FEZ1 interacts with CLASP2 and NEK1 through coiled-coil regions and their cellular colocalization suggests centrosomal functions and regulation by PKC

FEZ1 was initially described as a neuronal protein that influences axonal development and cell polarization. CLASP2 and NEK1 proteins are present in a centrosomal complex and participate in cell cycle and cell division mechanisms, but their functions were always described individually. Here, we report that NEK1 and CLASP2 colocalize with FEZ1 in a perinuclear region in mammalian cells, and observed that coiled-coil interactions occur between FEZ1/CLASP2 and FEZ1/NEK1 in vitro. These three proteins colocalize and interact with endogenous gamma-tubulin. Furthermore, we found that CLASP2 is phosphorylated and interacts with active PKC isoforms, and that FEZ1/CLASP2 colocalization is inhibited by PMA treatment. Our results provide evidence that these three proteins cooperate in centrosomal functions and open new directions for future studies.338416713545Fundacao de Amparo a Pesquisa do Estado Sao PauloConselho Nacional de Pesquisa e DesenvolvimentoLNL

Human FEZ1 Protein Forms a Disulfide Bond Mediated Dimer: Implications for Cargo Transport

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.9945954603Fundacao de Amparo a Pesquisa do Estado Sao PauloConselho Nacional de Pesquisa e DesenvolvimentoLNBio-CNPE

Late effects of chronic graft-vs.-host disease in minor salivary glands

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.34848649

A spectroscopic analysis of the interaction between the human regulatory proteins RACK1 and Ki-1/57

Ki-1/57 is a 57-kDa cytoplasmic and nuclear protein associated with protein kinase activity and is hyper-phosphorylated on Ser/Thr residues upon cellular activation. In previous studies we identified the receptor of activated kinase-1 (RACK1), a signaling adaptor protein that binds activated PKC, as a protein that interacts with Ki-1/57. Here we demonstrate that the far-UV circular dichroism spectrum of the WD repeat-containing RACK1 protein shows an unusual positive ellipticity at 229 nm, which in other proteins of the WD family has been attributed to surface tryptophans that are quenchable by N-bromo-succinimide (NBS). As well as NBS, in vitro binding of 6xHis-Ki-1/57(122-413) and 6xHis-Ki-1/57(264-413) can also quench the positive ellipticity of the RACK1 spectrum. We generated a model of RACK1 by homology modeling using a G protein beta subunit as template. Our model suggests the family-typical seven-bladed beta-propeller, with an aromatic cluster around the central tunnel that contains four Trp residues (17, 83, 150, 170), which are likely involved in the interaction with Ki-1/57.o TEXTO COMPLETO DESTE ARTIGO, ESTARÁ DISPONÍVEL À PARTIR DE AGOSTO DE 2015.387557758

Structural Analysis of Intermolecular Interactions in the Kinesin Adaptor Complex Fasciculation and Elongation Protein Zeta 1/Short Coiled-Coil Protein (FEZ1/SCOCO)

Cytoskeleton and protein trafficking processes, including vesicle transport to synapses, are key processes in neuronal differentiation and axon outgrowth. The human protein FEZ1 (fasciculation and elongation protein zeta 1 / UNC-76, in C. elegans), SCOCO (short coiled-coil protein / UNC-69) and kinesins (e. g. kinesin heavy chain / UNC116) are involved in these processes. Exploiting the feature of FEZ1 protein as a bivalent adapter of transport mediated by kinesins and FEZ1 protein interaction with SCOCO (proteins involved in the same path of axonal growth), we investigated the structural aspects of intermolecular interactions involved in this complex formation by NMR (Nuclear Magnetic Resonance), cross-linking coupled with mass spectrometry (MS), SAXS (Small Angle X-ray Scattering) and molecular modelling. The topology of homodimerization was accessed through NMR (Nuclear Magnetic Resonance) studies of the region involved in this process, corresponding to FEZ1 (92-194). Through studies involving the protein in its monomeric configuration (reduced) and dimeric state, we propose that homodimerization occurs with FEZ1 chains oriented in an anti-parallel topology. We demonstrate that the interaction interface of FEZ1 and SCOCO defined by MS and computational modelling is in accordance with that previously demonstrated for UNC-76 and UNC-69. SAXS and literature data support a heterotetrameric complex model. These data provide details about the interaction interfaces probably involved in the transport machinery assembly and open perspectives to understand and interfere in this assembly and its involvement in neuronal differentiation and axon outgrowth.810Fundacao de Amparo a Pesquisa do Estado Sao PauloConselho Nacional de Pesquisa e Desenvolvimento [471355/2010-0]Centro Nacional de Pesquisa em Energia e Materiais / Laboratorio Nacional de BiocienciasConselho Nacional de Pesquisa e Desenvolvimento [471355/2010-0

Risk factors and characteristics of ocular complications, and efficacy of autologous serum tears after haematopoietic progenitor cell transplantation.

The objective of the study was to evaluate the frequency and clinical characteristics of ocular complications and their risk factors, as well as autologous serum tears (AST) for the treatment of dry eye in these patients. Data from the files of 124 patients who had undergone allogeneic haematopoietic progenitor cell transplantation (HPCT) were evaluated. In addition, 33 HPCT patients were examined and their data were compared with controls. Analysis of tears and AST was performed. Dry eye manifestation occurred in 32% of patients and was positively correlated with age over 27 years (P = 0.05), peripheral blood progenitor cell transplant (P = 0.002), chronic graft-versus-host disease (P = 0.0027), and chronic or acute myeloid leukaemia (P = 0.001). Dry mouth and Schirmer test < 5 mm were predictive factors for dry eye in HPCT patients (P = 0.002 and odds ratio 3.9 and P = 0.007, odds ratio = 5.9, respectively). Microbiological analysis revealed that six of 11 AST samples were contaminated after 30 days of use. The present study supports the role of potential risk factors for ocular complications and key elements to detect alterations in the tear film from HPCT patients. In addition, AST contamination must be considered after longer periods of use.Journal Articleinfo:eu-repo/semantics/publishe