolism of fatty acids in a cAMP-dependent manner appears to be unparalleled in other biological organisms and is ideally suited to adapt to the complex environment that pathogenic mycobacteria experience in the host.

Antitumor and antioxidant status of Terminalia catappa against Ehrlich ascites carcinoma in Swiss albino mice

Objective: The present study was undertaken to evaluate the antitumor and antioxidant status of ethanol extract of Terminalia catappa leaves against Ehrlich ascites carcinoma (EAC) in Swiss albino mice. Materials and Methods: The leaves powder was extracted with Soxhlet apparatus and subjected to hot continuous percolation using ethanol (95% v/v). Tumor bearing animals was treated with 50 and 200 mg/kg of ethanol extract. EAC induced in mice by intraperitoneal injection of EAC cells 1 x 10(6) cells/mice. The study was assed using life span of EAC-bearing hosts, hematological parameters, volume of solid tumor mass and status of antioxidant enzymes such as lipid peroxidation (LPO), reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) activities. Total phenolics and flavonoids contents from the leaves extract were also determined. Results: Total phenolics and flavonoids contents from the leaves extract were found 354.02 and 51.67 mg/g extract. Oral administration of ethanol extract of T. catappa (50 and 200 mg/kg) increased the life span (27.82% and 60.59%), increased peritoneal cell count (8.85 +/- 0.20 and 10.37 +/- 0.26) and significantly decreased solid tumor mass (1.16 +/- 0.14 cm(2)) at 200 mg/kg as compared with EAC-tumor bearing mice (P < 0.01). Hematological profile including red blood cell count, white blood cell count, hemoglobin (11.91 +/- 0.47 % g) and protein estimation were found to be nearly normal levels in extract-treated mice compared with tumor bearing control mice. Treatment with T. catappa significantly decreased levels of LPO and GSH, and increased levels of SOD and CAT activity (P < 0.01). Conclusion: T. catappa exhibited antitumor effect by modulating LPO and augmenting antioxidant defense systems in EAC bearing mice. The phenolic and flavonoid components in this extract may be responsible for antitumor activity.

Modulation of redox regulatory molecules and electron transport chain activity in muscle of air breathing fish Heteropneustes fossilis under air exposure stress

Responses of redox regulatory system to long-term survival (> 18 h) of the catfish Heteropneustes fossilis in air are not yet understood. Lipid and protein oxidation level, oxidant (H2O2) generation, antioxidative status (levels of superoxide dismutase, catalase, glutathione peroxidase and reductase, ascorbic acid and non-protein sulfhydryl) and activities of respiratory complexes (I, II, III and IV) in mitochondria were investigated in muscle of H. fossilis under air exposure condition (0, 3, 6, 12 and 18 h at 25 A degrees C). The increased levels of both H2O2 and tissue oxidation were observed due to the decreased activities of antioxidant enzymes in muscle under water deprivation condition. However, ascorbic acid and non-protein thiol groups were the highest at 18 h air exposure time. A linear increase in complex II activity with air exposure time and an increase up to 12 h followed by a decrease in activity of complex I at 18 h were observed. Negative correlation was observed for complex III and V activity with exposure time. Critical time to modulate the above parameters was found to be 3 h air exposure. Dehydration induced oxidative stress due to modulation of electron transport chain and redox metabolizing enzymes in muscle of H. fossilis was clearly observed. Possible contribution of redox regulatory system in muscle tissue of the fish for long-term survival in air is elucidated. Results of the present study may be useful to understand the redox metabolism in muscle of fishes those are exposed to air in general and air breathing fishes in particular.

Modulation of glyceraldehyde-3-phosphate dehydrogenase activity by surface functionalized quantum dots

Enzymatic regulation is a fast and reliable diagnosis tool via identification and design of inhibitors for modulation of enzyme function. Previous reports on quantum dots (QDs)-enzyme interactions reveal a protein-surface recognition ability leading to promising applications in protein stabilization, protein delivery, bio-sensing and detection. However, the direct use of QDs to control enzyme inhibition has never been revealed to date. Here we show that a series of biocompatible surface-functionalized metal-chalcogenide QDs can be used as potent inhibitors for malignant cells through the modulation of enzyme activity, while normal cells remain unaffected. The in vitro activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an enzyme involved critically in the glycolysis of cancer cells, is inactivated selectively in a controlled way by the QDs at a significantly low concentration (nM). Cumulative kinetic studies delineate that the QDs undergo both reversible and irreversible inhibition mechanisms owing to the site-specific interactions, enabling control over the inhibition kinetics. These complementary loss-of-function probes may offer a novel route for rapid clinical diagnosis of malignant cells and biomedical applications.

A Novel C-Terminal Homologue of Aha1 Co-Chaperone Binds to Heat Shock Protein 90 and Stimulates Its ATPase Activity in Entamoeba histolytica

Cytosolic heat shock protein 90 (Hsp90) has been shown to be essential for many infectious pathogens and is considered a potential target for drug development. In this study, we have carried out biochemical characterization of Hsp90 from a poorly studied protozoan parasite of clinical importance, Entamoeba histolytica. We have shown that Entamoeba Hsp90 can bind to both ATP and its pharmacological inhibitor, 17-AAG (17-allylamino-17-demethoxygeldanamycin), with K-d values of 365.2 and 10.77 mu M, respectively, and it has a weak ATPase activity with a catalytic efficiency of 4.12 x 10(-4) min(-1) mu M-1. Using inhibitor 17-AAG, we have shown dependence of Entamoeba on Hsp90 for its growth and survival. Hsp90 function is regulated by various co-chaperones. Previous studies suggest a lack of several important co-chaperones in E. histolytica. In this study, we describe the presence of a novel homologue of co-chaperone Aha1 (activator of Hsp90 ATPase), EhAha1c, lacking a canonical Aha1 N-terminal domain. We also show that EhAha1c is capable of binding and stimulating ATPase activity of EhHsp90. In addition to highlighting the potential of Hsp90 inhibitors as drugs against amoebiasis, our study highlights the importance of E. histolytica in understanding the evolution of Hsp90 and its co-chaperone repertoire. (C) 2014 Elsevier Ltd. All rights reserved.

Residue specific contributions to stability and activity inferred from saturation mutagenesis and deep sequencing

Multiple methods currently exist for rapid construction and screening of single-site saturation mutagenesis (SSM) libraries in which every codon or nucleotide in a DNA fragment is individually randomized. Nucleotide sequences of each library member before and after screening or selection can be obtained through deep sequencing. The relative enrichment of each mutant at each position provides information on its contribution to protein activity or ligand-binding under the conditions of the screen. Such saturation scans have been applied to diverse proteins to delineate hot-spot residues, stability determinants, and for comprehensive fitness estimates. The data have been used to design proteins with enhanced stability, activity and altered specificity relative to wild-type, to test computational predictions of binding affinity, and for protein model discrimination. Future improvements in deep sequencing read lengths and accuracy should allow comprehensive studies of epistatic effects, of combinational variation at multiple sites, and identification of spatially proximate residues.

Bi-Domain Protein Tyrosine Phosphatases Reveal an Evolutionary Adaptation to Optimize Signal Transduction

Significance: The bi-domain protein tyrosine phosphatases (PTPs) exemplify functional evolution in signaling proteins for optimal spatiotemporal signal transduction. Bi-domain PTPs are products of gene duplication. The catalytic activity, however, is often localized to one PTP domain. The inactive PTP domain adopts multiple functional roles. These include modulation of catalytic activity, substrate specificity, and stability of the bi-domain enzyme. In some cases, the inactive PTP domain is a receptor for redox stimuli. Since multiple bi-domain PTPs are concurrently active in related cellular pathways, a stringent regulatory mechanism and selective cross-talk is essential to ensure fidelity in signal transduction. Recent Advances: The inactive PTP domain is an activator for the catalytic PTP domain in some cases, whereas it reduces catalytic activity in other bi-domain PTPs. The relative orientation of the two domains provides a conformational rationale for this regulatory mechanism. Recent structural and biochemical data reveal that these PTP domains participate in substrate recruitment. The inactive PTP domain has also been demonstrated to undergo substantial conformational rearrangement and oligomerization under oxidative stress. Critical Issues and Future Directions: The role of the inactive PTP domain in coupling environmental stimuli with catalytic activity needs to be further examined. Another aspect that merits attention is the role of this domain in substrate recruitment. These aspects have been poorly characterized in vivo. These lacunae currently restrict our understanding of neo-functionalization of the inactive PTP domain in the bi-domain enzyme. It appears likely that more data from these research themes could form the basis for understanding the fidelity in intracellular signal transduction.