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Klhl6 belongs to the KLHL gene family, which is composed of an N-terminal BTB-POZ domain and four to six Kelch motifs in tandem. Several of these proteins function as adaptors of the Cullin3 E3 ubiquitin ligase complex. In this article, we report that Klhl6 deficiency induces, as previously described, a 2-fold reduction in mature B cells. However, we find that this deficit is centered on the inability of transitional type 1 B cells to survive and to progress toward the transitional type 2 B cell stage, whereas cells that have passed this step generate normal germinal centers (GCs) upon a T-dependent immune challenge. Klhl6-deficient type 1 B cells showed a 2-fold overexpression of genes linked with cell proliferation, including most targets of the anaphase-promoting complex/cyclosome complex, a set of genes whose expression is precisely downmodulated upon culture of splenic transitional B cells in the presence of BAFF. These results thus suggest a delay in the differentiation process of Klhl6-deficient B cells between the immature and transitional stage. We further show, in the BL2 Burkitt's lymphoma cell line, that KLHL6 interacts with Cullin3, but also that it binds to HBXIP/Lamtor5, a protein involved in cell-cycle regulation and cytokinesis. Finally, we report that KLHL6, which is recurrently mutated in B cell lymphomas, is an off-target of the normal somatic hypermutation process taking place in GC B cells in both mice and humans, thus leaving open whether, despite the lack of impact of Klhl6 deficiency on GC B cell expansion, mutants could contribute to the oncogenic process., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Spermatogenesis is a dynamic process that is regulated by adhesive interactions between germ and Sertoli cells. Germ cells express the Junctional Adhesion Molecule-C (JAM-C, encoded by Jam3), which localizes to germ/Sertoli cell contacts. JAM-C is involved in germ cell polarity and acrosome formation. Using a proteomic approach, we demonstrated that JAM-C interacted with the Golgi reassembly stacking protein of 55 kDa (GRASP55, encoded by Gorasp2) in developing germ cells. Generation and study of Gorasp2-/- mice revealed that knock-out mice suffered from spermatogenesis defects. Acrosome formation and polarized localization of JAM-C in spermatids were altered in Gorasp2-/- mice. In addition, Golgi morphology of spermatocytes was disturbed in Gorasp2-/- mice. Crystal structures of GRASP55 in complex with JAM-C or JAM-B revealed that GRASP55 interacted via PDZ-mediated interactions with JAMs and induced a conformational change in GRASP55 with respect of its free conformation. An in silico pharmacophore approach identified a chemical compound called Graspin that inhibited PDZ-mediated interactions of GRASP55 with JAMs. Treatment of mice with Graspin hampered the polarized localization of JAM-C in spermatids, induced the premature release of spermatids and affected the Golgi morphology of meiotic spermatocytes.

The establishment and maintenance of apical-basal cell polarity is essential for the functionality of glandular epithelia. Cell polarity is often lost in advanced tumours correlating with acquisition of invasive and malignant properties. Despite extensive knowledge regarding the formation and maintenance of polarity, the mechanisms that deregulate polarity in metastasizing cells remain to be fully characterized. Here we show that AmotL2 expression correlates with loss of tissue architecture in tumours from human breast and colon cancer patients. We further show that hypoxic stress results in activation of c-Fos-dependent expression of AmotL2 leading to loss of polarity. c-Fos/hypoxia-induced p60 AmotL2 interacts with the Crb3 and Par3 polarity complexes retaining them in large vesicles and preventing them from reaching the apical membrane. The resulting loss of polarity potentiates the response to invasive cues in vitro and in vivo in mice. These data provide a molecular mechanism how hypoxic stress deregulates cell polarity during tumour progression.

Vertebrate genomes contain around 20,000 protein-encoding genes, of which a large fraction is still not associated with specific functions. A major task in future genomics will thus be to assign physiological roles to all open reading frames revealed by genome sequencing. Here we show that C2orf62, a highly conserved protein with little homology to characterized proteins, is strongly expressed in testis in zebrafish and mammals, and in various types of ciliated cells during zebrafish development. By yeast two hybrid and GST pull-down, C2orf62 was shown to interact with TTC17, another uncharacterized protein. Depletion of either C2orf62 or TTC17 in human ciliated cells interferes with actin polymerization and reduces the number of primary cilia without changing their length. Zebrafish embryos injected with morpholinos against C2orf62 or TTC17, or with mRNA coding for the C2orf62 C-terminal part containing a RII dimerization/docking (R2D2) - like domain show morphological defects consistent with imperfect ciliogenesis. We provide here the first evidence for a C2orf62-TTC17 axis that would regulate actin polymerization and ciliogenesis.

Vangl2 is one of the central proteins controlling the establishment of planar cell polarity in multiple tissues of different species. Previous studies suggest that the localization of the Vangl2 protein to specific intracellular microdomains is crucial for its function. However, the molecular mechanisms that control Vangl2 trafficking within a cell are largely unknown. Here, we identify Gipc1 (GAIP C-terminus interacting protein 1) as a new interactor for Vangl2, and we show that a myosin VI-Gipc1 protein complex can regulate Vangl2 traffic in heterologous cells. Furthermore, we show that in the cochlea of MyoVI mutant mice, Vangl2 presence at the membrane is increased, and that a disruption of Gipc1 function in hair cells leads to maturation defects, including defects in hair bundle orientation and integrity. Finally, stimulated emission depletion microscopy and overexpression of GFP-Vangl2 show an enrichment of Vangl2 on the supporting cell side, adjacent to the proximal membrane of hair cells. Altogether, these results indicate a broad role for Gipc1 in the development of both stereociliary bundles and cell polarization, and suggest that the strong asymmetry of Vangl2 observed in early postnatal cochlear epithelium is mostly a 'tissue' polarity readout.

Part of an ancestral bactericidal system, vertebrate C-type lysozyme targets the peptidoglycan moiety of bacterial cell walls. We report the crystal structure of a protein inhibitor of C-type lysozyme, the Escherichia coli Ivy protein, alone and in complex with hen egg white lysozyme. Ivy exhibits a novel fold in which a protruding five-residue loop appears essential to its inhibitory effect. This feature guided the identification of Ivy orthologues in other Gram-negative bacteria. The structure of the evolutionary distant Pseudomonas aeruginosa Ivy orthologue was also determined in complex with hen egg white lysozyme, and its antilysozyme activity was confirmed. Ivy expression protects porous cell-wall E. coli mutants from the lytic effect of lysozyme, suggesting that it is a response against the permeabilizing effects of the innate vertebrate immune system. As such, Ivy acts as a virulence factor for a number of Gram-negative bacteria-infecting vertebrates.

The tyrosine phosphatase r-PTPeta is able to suppress the malignant phenotype of rat thyroid tumorigenic cell lines. To identify r-PTPeta interacting proteins, a yeast two-hybrid screening was performed and an insert corresponding to the full-length syntenin cDNA was isolated. It encodes a protein containing two PDZ domains that mediates the binding of syntenin to proteins such as syndecan, proTGF-alpha, beta-ephrins and neurofascin. We show that r-PTPeta is able to interact with syntenin also in mammalian cells, and although syntenin is a tyrosine-phosphorylated protein it is not a substrate of r-PTPeta. The integrity of both PDZ domains of syntenin and the carboxy-terminal region of r-PTPeta are required for the interaction between syntenin and r-PTPeta.