NAD+ binding to the Escherichia coli K+-uptake protein TrkA and sequence similarity between TrkA and domains of a family of dehydrogenases suggest a role for NAD+ in bacterial transport

The nucleotide sequence of trkA, a gene encoding a surface component of the constitutive K⁺-uptake systems TrkG and TrkH from Escherichia coli, was determined. The structure of the TrkA protein deduced from the nucleotide sequence accords with the view that TrkA is peripherally bound to the inner side of the cytoplasmic membrane. Analysis by a dot matrix revealed that TrkA is composed of similar halves. The M-terminal part of each TrkA half (residues 1–130 and 234–355, respectively) is similar to the complete NAD⁺-binding domain of NAD⁺-dependent dehydrogenases. The C-terminal part of each TrkA half (residues 131–233 and 357–458, respectively) aligns with the first 100 residues of the catalytic domain of glyceraldehyde-3-phosphate dehydrogenase. Strong u.v. illumination at 252 nm led to cross-linking of NAD⁺ or NADH, but not of ATP to the isolated TrkA protein.     

The Ciliopathy Protein CC2D2A Associates with NINL and Functions in RAB8-MICAL3-Regulated Vesicle Trafficking

Ciliopathies are a group of human disorders caused by dysfunction of primary cilia, ubiquitous microtubule-based organelles involved in transduction of extra-cellular signals to the cell. This function requires the concentration of receptors and channels in the ciliary membrane, which is achieved by complex trafficking mechanisms, in part controlled by the small GTPase RAB8, and by sorting at the transition zone located at the entrance of the ciliary compartment. Mutations in the transition zone gene CC2D2A cause the related Joubert and Meckel syndromes, two typical ciliopathies characterized by central nervous system malformations, and result in loss of ciliary localization of multiple proteins in various models. The precise mechanisms by which CC2D2A and other transition zone proteins control protein entrance into the cilium and how they are linked to vesicular trafficking of incoming cargo remain largely unknown. In this work, we identify the centrosomal protein NINL as a physical interaction partner of CC2D2A. NINL partially co-localizes with CC2D2A at the base of cilia and ninl knockdown in zebrafish leads to photoreceptor outer segment loss, mislocalization of opsins and vesicle accumulation, similar to cc2d2a-/- phenotypes. Moreover, partial ninl knockdown in cc2d2a-/- embryos enhances the retinal phenotype of the mutants, indicating a genetic interaction in vivo, for which an illustration is found in patients from a Joubert Syndrome cohort. Similar to zebrafish cc2d2a mutants, ninl morphants display altered Rab8a localization. Further exploration of the NINL-associated interactome identifies MICAL3, a protein known to interact with Rab8 and to play an important role in vesicle docking and fusion. Together, these data support a model where CC2D2A associates with NINL to provide a docking point for cilia-directed cargo vesicles, suggesting a mechanism by which transition zone proteins can control the protein content of the ciliary compartment.     

Identification of multiple cell cycle regulatory functions of p57Kip2 in human T lymphocytes

The specific functions of p57(Kip2) in lymphocytes have not yet been fully elucidated. In this study, it is shown that p57(Kip2), which is a member of the Cip/Kip family of cyclin-dependent kinase inhibitors, is present in the nuclei of normal resting (G(0)) T cells from peripheral blood and in the nuclei of the T cell-derived Jurkat cell line. Activation through the TCR results in rapid transport of cytoplasmic cyclin-dependent kinase 6 (cdk6) to nuclei, where it associates with cyclin D and p57(Kip2) in active enzyme complexes. Using purified recombinant proteins, it was shown in vitro that addition of p57(Kip2) protein to a mixture of cyclin D2 and cdk6 enhanced the association of the latter two proteins and resulted in phosphorylation of p57(Kip2). To probe further the function of p57(Kip2), Jurkat cells stably transfected with a plasmid encoding p57(Kip2) under control of an inducible (tetracycline) promoter were made. Induction of p57(Kip2) resulted in increased association of cdk6 with cyclin D3, without receptor-mediated T cell stimulation. The overall amounts of cdk6 and cyclin D3, and also of cdk4 and cyclin E, remained unchanged. Most notably, increased p57(Kip2) levels resulted in marked inhibition of both cyclin E- and cyclin A-associated cdk2 kinase activities and a decrease in cyclin A amounts. Therefore, although facilitating activation of cdk6, the ultimate outcome of p57(Kip2) induction was a decrease in DNA synthesis and cell proliferation. The results indicate that p57(Kip2) is involved in the regulation of several aspects of the T cell cycle.     

Intraspecific sexual selection on a speciation trait, male coloration, in the Lake Victoria cichlid Pundamilia nyererei

The haplochromine cichlids of Lake Victoria constitute a classical example of explosive speciation. Extensive intra- and interspecific variation in male nuptial coloration and female mating preferences, in the absence of postzygotic isolation between species, has inspired the hypothesis that sexual selection has been a driving force in the origin of this species flock. This hypothesis rests on the premise that the phenotypic traits that underlie behavioural reproductive isolation between sister species diverged under sexual selection within a species. We test this premise in a Lake Victoria cichlid, by using laboratory experiments and field observations. We report that a male colour trait, which has previously been shown to be important for behavioural reproductive isolation between this species and a close relative, is under directional sexual selection by female mate choice within this species. This is consistent with the hypothesis that female choice has driven the divergence in male coloration between the two species. We also find that male territoriality is vital for male reproductive success and that multiple mating by females is common.     

Airway hyperresponsiveness through synergy of gammadelta} T cells and NKT cells

Mice sensitized and challenged with OVA were used to investigate the role of innate T cells in the development of allergic airway hyperresponsiveness (AHR). AHR, but not eosinophilic airway inflammation, was induced in T cell-deficient mice by small numbers of cotransferred gammadelta T cells and invariant NKT cells, whereas either cell type alone was not effective. Only Vgamma1+Vdelta5+ gammadelta T cells enhanced AHR. Surprisingly, OVA-specific alphabeta T cells were not required, revealing a pathway of AHR development mediated entirely by innate T cells. The data suggest that lymphocytic synergism, which is key to the Ag-specific adaptive immune response, is also intrinsic to T cell-dependent innate responses.     

Activation and inactivation of antiviral CD8 T cell responses during murine pneumovirus infection

Pneumonia virus of mice (PVM) is a natural pathogen of mice and has been proposed as a tractable model for the replication of a pneumovirus in its natural host, which mimics human infection with human respiratory syncytial virus (RSV). PVM infection in mice is highly productive in terms of virus production compared with the situation seen with RSV in mice. Because RSV suppresses CD8 T cell effector function in the lungs of infected mice, we have investigated the nature of PVM-induced CD8 T cell responses to study pneumovirus-induced T cell responses in a natural virus-host setting. PVM infection was associated with a massive influx of activated CD8 T cells into the lungs. After identification of three PVM-specific CD8 T cell epitopes, pulmonary CD8 T cell responses were enumerated. The combined frequency of cytokine-secreting CD8 T cells specific for the three epitopes was much smaller than the total number of activated CD8 T cells. Furthermore, quantitation of the CD8 T cell response against one of these epitopes (residues 261-270 from the phosphoprotein) by MHC class I pentamer staining and by in vitro stimulation followed by intracellular IFN-gamma and TNF-alpha staining indicated that the majority of pulmonary CD8 specific for the P261 epitope were deficient in cytokine production. This deficient phenotype was retained up to 96 days postinfection, similar to the situation in the lungs of human RSV-infected mice. The data suggest that PVM suppresses T cell effector functions in the lungs.     

Cloning, genomic structure, and expression profiles of TULIP1 (GARNL1), a brain-expressed candidate gene for 14q13-linked neurological phenotypes, and its murine homologue

Previously, we have described the clinical and molecular characterization of a de novo 14q13.1-q21.1 microdeletion, less than 3.5 Mb in size, in a patient with severe microcephaly, psychomotor retardation, and other clinical anomalies. Here we report the characterization of the genomic structure of the human tuberin-like protein gene 1 (TULIP1; approved gene symbol GARNL1), a CpGisland-associated, brain-expressed candidate gene for the neurological findings in our patient, and its murine homologue. The human TULIP1 gene was mapped to chromosome band 14q13.2 by fluorescence in situ hybridization of BAC clone RP11-355C3 (GenBank Accession No. AL160231), containing the 3' region of the gene. TULIP1 spans about 271 kb of human genomic DNA and is divided into 41 exons. An untranscribed, processed pseudogene of TULIP1 was found on human chromosome band 9q31.1. The active locus TULIP1, encoding a predicted protein of 2036 amino acids, is expressed ubiquitously in pre- and postnatal human tissues. The murine homologue Tulip1 spans about 220 kb of mouse genomic DNA and is also divided into 41 exons, encoding a predicted protein of 2035 amino acids. No pseudogene could be found in the available mouse sequence data. Several splicing variants were found. Considering the location, expression profile, and predicted function, TULIP1 is a strong candidate for several neurological features seen in 14q deletion patients. Additionally we searched for mutations in the coding region of TULIP1 in subjects from a family with idiopathic basal ganglia calcification (IBGC; Fahr disease), previously linked to chromosome 14q. We identified two novel SNPs in the intron-exon boundaries; however, they did not segregate only with affected subjects in the predicted model of an autosomal dominant disease such as IBGC.     

1H, 13C and 15N assignments of a camelid nanobody directed against human α-synuclein

Nanobodies are single chain antibodies that are uniquely produced in Camelidae, e.g. camels and llamas. They have the desirable features of small sizes (Mw < 14 kDa) and high affinities against antigens (Kd ~ nM), making them ideal as structural probes for biomedically relevant motifs both in vitro and in vivo. We have previously shown that nanobody binding to amyloidogenic human lysozyme variants can effectively inhibit their aggregation, the process that is at the origin of systemic amyloid disease. Here we report the NMR assignments of a new nanobody, termed NbSyn2, which recognises the C-terminus of the intrinsically disordered protein, human α-synuclein (aS), whose aberrant self-association is implicated in Parkinson’s disease.