Structural and Functional Effects of Cytochrome b5 Interactions with Human Cytochrome P450 Enzymes [Enzymology]

October 27th, 2017 by Aaron G. Bart, Emily E. Scott

The small heme-containing protein cytochrome b5 can facilitate, inhibit, or have no effect on cytochrome P450 catalysis, often in a P450-dependent and substrate-dependent manner that is not well understood. Herein solution NMR was used to identify b5 residues interacting with different human drug-metabolizing P450 enzymes. NMR results revealed that P450 enzymes bound to either b5 α4-5 (CYP2A6 and CYP2E1) or this region and α2-3 (CYP2D6 and CYP3A4) and suggested variation in the affinity for b5. Mutations of key b5 residues suggest that not only are different b5 surfaces responsible for binding different P450 enzymes, but that these different complexes are relevant to the observed effects on P450 catalysis.

Structural analyses of the bacterial primosomal protein DnaB reveal that it is a tetramer and forms a complex with a primosomal re-initiation protein [Protein Structure and Folding]

August 14th, 2017 by Yi-Ching Li, Vankadari Naveen, Min-Guan Lin, Chwan-Deng Hsiao

The DnaB primosomal protein from Gram-positive bacteria plays a key role in DNA replication and restart as a loader protein for the recruitment of replisome cascade proteins. Previous investigations have established that DnaB is composed of an N-terminal domain, a middle domain, and a C-terminal domain. However, structural evidence for how DnaB functions at the atomic level is lacking. Here, we report the crystal structure of DnaB, encompassing the N-terminal and middle domains (residues 1-300), from Geobacillus stearothermophilus (GstDnaB1-300) at 2.8 Å resolution. Our structure revealed that GstDnaB1-300 forms a tetramer with two basket-like architecture, a finding s consistent with those from solution studies using analytical ultracentrifugation. Furthermore, our results from both GST pull-down assays and analytical ultracentrifugation show that GstDnaB1-300 is sufficient to form a complex with PriA, the primosomal re-initiation protein. Moreover, with the aid of small angle X-ray scattering (SAXS) experiments, we also determined the structural envelope of full-length DnaB (GstDnaBFL) in solution. These SAXS studies indicated that GstDnaBFL has an elongated conformation and that the protruding density envelopes originating from GstDnaB1-300 could completely accommodate the GstDnaB C-terminal domain (residues 301-461) . Taken together with biochemical assays, our results suggest that GstDnaB uses different domains to distinguish the PriA-interaction and ssDNA-binding. This finding can further extend our understanding of primosomal assembly in replication restart.
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Efficient reduction of CO2 by the molybdenum-containing formate dehydrogenase from Cupriavidus necator (Ralstonia eutropha). [Molecular Biophysics]

August 7th, 2017 by Xuejun Yu, Dimitri Niks, Ashok Mulchandani, Russ Hille

The ability of the FdsABG formate dehydrogenase from Cupriavidus necator (formerly known as Ralstonia eutropha) to catalyze the reverse of the physiological reaction, the reduction of CO2 to formate utilizing NADH as electron donor, has been investigated. Contrary to previous studies of this enzyme, we demonstrate that it is in fact effective in catalyzing the reverse reaction, with a kcat of 11 ± 0.4 s-1. We also quantify the stoichiometric accumulation of formic acid as the product of the reaction and demonstrate that the observed kinetic parameters for catalysis in the forward and reverse reaction are thermodynamically consistent, complying with the expected Haldane relationships. Finally, we demonstrate the reaction conditions necessary for gauging the ability of a given formate dehydrogenase or other CO2-utilizing enzyme to catalyze the reverse direction so as to avoid false negative results. In conjunction with our earlier studies on the reaction mechanism of this enzyme (Niks et al. (2016) J. Biol. Chem. 291, 1162- 1174), and on the basis of the present work we conclude that all molybdenum- and tungsten-containing formate dehydrogenases and related enzymes likely operate via a simple hydride transfer mechanism and are effective in catalysing the reversible interconversion of CO2 and formate under the appropriate experimental conditions.
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Palmitoylation is a prerequisite for dimerization-dependent raftophilicity of rhodopsin [Membrane Biology]

July 26th, 2017 by Keiji Seno, Fumio Hayashi

The visual photopigment rhodopsin (Rh) is a prototypical G protein-coupled receptor (GPCR) responsible for initiation of the phototransduction cascade in rod photoreceptors. Similar to other GPCRs, Rh can form dimers or even higher oligomers, and tends to have a supramolecular organization that is likely important in the dim light response. Rh also exhibits high affinity for lipid rafts (raftophilicity) upon light-dependent binding with cognate G protein transducin (Gt), suggesting the presence of lipid raft-like domains in the retinal disk membrane and their importance in phototransduction. However, the relationship between Rh oligomerization and lipid rafts in the disk membrane remains to be explored. Given previous findings that Gt binds to dimeric Rh and Rh is post-translationally modified with two highly raftophilic palmitoyl moieties, we hypothesized that Rh becomes raftophilic upon dimerization. Here, we tested this hypothesis biochemically. First, we found that Rh*-Gt complexes in the detergent-resistant membrane (DRM) are partially resistant to cholesterol depletion by methyl-β-cyclodextrin (MCD), and the stoichiometry of Rh to Gt in this MCD-resistant complex is 2:1. We then found that IgG-crosslinking renders Rh highly raftophilic, supporting the premise that Rh becomes raftophilic upon dimerization. Depalmitoylation of Rh with the reduction of thioester linkages by dithiothreitol blocked the translocation of IgG-crosslinked Rh to the DRM, highlighting the importance of the two palmitoyl moieties in the dimerization-dependent raftophilicity of Rh. These results indicate that palmitoylated GPCRs, such as Rh, may acquire raftophilicity upon G protein-stabilized dimerization and thereby organize receptor-cluster rafts by recruiting raftophilic lipids.

Iron Transport Proteins: Gateways of Cellular and Systemic Iron Homeostasis [Cell Biology]

June 16th, 2017 by Mitchell D. Knutson

Cellular iron homeostasis is maintained by iron and heme transport proteins that work in concert with ferrireductases, ferroxidases, and chaperones to direct the movement of iron into, within, and out of cells. Systemic iron homeostasis is regulated by the liver-derived peptide hormone, hepcidin. The interface between cellular and systemic iron homeostasis is readily observed in the highly dynamic iron handling of four main cell types: duodenal enterocytes, erythrocyte precursors, macrophages, and hepatocytes. This review provides an overview of how these cell types handle iron, highlighting how iron and heme transporters mediate the exchange and distribution of body iron in health and disease.

A ciliary opsin in the brain of a marine annelid zooplankton is UV-sensitive and the sensitivity is tuned by a single amino acid residue [Molecular Biophysics]

June 16th, 2017 by Hisao Tsukamoto, I-Shan Chen, Yoshihiro Kubo, Yuji Furutani

Ciliary opsins were classically thought to function only in vertebrates for vision, but they have been recently identified also in invertebrates for non-visual photoreception. Larvae of the annelid Platynereis dumerilii are used as a zooplankton model, and this zooplankton species possesses a "vertebrate-type" ciliary opsin (named c-opsin) in the brain. Platynereis c-opsin is suggested to relay light signals to melatonin production and circadian behaviors. Thus, the spectral and biochemical characteristics of this c-opsin would be directly related to non-visual photoreception in this zooplankton model. Here, we demonstrate that the c-opsin can sense UV to activate intracellular signaling cascades, and that it can directly bind exogenous all-trans-retinal. These results suggest that this c-opsin regulates circadian signaling in a UV-dependent manner and that it does not require supply of 11-cis-retinal for photoreception. Avoidance of damaging UV irradiation is a major cause of a large-scale daily zooplankton movement, and the observed capability of the c-opsin to transmit UV signals and bind all-trans-retinal is ideally suited for sensing UV radiation in the brain, which presumably lacks enzymes producing 11-cis-retinal. Mutagenesis analyses indicated that a unique amino acid residue (Lys-94) is responsible for c-opsin-mediated UV sensing in the Platynereis brain. We therefore propose that acquisition of the lysine residue in the c-opsin would be a critical event in the evolution of Platynereis to enable detection of ambient UV. In summary, our findings indicate that the c-opsin possesses spectral and biochemical properties suitable for UV sensing by the zooplankton model.
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Endoplasmic Reticulum Stress-induced Degradation of DNAJB12 Stimulates BOK Accumulation and Primes Cancer Cells for Apoptosis [Cell Biology]

May 23rd, 2017 by Pattarawut Sopha, Hong Yu Ren, Diane E. Grove, Douglas M Cyr

DNAJB12 (JB12) is an endoplasmic reticulum (ER)-associated Hsp40 family protein that recruits Hsp70 to the ER surface to coordinate the function of ER-associated and cytosolic chaperone systems in protein quality control. Hsp70 is stress inducible, but paradoxically, we report here that JB12 was degraded by the proteasome during severe ER stress. Destabilized JB12 was degraded by ER-associated degradation (ERAD) complexes that contained HERP, Sel1L, and gp78. JB12 was the only ER-associated chaperone that was destabilized by reductive stress. JB12 knockdown by siRNA led to the induction of Caspase processing, but not the unfolded protein response. ER stress-induced apoptosis is regulated by the highly labile and ER associated BCL-2 family member BOK, which is controlled at the level of protein stability by ERAD components. We found that JB12 was required in Huh-7 liver cancer cells to maintain BOK at low levels and BOK was detected in complexes with JB12 and gp78. Depletion of JB12 during reductive stress or by shRNA from Huh-7 cells was associated with accumulation of BOK, and activation of Caspase 3, 7, and 9. Absence of JB12 sensitized Huh-7 to death caused by proteotoxic agents and the proapoptotic chemotherapeutic LCL-161. In summary, JB12 is a stress sensitive Hsp40 whose degradation during severe ER stress provides a mechanism to promote BOK accumulation and induction of apoptosis.

The sialate O-acetylesterase EstA from gut Bacteroidetes species enables sialidase-mediated cross-species foraging of 9-O-acetylated sialoglycans [Microbiology]

May 19th, 2017 by Lloyd S Robinson, Warren G Lewis, Amanda L Lewis

The gut harbors many symbiotic, commensal, and pathogenic microbes that engage in the breakdown and metabolism of host carbohydrates. Sialic acids are prominent outermost carbohydrates on mucins and protect underlying glycan chains from enzymatic degradation. Sialidases produced by some members of the colonic microbiota have been shown to promote the expansion of several potential pathogens (e.g. Clostridium difficile, Salmonella, Escherichia coli) that do not produce sialidases. O-acetyl ester modifications of sialic acids help resist the action of many sialidases and are found at high levels in the mammalian colon. However, some gut bacteria, in turn, produce sialylate-O-acetyl esterases to remove them. Here we investigated O-acetylation as a shield against the release of sialic acids by Bacteroidetes sialidases and the subsequent utilization of host sialic acids by commensal and pathogenic strains of E. coli. In vitro foraging studies demonstrated that sialidase-dependent E. coli outgrowth on mucin is enabled by Bacteroides EstA, which acts on glycosidically-linked sialylate-O-acety-esterase substrates, particularly at neutral pH. Biochemical studies suggest that spontaneous migration of O-acetyl esters on the side chain of sialic acid, which can occur at colonic pH, may serve as a switch controlling EstA-assisted sialic acid liberation. Specifically, EstA does not act on O-acetyl esters in their initial 7-position. But, following migration to the 9-position, glycans with O-acetyl esters become susceptible to the sequential enzyme action of bacterial esterases and sialidases. Thus, EstA specifically unlocks the nutritive potential of 9-O-acetylated mucus sialic acids for foraging by bacteria that otherwise lack the means to access this potential carbon source.
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Functional regions of the peroxin Pex19 necessary for peroxisome biogenesis [Membrane Biology]

May 19th, 2017 by Gaurav Agrawal, Helen H. Shang, Zhi-Jie Xia, Suresh Subramani

The peroxins Pex19 and Pex3 play an indispensable role in peroxisomal membrane protein (PMP) biogenesis, peroxisome division and inheritance. Pex19 plays multiple roles in these processes, but how these functions relate to the structural organization of the Pex19 domains is unresolved. To this end, using deletion mutants, we mapped the Pex19 regions required for peroxisome biogenesis in the yeast Pichia pastoris. Surprisingly, import-competent peroxisomes still formed when Pex19 domains previously believed to be required for biogenesis were deleted, although the peroxisome size was larger than that in wild-type cells. Moreover, these mutants exhibited a delay of 14-24 h in peroxisome biogenesis. The shortest functional N-terminal (NTCs) and C-terminal constructs (CTCs) were Pex19 (aa 1-150) and Pex19 (aa 89-300), respectively. Deletions of the N-terminal Pex3-binding site disrupted direct interactions of Pex19 with Pex3, but preserved interactions with a membrane peroxisomal targeting signal (mPTS)-binding PMP, Pex10. In contrast, deletion of the C-terminal mPTS-binding domain of Pex19 disrupted its interaction with Pex10, while leaving the Pex19-Pex3 interactions intact. However, Pex11 and Pex25 retained their interactions with both N- and C-terminal deletion mutants. NTC-CTC co-expression improved growth and reverted the larger-than-normal peroxisome size observed with the single deletions. Pex25 was critical for peroxisome formation with the CTC variants, and its overexpression enhanced their interactions with Pex3 and aided the growth of both NTC and CTC Pex19 variants. In conclusion, physical segregation of the Pex3 and PMP-binding domains of Pex19 has provided novel insights into the modular architecture of Pex19. We define the minimum region of Pex19 required for peroxisome biogenesis and a unique role for Pex25 in this process.

H2S oxidation by nanodisc-embedded human sulfide quinone oxidoreductase [Enzymology]

May 16th, 2017 by Aaron P Landry, David P Ballou, Ruma Banerjee

Buildup of hydrogen sulfide (H2S), which functions as a signaling molecule but is toxic at high concentrations, is averted by its efficient oxidation by the mitochondrial sulfide oxidation pathway. The first step in this pathway is catalyzed by a flavoprotein, sulfide quinone oxidoreductase (SQR), which converts H2S to a persulfide and transfers electrons to coenzyme Q via a flavin cofactor. All previous studies on human SQR have used detergent-solubilized protein. Here, we embedded human SQR in nanodiscs (ndSQR) and studied highly homogenous preparations by steady-state and rapid kinetics techniques. ndSQR exhibited higher catalytic rates in its membranous environment than in its solubilized state. Stopped-flow spectroscopic data revealed that transfer of the sulfane sulfur from an SQR-bound cysteine persulfide intermediate to a small-molecule acceptor is the rate-limiting step. The physiological acceptor of sulfane sulfur from SQR has been the subject of controversy; we report that the kinetic analysis of ndSQR is consistent with glutathione rather than sulfite being the predominant acceptor at physiologically relevant concentrations of the respective metabolites. The identity of the acceptor has an important bearing on how the sulfide oxidation pathway is organized. Our data are more consistent with the following reaction sequence for sulfide oxidation: H2S→glutathione persulfide→sulfite→sulfate, than with a more convoluted route that would result if sulfite were the primary acceptor of sulfane sulfur. In summary, nanodisc-incorporated human SQR exhibits enhanced catalytic performance, and pre-steady state kinetic characterization of the complete SQR catalytic cycle indicates that GSH serves as the physiologically relevant sulfur acceptor.