Structural/functional analysis of the human OXR1 protein: identification of exon 8 as the anti-oxidant encoding function

ABSTRACT: BACKGROUND: The human OXR1 gene belongs to a class of genes with conserved functions that protect cells from reactive oxygen species (ROS). The gene was found using a screen of a human cDNA library by its ability to suppress the spontaneous mutator phenotype of an E. coli mutH nth strain. The function of OXR1 is unknown. The human and yeast genes are induced by oxidative stress and targeted to the mitochondria; the yeast gene is required for resistance to hydrogen peroxide. Multiple spliced isoforms are expressed in a variety of human tissues, including brain. RESULTS: In this report, we use a papillation assay that measures spontaneous mutagenesis of an E. coli mutM mutY strain, a host defective for oxidative DNA repair. Papillation frequencies with this strain are dependent upon a G->T transversion in the lacZ gene (a mutation known to occur as a result of oxidative damage) and are suppressed by in vivo expression of human OXR1. N-terminal, C-terminal and internal deletions of the OXR1 gene were constructed and tested for suppression of the mutagenic phenotype of the mutM mutY strain. We find that the TLDc domain, encoded by the final four exons of the OXR1 gene, is not required for papillation suppression in E. coli. Instead, we show that the protein segment encoded by exon 8 of OXR1 is responsible for the suppression of oxidative damage in E. coli. CONCLUSION: The protein segment encoded by OXR1 exon 8 plays an important role in the anti-oxidative function of the human OXR1 protein. This result suggests that the TLDc domain, found in OXR1 exons 12-16 and common in many proteins with nuclear function, has an alternate (undefined) role other than oxidative repair.     

Crystal structure of the TLDc domain of oxidation resistance protein 2 from zebrafish

The oxidation resistance proteins (OXR) help to protect eukaryotes from reactive oxygen species. The sole C‐terminal domain of the OXR, named TLDc is sufficient to perform this function. However, the mechanism by which oxidation resistance occurs is poorly understood. We present here the crystal structure of the TLDc domain of the oxidation resistance protein 2 from zebrafish. The structure was determined by X‐ray crystallography to atomic resolution (0.97Å) and adopts an overall globular shape. Two antiparallel β‐sheets form a central β‐sandwich, surrounded by two helices and two one‐turn helices. The fold shares low structural similarity to known structures. Proteins 2012. © 2012 Wiley Periodicals, Inc.     

The Anopheles gambiae Oxidation Resistance 1 (OXR1) Gene Regulates Expression of Enzymes That Detoxify Reactive Oxygen Species

Background

OXR1 is an ancient gene, present in all eukaryotes examined so far that confers protection from oxidative stress by an unknown mechanism. The most highly conserved region of the gene is the carboxyl-terminal TLDc domain, which has been shown to be sufficient to prevent oxidative damage.

Methodology/Principal Findings

OXR1 has a complex genomic structure in the mosquito A. gambiae, and we confirm that multiple splice forms are expressed in adult females. Our studies revealed that OXR1 regulates the basal levels of catalase (CAT) and glutathione peroxidase (Gpx) expression, two enzymes involved in detoxification of hydrogen peroxide, giving new insight into the mechanism of action of OXR1. Gene silencing experiments indicate that the Jun Kinase (JNK) gene acts upstream of OXR1 and also regulates expression of CAT and GPx. Both OXR1 and JNK genes are required for adult female mosquitoes to survive chronic oxidative stress. OXR1 silencing decreases P. berghei oocyst formation. Unexpectedly, JNK silencing has the opposite effect and enhances Plasmodium infection in the mosquito, suggesting that JNK may also mediate some, yet to be defined, antiparasitic response.

Conclusion

The JNK pathway regulates OXR1 expression and OXR1, in turn, regulates expression of enzymes that detoxify reactive oxygen species (ROS) in Anopheles gambiae. OXR1 silencing decreases Plasmodium infection in the mosquito, while JNK silencing has the opposite effect and enhances infection.     

TBC proteins: GAPs for mammalian small GTPase Rab?

The TBC (Tre-2/Bub2/Cdc16) domain was originally identified as a conserved domain among the tre-2 oncogene product and the yeast cell cycle regulators Bub2 and Cdc16, and it is now widely recognized as a conserved protein motif that consists of approx. 200 amino acids in all eukaryotes. Since the TBC domain of yeast Gyps [GAP (GTPase-activating protein) for Ypt proteins] has been shown to function as a GAP domain for small GTPase Ypt/Rab, TBC domain-containing proteins (TBC proteins) in other species are also expected to function as a certain Rab-GAP. More than 40 different TBC proteins are present in humans and mice, and recent accumulating evidence has indicated that certain mammalian TBC proteins actually function as a specific Rab-GAP. Some mammalian TBC proteins {e.g. TBC1D1 [TBC (Tre-2/Bub2/Cdc16) domain family, member 1] and TBC1D4/AS160 (Akt substrate of 160 kDa)} play an important role in homoeostasis in mammals, and defects in them are directly associated with mouse and human diseases (e.g. leanness in mice and insulin resistance in humans). The present study reviews the structure and function of mammalian TBC proteins, especially in relation to Rab small GTPases.     

TBC蛋白家族成员在人类疾病发生发展中的作用

TBC(Tre-2/Bub2/Cdc16)是真核生物中普遍存在的一种由200个氨基酸残基组成的保守性蛋白质结构域,含有该结构域的蛋白质被称为TBC蛋白。TBC蛋白具有GTPase激活活性,可促进小G蛋白Rab-GTP水解为Rab-GDP,从而参与特异的胞内转运过程。在哺乳动物中,部分TBC蛋白具有十分重要的作用,其功能异常与人类疾病的发生发展密切相关。本文主要介绍了哺乳动物TBC蛋白的结构和功能,以及近年来TBC蛋白在人类疾病发生发展中的作用,以期为深入解析TBC蛋白的致病机制提供参考。     

An Ancient Autoproteolytic Domain Found in GAIN,ZU5 and Nucleoporin98

A large family of G protein-coupled receptors (GPCRs) involved in cell adhesion has a characteristic autoproteolysis motif of HLT/S known as the GPCR proteolysis site (GPS). GPS is also shared by polycystic kidney disease proteins and it precedes the first transmembrane segment in both families. Recent structural studies have elucidated the GPS to be part of a larger domain named GPCR autoproteolysis inducing (GAIN) domain. Here we demonstrate the remote homology relationships of GAIN domain to ZU5 domain and Nucleoporin98 (Nup98) C-terminal domain by structural and sequence analysis. Sequence homology searches were performed to extend ZU5-like domains to bacteria and archaea, as well as new eukaryotic families. We found that the consecutive ZU5-UPA-death domain domain organization is commonly used in human cytoplasmic proteins with ZU5 domains, including CARD8 (caspase recruitment domain-containing protein 8) and NLRP1 (NACHT, LRR and PYD domain-containing protein 1) from the FIIND (Function to Find) family. Another divergent family of extracellular ZU5-like domains was identified in cartilage intermediate layer proteins and FAM171 proteins. Current diverse families of GAIN domain subdomain B, ZU5 and Nup98 C-terminal domain likely evolved from an ancient autoproteolytic domain with an HFS motif. The autoproteolytic site was kept intact in Nup98, p53-induced protein with a death domain and UNC5C-like, deteriorated in many ZU5 domains and changed in GAIN and FIIND. Deletion of the strand after the cleavage site was observed in zonula occluden-1 and some Nup98 homologs. These findings link several autoproteolytic domains, extend our understanding of GAIN domain origination in adhesion GPCRs and provide insights into the evolution of an ancient autoproteolytic domain.     

Stress induction and mitochondrial localization of Oxr1 proteins in yeast and humans

Reactive oxygen species (ROS) are critical molecules produced as a consequence of aerobic respiration. It is essential for cells to control the production and activity of such molecules in order to protect the genome and regulate cellular processes such as stress response and apoptosis. Mitochondria are the major source of ROS within the cell, and as a result, numerous proteins have evolved to prevent or repair oxidative damage in this organelle. The recently discovered OXR1 gene family represents a set of conserved eukaryotic genes. Previous studies of the yeast OXR1 gene indicate that it functions to protect cells from oxidative damage. In this report, we show that human and yeast OXR1 genes are induced by heat and oxidative stress and that their proteins localize to the mitochondria and function to protect against oxidative damage. We also demonstrate that mitochondrial localization is required for Oxr1 protein to prevent oxidative damage.     

Phosphorylation of the LIR Domain of SCOC Modulates ATG8 Binding Affinity and Specificity

Autophagy is a highly conserved degradative pathway, essential for cellular homeostasis and implicated in diseases including cancer and neurodegeneration. Autophagy-related 8 (ATG8) proteins play a central role in autophagosome formation and selective delivery of cytoplasmic cargo to lysosomes by recruiting autophagy adaptors and receptors. The LC3-interacting region (LIR) docking site (LDS) of ATG8 proteins binds to LIR motifs present in autophagy adaptors and receptors. LIR-ATG8 interactions can be highly selective for specific mammalian ATG8 family members (LC3A-C, GABARAP, and GABARAPL1-2) and how this specificity is generated and regulated is incompletely understood.We have identified a LIR motif in the Golgi protein SCOC (short coiled-coil protein) exhibiting strong binding to GABARAP, GABARAPL1, LC3A and LC3C. The residues within and surrounding the core LIR motif of the SCOC LIR domain were phosphorylated by autophagy-related kinases (ULK1-3, TBK1) increasing specifically LC3 family binding. More distant flanking residues also contributed to ATG8 binding. Loss of these residues was compensated by phosphorylation of serine residues immediately adjacent to the core LIR motif, indicating that the interactions of the flanking LIR regions with the LDS are important and highly dynamic.Our comprehensive structural, biophysical and biochemical analyses support and provide novel mechanistic insights into how phosphorylation of LIR domain residues regulates the affinity and binding specificity of ATG8 proteins towards autophagy adaptors and receptors.     

Evolutionary and Functional Analysis of the Invariant SWIM Domain in the Conserved Shu2/SWS1 Protein Family from Saccharomyces cerevisiae to Homo sapiens

The Saccharomyces cerevisiae Shu2 protein is an important regulator of Rad51, which promotes homologous recombination (HR). Shu2 functions in the Shu complex with Shu1 and the Rad51 paralogs Csm2 and Psy3. Shu2 belongs to the SWS1 protein family, which is characterized by its SWIM domain (CXC...X_n...CXH), a zinc-binding motif. In humans, SWS1 interacts with the Rad51 paralog SWSAP1. Using genetic and evolutionary analyses, we examined the role of the Shu complex in mitotic and meiotic processes across eukaryotic lineages. We provide evidence that the SWS1 protein family contains orthologous genes in early-branching eukaryote lineages (e.g., Giardia lamblia), as well as in multicellular eukaryotes including Caenorhabditis elegans and Drosophila melanogaster. Using sequence analysis, we expanded the SWIM domain to include an invariant alanine three residues after the terminal CXH motif (CXC…X_n…CXHXXA). We found that the SWIM domain is conserved in all eukaryotic orthologs, and accordingly, in vivo disruption of the invariant residues within the canonical SWIM domain inhibits DNA damage tolerance in yeast and protein-protein interactions in yeast and humans. Furthermore, using evolutionary analyses, we found that yeast and Drosophila Shu2 exhibit strong coevolutionary signatures with meiotic proteins, and in yeast, its disruption leads to decreased meiotic progeny. Together our data indicate that the SWS1 family is an ancient and highly conserved eukaryotic regulator of meiotic and mitotic HR.     

The Bin/Amphiphysin/Rvs (BAR) Domain Protein Endophilin B2 Interacts with Plectin and Controls Perinuclear Cytoskeletal Architecture

Proteins of the Bin/amphiphysin/Rvs (BAR) domain superfamily are essential in controlling the shape and dynamics of intracellular membranes. Here, we present evidence for the unconventional function of a member of the endophilin family of BAR and Src homology 3 domain-containing proteins, namely endophilin B2, in the perinuclear organization of intermediate filaments. Using mass spectrometry analysis based on capturing endophilin B2 partners in in situ pre-established complexes in cells, we unravel the interaction of endophilin B2 with plectin 1, a variant of the cytoskeleton linker protein plectin as well as with vimentin. Endophilin B2 directly binds the N-terminal region of plectin 1 via Src homology 3-mediated interaction and vimentin indirectly via plectin-mediated interaction. The relevance of these interactions is strengthened by the selective and drastic reorganization of vimentin around nuclei upon overexpression of endophilin B2 and by the extensive colocalization of both proteins in a meshwork of perinuclear filamentous structures. By generating mutants of the endophilin B2 BAR domain, we show that this phenotype requires the BAR-mediated membrane binding activity of endophilin B2. Plectin 1 or endophilin B2 knockdown using RNA interference disturbed the perinuclear organization of vimentin. Altogether, these data suggest that the endophilin B2-plectin 1 complex functions as a membrane-anchoring device organizing and stabilizing the perinuclear network of vimentin filaments. Finally, we present evidence for the involvement of endophilin B2 and plectin 1 in nuclear positioning in individual cells. This points to the potential importance of the endophilin B2-plectin complex in the biological functions depending on nuclear migration and positioning.     

RAB3GAP1 and RAB3GAP2 modulate basal and rapamycin-induced autophagy

Macroautophagy is a degradative pathway that sequesters and transports cytosolic cargo in autophagosomes to lysosomes, and its deterioration affects intracellular proteostasis. Membrane dynamics accompanying autophagy are mostly elusive and depend on trafficking processes. RAB GTPase activating proteins (RABGAPs) are important factors for the coordination of cellular vesicle transport systems, and several TBC (TRE2-BUB2-CDC16) domain-containing RABGAPs are associated with autophagy. Employing C. elegans and human primary fibroblasts, we show that RAB3GAP1 and RAB3GAP2, which are components of the TBC domain-free RAB3GAP complex, influence protein aggregation and affect autophagy at basal and rapamycin-induced conditions. Correlating the activity of RAB3GAP1/2 with ATG3 and ATG16L1 and analyzing ATG5 punctate structures, we illustrate that the RAB3GAPs modulate autophagosomal biogenesis. Significant levels of RAB3GAP1/2 colocalize with members of the Atg8 family at lipid droplets, and their autophagy modulatory activity depends on the GTPase-activating activity of RAB3GAP1 but is independent of the RAB GTPase RAB3. Moreover, we analyzed RAB3GAP1/2 in relation to the previously reported suppressive autophagy modulators FEZ1 and FEZ2 and demonstrate that both reciprocally regulate autophagy. In conclusion, we identify RAB3GAP1/2 as novel conserved factors of the autophagy and proteostasis network.     

YybT Is a Signaling Protein That Contains a Cyclic Dinucleotide Phosphodiesterase Domain and a GGDEF Domain with ATPase Activity

The cyclic dinucleotide c-di-GMP synthesized by the diadenylate cyclase domain was recently discovered as a messenger molecule for signaling DNA breaks in Bacillus subtilis. By searching bacterial genomes, we identified a family of DHH/DHHA1 domain proteins (COG3387) that co-occur with a subset of the diadenylate cyclase domain proteins. Here we report that the B. subtilis protein YybT, a member of the COG3387 family proteins, exhibits phosphodiesterase activity toward cyclic dinucleotides. The DHH/DHHA1 domain hydrolyzes c-di-AMP and c-di-GMP to generate the linear dinucleotides 5′-pApA and 5′-pGpG. The data suggest that c-di-AMP could be the physiological substrate for YybT given the physiologically relevant Michaelis-Menten constant (K_m) and the presence of YybT family proteins in the bacteria lacking c-di-GMP signaling network. The bacterial regulator ppGpp was found to be a strong competitive inhibitor of the DHH/DHHA1 domain, suggesting that YybT is under tight control during stringent response. In addition, the atypical GGDEF domain of YybT exhibits unexpected ATPase activity, distinct from the common diguanylate cyclase activity for GGDEF domains. We further demonstrate the participation of YybT in DNA damage and acid resistance by characterizing the phenotypes of the ΔyybT mutant. The novel enzymatic activity and stress resistance together point toward a role for YybT in stress signaling and response.     

Silencing of TBC1D15 promotes RhoA activation and membrane blebbing

Membrane blebs are round-shaped dynamic membrane protrusions that occur under many physiological conditions. Membrane bleb production is primarily controlled by actin cytoskeletal rearrangements mediated by RhoA. Tre2–Bub2–Cdc16 (TBC) domain-containing proteins are negative regulators of the Rab family of small GTPases and contain a highly conserved TBC domain. In this report, we show that the expression of TBC1D15 is associated with the activity of RhoA and the production of membrane blebs. Depletion of TBC1D15 induced activation of RhoA and membrane blebbing, which was abolished by the addition of an inhibitor for RhoA signaling. In addition, we show that TBC1D15 is required for the accumulation of RhoA at the equatorial cortex for the ingression of the cytokinetic furrow during cytokinesis. Our results demonstrate a novel role for TBC1D15 in the regulation of RhoA during membrane blebbing and cytokinesis.     

ELMO Domains,Evolutionary and Functional Characterization of a Novel GTPase-activating Protein (GAP) Domain for Arf Protein Family GTPases

The human family of ELMO domain-containing proteins (ELMODs) consists of six members and is defined by the presence of the ELMO domain. Within this family are two subclassifications of proteins, based on primary sequence conservation, protein size, and domain architecture, deemed ELMOD and ELMO. In this study, we used homology searching and phylogenetics to identify ELMOD family homologs in genomes from across eukaryotic diversity. This demonstrated not only that the protein family is ancient but also that ELMOs are potentially restricted to the supergroup Opisthokonta (Metazoa and Fungi), whereas proteins with the ELMOD organization are found in diverse eukaryotes and thus were likely the form present in the last eukaryotic common ancestor. The segregation of the ELMO clade from the larger ELMOD group is consistent with their contrasting functions as unconventional Rac1 guanine nucleotide exchange factors and the Arf family GTPase-activating proteins, respectively. We used unbiased, phylogenetic sorting and sequence alignments to identify the most highly conserved residues within the ELMO domain to identify a putative GAP domain within the ELMODs. Three independent but complementary assays were used to provide an initial characterization of this domain. We identified a highly conserved arginine residue critical for both the biochemical and cellular GAP activity of ELMODs. We also provide initial evidence of the function of human ELMOD1 as an Arf family GAP at the Golgi. These findings provide the basis for the future study of the ELMOD family of proteins and a new avenue for the study of Arf family GTPases.     

Systematic Analysis of Bacterial Effector-Postsynaptic Density 95/Disc Large/Zonula Occludens-1 (PDZ) Domain Interactions Demonstrates Shigella OspE Protein Promotes Protein Kinase C Activation via PDLIM Proteins

Diseases caused by many Gram-negative bacterial pathogens depend on the activities of bacterial effector proteins that are delivered into eukaryotic cells via specialized secretion systems. Effector protein function largely depends on specific subcellular targeting and specific interactions with cellular ligands. PDZ domains are common domains that serve to provide specificity in protein-protein interactions in eukaryotic systems. We show that putative PDZ-binding motifs are significantly enriched among effector proteins delivered into mammalian cells by certain bacterial pathogens. We use PDZ domain microarrays to identify candidate interaction partners of the Shigella flexneri effector proteins OspE1 and OspE2, which contain putative PDZ-binding motifs. We demonstrate in vitro and in cells that OspE proteins interact with PDLIM7, a member of the PDLIM family of proteins, which contain a PDZ domain and one or more LIM domains, protein interaction domains that participate in a wide variety of functions, including activation of isoforms of protein kinase C (PKC). We demonstrate that activation of PKC during S. flexneri infection is attenuated in the absence of PDLIM7 or OspE proteins and that the OspE PDZ-binding motif is required for wild-type levels of PKC activation. These results are consistent with a model in which binding of OspE to PDLIM7 during infection regulates the activity of PKC isoforms that bind to the PDLIM7 LIM domain.     

Identification of a Novel Sequence Motif Recognized by the Ankyrin Repeat Domain of zDHHC17/13 S-Acyltransferases

S-Acylation is a major post-translational modification affecting several cellular processes. It is particularly important for neuronal functions. This modification is catalyzed by a family of transmembrane S-acyltransferases that contain a conserved zinc finger DHHC (zDHHC) domain. Typically, eukaryote genomes encode for 7–24 distinct zDHHC enzymes, with two members also harboring an ankyrin repeat (AR) domain at their cytosolic N termini. The AR domain of zDHHC enzymes is predicted to engage in numerous interactions and facilitates both substrate recruitment and S-acylation-independent functions; however, the sequence/structural features recognized by this module remain unknown. The two mammalian AR-containing S-acyltransferases are the Golgi-localized zDHHC17 and zDHHC13, also known as Huntingtin-interacting proteins 14 and 14-like, respectively; they are highly expressed in brain, and their loss in mice leads to neuropathological deficits that are reminiscent of Huntington''s disease. Here, we report that zDHHC17 and zDHHC13 recognize, via their AR domain, evolutionary conserved and closely related sequences of a [VIAP][VIT]XXQP consensus in SNAP25, SNAP23, cysteine string protein, Huntingtin, cytoplasmic linker protein 3, and microtubule-associated protein 6. This novel AR-binding sequence motif is found in regions predicted to be unstructured and is present in a number of zDHHC17 substrates and zDHHC17/13-interacting S-acylated proteins. This is the first study to identify a motif recognized by AR-containing zDHHCs.     

Identification of phosphotyrosine binding domain-containing proteins as novel downstream targets of the EphA8 signaling function

Eph receptors and ephrins have been implicated in a variety of cellular processes, including morphology and motility, because of their ability to modulate intricate signaling networks. Here we show that the phosphotyrosine binding (PTB) domain-containing proteins AIDA-1b and Odin are tightly associated with the EphA8 receptor in response to ligand stimulation. Both AIDA-1b and Odin belong to the ankyrin repeat and sterile alpha motif domain-containing (Anks) protein family. The PTB domain of Anks family proteins is crucial for their association with the juxtamembrane domain of EphA8, whereas EphA8 tyrosine kinase activity is not required for this protein-protein interaction. In addition, we found that Odin is a more physiologically relevant partner of EphA8 in mammalian cells. Interestingly, overexpression of the Odin PTB domain alone attenuated EphA8-mediated inhibition of cell migration in HEK293 cells, suggesting that it acts as a dominant-negative mutant of the endogenous Odin protein. More importantly, small interfering RNA-mediated Odin silencing significantly diminished ephrinA5-induced EphA8 signaling effects, which inhibit cell migration in HEK293 cells and retract growing neurites of Neuro2a cells. Taken together, our findings support a possible function for Anks family proteins as scaffolding proteins of the EphA8 signaling pathway.