RESUMO
Myosin Va (MyoVa) is an actin-based molecular motor abundantly found at the centrosome. However, the role of MyoVa at this organelle has been elusive due to the lack of evidence on interacting partners or functional data. Herein, we combined yeast two-hybrid screen, biochemical studies and cellular assays to demonstrate that MyoVa interacts with RPGRIP1L, a cilia-centrosomal protein that controls ciliary signaling and positioning. MyoVa binds to the C2 domains of RPGRIP1L via residues located near or in the Rab11a-binding site, a conserved site in the globular tail domain (GTD) from class V myosins. According to proximity ligation assays, MyoVa and RPGRIP1L can interact near the cilium base in ciliated RPE cells. Furthermore, we showed that RPE cells expressing dominant-negative constructs of MyoVa are mostly unciliated, providing the first experimental evidence about a possible link between this molecular motor and cilia-related processes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Sítios de Ligação , Centrossomo/metabolismo , Cílios/genética , Cílios/metabolismo , Sequência Conservada , Humanos , Modelos Moleculares , Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/química , Miosina Tipo V/genética , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas RecombinantesRESUMO
The human proteins FEZ1 (fasciculation and elongation protein zeta 1) and FEZ2 are orthologs of the protein UNC-76 from C. elegans, involved in the growth and fasciculation of the worms axon. Pull down assays showed that the protein FEZ1 interacts with other proteins (e.g., the protein SCOCO, short coiled-coil protein), mitochondria, and vesicles. These components may therefore represent cargoes to be transported along the microtubule, and the transport may be mediated through FEZ1 reported binding to kinesins (KIF3A). We previously showed that FEZ1 dimerizes in its N-terminal region and interacts with other proteins, including the candidate cargoe proteins, through its C-terminus. Here, we studied the fragment FEZ1(92-194) as well as full-length 6xHis-FEZ1 (1-392) in vitro and endogenous FEZ1 isolated from HEK 293 cells and were able to demonstrate the formation of an intermolecular disulfide bond through FEZ1 Cys-133, which appears to be essential for dimerization. This disulfide bond may be important for the FEZ1 role as a dimeric and bivalent transport adaptor molecule, since it establishes a strong link between the monomers, which could be a prerequisite for the simultaneous binding of two cargoes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Dissulfetos/química , Proteínas do Tecido Nervoso/química , Proteômica/métodos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Sequência Conservada , Dissulfetos/metabolismo , Humanos , Espectrometria de Massas , Modelos Moleculares , Anotação de Sequência Molecular , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Multimerização Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Espalhamento a Baixo Ângulo , Alinhamento de Sequência , Difração de Raios XRESUMO
BACKGROUND: The established pathologic criteria for minor salivary gland (MSG) involvement in chronic graft-vs.-host disease (cGVHD) could play a role in monitoring response to therapy. METHODS: We evaluated MSG sequential biopsies during cGVHD therapy in 14 allogeneic bone marrow transplantation (BMT) patients. Nine patients that did not develop GVHD after BMT entered the control group. Biopsies were examined using hematoxylin-eosin, Periodic acid-Schiff (PAS) and leukocyte common antigen staining. RESULTS: A significant loss of PAS+ acinar volume was observed at the diagnosis of cGVHD as much as at the end of treatment when compared with the control group. In the second evaluation, the inflammatory infiltrate was still greater than control group. CONCLUSIONS: The results suggest that persistent xerostomia after cGVHD treatment is because of maintenance of lymphocytic infiltrate and consequent absence of MSG secretory unit recovery. This data may be useful to provide improved insight into the histopathology of this organ involvement.
