Identification and Characterization of RBEL1 Subfamily of GTPases in the Ras Superfamily Involved in Cell Growth Regulation

Recently, we reported the identification of a novel gene named RBEL1 (Rab-like protein 1) and characterized its two encoded isoforms, RBEL1A and RBEL1B, that function as novel GTPases of Ras superfamily. Here we report the identification of two additional splice variants of RBEL1 that we have named RBEL1C and -D. All four RBEL1 isoforms (A, B, C, and D) have identical N termini harboring the Rab-like GTPase domains but contain variable C termini. Although all isoforms can be detected in both cytoplasm and nucleus, RBEL1A is predominantly cytoplasmic, whereas RBEL1B is mostly nuclear. RBEL1C and -D, by contrast, are evenly distributed between the cytoplasm and nucleus. Furthermore, all four RBEL1 proteins are also capable of associating with cellular membrane. The RBEL1 proteins also exhibit a unique nucleotide-binding potential and, whereas the larger A and B isoforms are mainly GTP-bound, the smaller C and D variants bind to both GTP and GDP. Furthermore, a regulatory region at amino acid position 236–302 immediately adjacent to the GTP-binding domain is important for GTP-binding potential of RBEL1A, because deletion of this region converts RBEL1A from predominantly GTP-bound to GDP-bound. RBEL1 knockdown via RNA interference results in marked cell growth suppression, which is associated with morphological and biochemical features of apoptosis as well as inhibition of extracellular signal-regulated kinase phosphorylation. Taken together, our results indicate that RBEL1 proteins are linked to cell growth and survival and possess unique biochemical, cellular, and functional characteristics and, therefore, appear to form a novel subfamily of GTPases within the Ras superfamily.The Ras superfamily is known to comprise five structurally distinct subfamilies of small GTPases, including Ras, Rho, Rab, Sar1/Arf, and Ran, and each subfamily of these GTPases possess distinct functions in the regulation of a variety of cellular processes such as cell proliferation, cell differentiation, cytoskeletal organization, protein transport, and trafficking (1–4). The Ras subfamily of GTPases (N-, H-, and K-Ras) function predominantly in relaying signals from receptors at the plasma membrane and modulating cell signaling pathways that regulate cell proliferation, differentiation, and survival (5). Ran GTPase, on other hand, is a key regulator of nucleocytoplasmic transport that regulates protein transport across the nuclear pore complex (6, 7). The Rab subfamily is the largest subfamily among the Ras superfamily and contains more than 60 members. The key functions of the Rab GTPases are to regulate protein exocytic and endocytic pathways and modulate intracellular protein transport/trafficking (8–13).In general, the Ras superfamily GTPases cycle between an active GTP-bound state and an inactive GDP-bound state. There are five N-terminal motifs involved in the binding and hydrolysis of GTP that are highly conserved among all GTPases: G1 (GXXXXGK(S/T)), G2 (T), G3 (DXXG), G4 ((N/T)(K/Q)XD), and G5 (EXSAX). Each sequence has particular functions involved in binding nucleotides (GTP or GDP) and facilitating hydrolysis (4, 14, 15). In general, the intrinsic GTPase activity (converting GTP to GDP) and exchange of GDP for GTP are slow processes for these GTPases and thus require regulatory proteins such as GTPase-activating proteins and GDP/GTP exchange factors to facilitate these processes (16–18).For the last two decades, the Ras superfamily has been a major focus in the cancer field as many of the members are either mutated or dysregulated in cancer. The founding members of the Ras superfamily, H-Ras and K-Ras, were first identified as viral oncogenes (1, 4). Later studies demonstrated that mutations of the Ras proteins (H-, N-, and K-Ras) occur frequently in human cancers, and the mutations identified are mostly clustered within the GTP-binding domains of the proteins thus locking Ras proteins in a GTP-bound configuration. GTP-bound Ras is constitutively active; it constantly activates its effector proteins to transduce cell proliferative signals (1, 4). Unlike Ras subfamily genes, mutations occurring in Rab and Rab-like genes are less common, yet alterations in gene expression of a number of Rab genes have been reported in multiple human malignancies. For example, Rab25 overexpression has been linked to prostate cancer progression (19). Rab2 overexpression has been found in lung adenomas and adenocarcinomas (20). In addition, alterations in Rab gene expression have also been linked to cancer drug resistance. For instance, resistance to the anticancer drug doxorubicin in MCF-7 cells has been linked with reduced expression of Rab6C, and introduction of exogenous Rab6C restores drug sensitivity (21).We have recently reported the identification two novel Ras superfamily GTPases, RBEL1A and RBEL1B (22). RBEL1A and RBEL1B are two splice variants of the RBEL1 gene and are highly homologous to the Rab and Ran GTPases within their N-terminal GTP-binding domains (22). Our studies show that both RBEL1A and -B predominantly bind to GTP. A single point mutation (T57N) in the GTP-binding domain of RBEL1A and -B abolishes their ability to bind to both GTP and GDP. Both RBEL1A and RBEL1B localize in the nucleus as well as in the cytosol. Whereas RBEL1A is predominantly cytosolic, RBEL1B is primarily nuclear. Interestingly, our studies also suggested that nucleotide (GTP or GDP)-binding could be important for the nuclear distribution of RBEL1B, because the nucleotide binding-deficient mutant form (T57N) of RBEL1B did not reside in the nucleus but rather became largely cytosolic (22).In our continuous efforts to fully elucidate the function of RBEL1, we have identified two additional splice variants that we have named RBEL1C and RBEL1D. Here we report further characterization of all four RBEL1 splice variants in terms of their GTPase activities, subcellular localizations, regulations, and potential functions. Our results indicate that RBEL1 GTPases, although sharing some common features with other Ras superfamily members, also harbor unique characteristics that are significantly different from other Ras superfamily GTPases. Based on our findings, we suggest that RBEL1 proteins appear to form a novel subfamily of GTPases within the Ras superfamily.     

A comparison of random vs. chemotaxis-driven contacts of T cells with dendritic cells during repertoire scanning

Generating adaptive immunity after infection or immunization requires physical interactions within a lymph node (LN) T-zone between antigen-bearing dendritic cells (DCs) that arrive from peripheral tissues and rare cognate T cells entering via high endothelial venules (HEVs). This interaction results in activation of cognate T cells, expansion of that T cell lineage and their exit from the LN T-zone via efferent lymphatics (ELs). How antigen-specific T cells locate DCs within this complex environment is controversial, and both random T cell migration and chemotaxis have been proposed. We developed an agent-based computational model of a LN that captures many features of T cell and DC dynamics observed by two-photon microscopy. Our simulations matched in vivo two-photon microscopy data regarding T cell speed, short-term directional persistence of motion and cell motility. We also obtained in vivo data regarding density of T cells and DCs within a LN and matched our model environment to measurements of the distance from HEVs to ELs. We used our model to compare chemotaxis with random motion and showed that chemotaxis increased total number of T cell DC contacts, but decreased unique contacts, producing fewer activated T cells. Our results suggest that, within a LN T-zone, a random search strategy is optimal for a rare cognate T cell to find its DC match and maximize production of activated T cells.     

Hormonal regulation of estradiol biosynthesis, aromatase activity, and aromatase mRNA in rat ovarian follicles and corpora lutea

To determine the molecular basis for changes in aromatase (P450arom) activity in rat ovarian follicles and corpora lutea, seven clones for rat P450arom cDNA have been identified and isolated from a rat granulosa cell λgtll cDNA expression library using a 62 mer deoxyoligonucleotide probe (derived from an amino acid sequence of purified human placental aromatase) and a human placental P450arom cDNA probe. One of the rat P450arom cDNA clones contained an insert 1.2 kb in size. Both the human 1.8 kb cDNA and the rat 1.2 kb cDNA probes hybridized to a single species of P450arom mRNA that was 2.6 kb in size. Northern blot analysis revealed that corpora lutea isolated on day 15 of pregnancy contained high amounts of P450arom mRNA, whereas granulosa cells of antral follicles of hormonally primed, hypophysectomized rats (i.e., those from which mRNA was isolated to construct the cDNA library) contained only low amounts of P450arom mRNA. The lower amounts of P450arom in granulosa cells of preovulatory follicles in the estradiol-follicle-stimulating hormone primed hypophysectomized rats were unexpected because follicles incubated in medium containing testosterone substrate produce more estradiol than do corpora lutea isolated on day 15 of pregnancy and incubated under similar conditions. Additional studies will determine the hormonal events responsible for the elevated amounts and constitutive maintenance of P450arom mRNA and aromatase activity in luteal cells in vivo and in vitro.     

Purification and characterization of an enzymically active cleavage product of pig kidney aldehyde reductase

During the purification of pig kidney aldehyde reductase by an established procedure [Flynn, Cromlish & Davidson (1982) Methods Enzymol. 89, 501-506] a second enzyme with aldehyde reductase activity may be purified. When the procedure was performed in the presence of 5 mM-EDTA, only traces of the second reductase, pig kidney aldehyde reductase (minor form), were present. By the criterion of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, pig kidney aldehyde reductase (minor form) had Mr 35 000, in comparison with Mr 40 200 found for pig kidney aldehyde reductase. Amino acid analysis of both enzymes and tryptic-peptide-map comparisons indicated differences in primary structure. The N-terminus of pig kidney aldehyde reductase (minor form) had the sequence Lys-Val-Leu, in contrast with the blocked (acetylated) N-terminus of pig kidney aldehyde reductase. The C-terminal sequence of both enzymes was the same. Both reductases were immunologically identical by double immunodiffusion and rocket immunoelectrophoresis. Pig kidney aldehyde reductase (minor form) had 50% of the specific activity of pig kidney aldehyde reductase when tested with a variety of aldehyde substrates. Michaelis constants of both enzymes for these substrates and for NADPH were similar, but values for kcat. and kcat./Km indicated that catalytically pig kidney aldehyde reductase was the more efficient enzyme. Typical aldehyde reductase inhibitors, such as phenobarbital and sodium valproate, had the same effect on both enzymes. It was concluded that pig kidney aldehyde reductase (minor form) is an enzymically active cleavage product of pig kidney aldehyde reductase which is formed when the latter is purified in the absence of the metalloproteinase inhibitor EDTA.     

Mutational analysis of upstream AUG codons of poliovirus RNA.

The 5' untranslated region of poliovirus type 2 Lansing RNA consists of 744 nucleotides containing seven AUG codons which are followed by in-frame termination codons, thus forming short open reading frames (ORFs). To determine the biological significance of these small ORFs, all of the upstream AUG codons were mutated to UUG. The point mutations were introduced into an infectious poliovirus cDNA clone, and RNA transcribed in vitro from the altered cDNA was transfected into HeLa cells to recover the virus. Mutation of AUG 7 resulted in a virus (called R2-5NC-14) with a small-plaque phenotype, whereas mutation of the other six AUG codons produced virus with a wild-type plaque morphology. To determine whether the small-plaque phenotype of R2-5NC-14 was due to altered translational efficiency of the viral mRNA, we constructed chimeric mRNAs containing the 5' noncoding region of poliovirus mRNA fused to the chloramphenicol acetyltransferase (CAT) coding sequence. mRNA containing a mutated AUG 7 codon showed decreased translational efficiency in vitro. The results indicate that the upstream ORFs of poliovirus RNA are not essential for viral replication and do not act as barriers to the translation of poliovirus mRNA. AUG 7 and flanking sequences may play a positive acting role in poliovirus RNA translation.     

Characterization of liver disease and lipid metabolism in the Niemann-Pick C1 mouse

Niemann-Pick type C1 (NPC1) disease is an autosomal-recessive cholesterol-storage disorder characterized by liver dysfunction, hepatosplenomegaly, and progressive neurodegeneration. The NPC1 gene is expressed in every tissue of the body, with liver expressing the highest amounts of NPC1 mRNA and protein. A number of studies have now indicated that the NPC1 protein regulates the transport of cholesterol from late endosomes/lysosomes to other cellular compartments involved in maintaining intracellular cholesterol homeostasis. The present study characterizes liver disease and lipid metabolism in NPC1 mice at 35 days of age before the development of weight loss and neurological symptoms. At this age, homozygous affected (NPC1(-/-)) mice were characterized with mild hepatomegaly, an elevation of liver enzymes, and an accumulation of liver cholesterol approximately four times that measured in normal (NPC1(+/+)) mice. In contrast, heterozygous (NPC1(+/-)) mice were without hepatomegaly and an elevation of liver enzymes, but the livers had a significant accumulation of triacylglycerol. With respect to apolipoprotein and lipoprotein metabolism, the results indicated only minor alterations in NPC1(-/-) mouse serum. Finally, compared to NPC1(+/+) mouse livers, the amount and processing of SREBP-1 and -2 proteins were significantly increased in NPC1(-/-) mouse livers, suggesting a relative deficiency of cholesterol at the metabolically active pool of cholesterol located at the endoplasmic reticulum. The results from this study further support the hypothesis that an accumulation of lipoprotein-derived cholesterol within late endosomes/lysosomes, in addition to altered intracellular cholesterol homeostasis, has a key role in the biochemical and cellular pathophysiology associated with NPC1 liver disease.     

Commensal microbiota stimulate systemic neutrophil migration through induction of Serum amyloid A

Neutrophils serve critical roles in inflammatory responses to infection and injury, and mechanisms governing their activity represent attractive targets for controlling inflammation. The commensal microbiota is known to regulate the activity of neutrophils and other leucocytes in the intestine, but the systemic impact of the microbiota on neutrophils remains unknown. Here we utilized in vivo imaging in gnotobiotic zebrafish to reveal diverse effects of microbiota colonization on systemic neutrophil development and function. The presence of a microbiota resulted in increased neutrophil number and myeloperoxidase expression, and altered neutrophil localization and migratory behaviours. These effects of the microbiota on neutrophil homeostasis were accompanied by an increased recruitment of neutrophils to injury. Genetic analysis identified the microbiota‐induced acute phase protein serum amyloid A (Saa) as a host factor mediating microbial stimulation of tissue‐specific neutrophil migratory behaviours. In vitro studies revealed that zebrafish cells respond to Saa exposure by activating NF‐κB, and that Saa‐dependent neutrophil migration requires NF‐κB‐dependent gene expression. These results implicate the commensal microbiota as an important environmental factor regulating diverse aspects of systemic neutrophil development and function, and reveal a critical role for a Saa‐NF‐κB signalling axis in mediating neutrophil migratory responses.     

Generation of a cytotoxic T-lymphocyte response using a Salmonella antigen-delivery system

We have constructed a general-use vector for the cloning and stable expression of foreign genes in the chromosome of attenuated Salmonella typhimurium. Using this chromosomal expression vector (CEV), we expressed the circumsporozoite (CS) gene of the mouse malaria Plasmodium yoelii in an aroA S. typhimurium strain. Mice immunized with CS-expressing Salmonella recombinants mount a CS-specific cytotoxic T-lymphocyte (CTL) response. This is the first demonstration that attenuated Salmonella can elicit a specific CTL response to a foreign protein in mice. The ability to easily and stably express foreign genes from the Salmonella chromosome and the generation of specific CTL greatly expands the potential of Salmonella as an antigen-delivery system.