BLOC-2, AP-3, and AP-1 proteins function in concert with Rab38 and Rab32 proteins to mediate protein trafficking to lysosome-related organelles

Lysosome-related organelles (LROs) are synthesized in specialized cell types where they largely coexist with conventional lysosomes. Most of the known cellular transport machinery involved in biogenesis are ubiquitously expressed and shared between lysosomes and LROs. Examples of common components are the adaptor protein complex-3 (AP-3) and biogenesis of lysosome-related organelle complex (BLOC)-2. These protein complexes control sorting and transport of newly synthesized integral membrane proteins from early endosomes to both lysosomes and LROs such as the melanosome. However, it is unknown what factors cooperate with the ubiquitous transport machinery to mediate transport to LROs in specialized cells. Focusing on the melanosome, we show that the ubiquitous machinery interacts with cell type-specific Rab proteins, Rab38 and Rab32, to facilitate transport to the maturing organelle. BLOC-2, AP-3, and AP-1 coimmunoprecipitated with Rab38 and Rab32 from MNT-1 melanocytic cell extracts. BLOC-2, AP-3, AP-1, and clathrin partially colocalized with Rab38 and Rab32 by confocal immunofluorescence microscopy in MNT-1 cells. Rab38- and Rab32-deficient MNT-1 cells displayed abnormal trafficking and steady state levels of known cargoes of the BLOC-2, AP-3, and AP-1 pathways, the melanin-synthesizing enzymes tyrosinase and tyrosinase-related protein-1. These observations support the idea that Rab38 and Rab32 are the specific factors that direct the ubiquitous machinery to mediate transport from early endosomes to maturing LROs. Additionally, analysis of tyrosinase-related protein-2 and total melanin production indicates that Rab32 has unique functions that cannot be carried out by Rab38 in melanosome biogenesis.     

Role of Notch pathway in terminal follicle cell differentiation during Drosophila oogenesis

 During Drosophila oogenesis the body axes are determined by signaling between the oocyte and the somatic follicle cells that surround the egg chamber. A key event in the establishment of oocyte anterior-posterior polarity is the differential patterning of the follicle cell epithelium along the anterior-posterior axis. Both the Notch and epithelial growth factor (EGF) receptor pathways are required for this patterning. To understand how these pathways act in the process we have analyzed markers for anterior and posterior follicle cells accompanying constitutive activation of the EGF receptor, loss of Notch function, and ectopic expression of Delta. We find that a constitutively active EGF receptor can induce posterior fate in anterior but not in lateral follicle cells, showing that the EGF receptor pathway can act only on predetermined terminal cells. Furthermore, Notch function is required at both termini for appropriate expression of anterior and posterior markers, while loss of both the EGF receptor and Notch pathways mimic the Notch loss-of-function phenotype. Ectopic expression of the Notch ligand, Delta, disturbs EGF receptor dependent posterior follicle cell differentiation and anterior-posterior polarity of the oocyte. Our data are consistent with a model in which the Notch pathway is required for early follicle cell differentiation at both termini, but is then repressed at the posterior for proper determination of the posterior follicle cells by the EGF receptor pathway. Received: 5 November 1998 / Accepted: 14 December 1998     

Listen to one another and act in concert (the Renewal Project)

The organization of work on the project "Harm Reduction for Intravenous Drug Users in Kazan" is substantiated. Difficulties in the realization of such projects due to the lack of coordination between different services, also taking part in controlling the spread of drug addiction and ensuring the treatment of addicts, are emphasized. The coordination of actions of various state structures will make it possible to enhance the effectiveness of programs of harm reduction. The expediency of the development of the general state program of primary prophylaxis, realized by trained specialists with the use of effective technology, is discussed.     

Refined characterization of corneodesmosin proteolysis during terminal differentiation of human epidermis and its relationship to desquamation

Corneodesmosin is a putative adhesion glycoprotein located in the extracellular part of the desmosomes in the upper layers of the epidermis. Synthesized by granular keratinocytes as a 52-56-kDa protein, corneodesmosin is progressively proteolysed during corneocyte maturation. This processing is a prerequisite for desquamation. Two glycine- and serine-rich domains of the protein might take on the conformation of adhesive secondary structures similar to glycine loops. Corneodesmosin proteolysis was further characterized. Deglycosylation experiments and reactivity with lectins demonstrated that the corneodesmosin carbohydrate moiety does not prevent the proteolysis. Immunoblotting, immunohistochemistry, and immunoelectron microscopy experiments using affinity-purified anti-peptide antibodies raised to four of the five structural domains of corneodesmosin and a monoclonal antibody against its fifth central domain showed that the first step in corneodesmosin processing is the cleavage of its extremities and probably occurs before its incorporation into desmosomes. Then the glycine loop-related domains are cleaved, first the N-terminal and then part of the C-terminal domain. At the epidermis surface, the multistep proteolytic cleavage leaves intact only the central domain, which was detected on exfoliated corneocytes and probably lacks adhesive properties. Importantly, corneodesmosin was demonstrated to be a preferred substrate of two serine proteases involved in desquamation, the stratum corneum tryptic and chymotryptic enzymes.     

Plk1 and CK2 act in concert to regulate Rad51 during DNA double strand break repair

Homologous recombination (HR) plays an important role in the maintenance of genome integrity. HR repairs broken DNA during S and G2 phases of the cell cycle but its regulatory mechanisms remain elusive. Here, we report that Polo-like kinase 1 (Plk1), which is vital for cell proliferation and is frequently upregulated in cancer cells, phosphorylates the essential Rad51 recombinase at serine 14 (S14) during the cell cycle and in response to DNA damage. Strikingly, S14 phosphorylation licenses subsequent Rad51 phosphorylation at threonine 13 (T13) by casein kinase 2 (CK2), which in turn triggers direct binding to the Nijmegen breakage syndrome gene product, Nbs1. This mechanism facilitates Rad51 recruitment to damage sites, thus enhancing cellular resistance to genotoxic stresses. Our results uncover a role of Plk1 in linking DNA damage recognition with HR repair and suggest a molecular mechanism for cancer development associated with elevated activity of Plk1.     

Common single-nucleotide polymorphisms act in concert to affect plasma levels of high-density lipoprotein cholesterol

The identification of DNA sequence variants underlying human complex phenotypes remains a significant challenge for several reasons: individual variants can have small phenotypic effects or low population frequencies, and multiple allelic variants may act in concert to affect a trait. We evaluated the combined effect of allelic variants in seven genes involved in high-density lipoprotein (HDL) metabolism, using forward stepwise regression. Analysis of all known common single-nucleotide polymorphisms (SNPs) in the seven candidate genes revealed four variants that were associated with incremental changes in HDL cholesterol levels in three independent samples. Conversely, analysis of 660 polymorphisms in eight genes that do not appear to be involved in HDL metabolism did not identify any associations with plasma HDL-cholesterol levels. These data indicate that several common SNPs act in concert to influence plasma levels of HDL cholesterol.     

The Hop2 and Mnd1 proteins act in concert with Rad51 and Dmc1 in meiotic recombination

During the first meiotic division, homologous chromosomes (homologs) have to separate to opposite poles of the cell to ensure the right complement in the progeny. Homologous recombination provides a mechanism for a genome-wide homology search and physical linkage among the homologs before their orderly segregation. Rad51 and Dmc1 recombinases are the major players in these processes. Disruption of meiosis-specific HOP2 or MND1 genes leads to severe defects in homologous synapsis and an early-stage recombination failure resulting in sterility. Here we show that mouse Hop2 can efficiently form D-loops, the first recombination intermediates, but this activity is abrogated upon association with Mnd1. Furthermore, the Hop2-Mnd1 heterodimer physically interacts with Rad51 and Dmc1 recombinases and stimulates their activity up to 35-fold. Our data reveal an interplay among Hop2, Mnd1 and Rad51 and Dmc1 in the formation of the first recombination intermediates during meiosis.     

Ligand-receptor and receptor-receptor interactions act in concert to activate signaling in the Drosophila toll pathway

In Drosophila, the signaling pathway mediated by the Toll receptor is critical for the establishment of embryonic dorso-ventral pattern and for innate immune responses to bacterial and fungal pathogens. Toll is activated by high affinity binding of the cytokine Sp?tzle, a dimeric ligand of the cystine knot family. In vertebrates, a related family of Toll-like receptors play a critical role in innate immune responses. Despite the importance of this family of receptors, little is known about the biochemical events that lead to receptor activation and signaling. Here, we show that Sp?tzle binds to the N-terminal region of Toll and, using biophysical methods, that the binding is complex. The two binding events that cause formation of the cross-linked complex are non-equivalent: the first Toll ectodomain binds Sp?tzle with an affinity 3-fold higher than the second molecule suggesting that pathway activation involves negative cooperativity. We further show that the Toll ectodomains are able to form low affinity dimers in solution and that juxtamembrane sequences of Toll are critical for the activation or derepression of the pathway. These results, taken together, suggest a mechanism of signal transduction that requires both ligand-receptor and receptor-receptor interactions.     

Four cysteines of the membrane protein DsbB act in concert to oxidize its substrate DsbA

Protein disulfide bond formation in Escherichia coli is catalyzed by the periplasmic protein DsbA. A cytoplasmic membrane protein DsbB maintains DsbA in the oxidized state by transferring electrons from DsbA to quinones in the respiratory chain. Here we show that DsbB activity can be reconstituted by co-expression of N- and C-terminal fragments of the protein, each containing one of its redox-active disulfide bonds. This system has allowed us (i) to demonstrate that the two DsbB redox centers interact directly through a disulfide bond formed between the two DsbB domains and (ii) to identify the specific cysteine residues involved in this covalent interaction. Moreover, we are able to capture an intermediate in the process of electron transfer from one redox center to the other. These results lead us to propose a model that describes how the cysteines cooperate in the early stages of oxidation of DsbA. DsbB appears to adopt a novel mechanism to oxidize DsbA, using its two pairs of cysteines in a coordinated reaction to accept electrons from the active cysteines in DsbA.     

Biochemical identification and immunological localization of two non-keratin polypeptides associated with the terminal differentiation of avian scale epidermis

Summary The expression of two previously uncharacterized polypeptides produced in epidermal cells of chick reticulate and scutate scales during late embryonic scale histogenesis and in hatchling birds has been studied biochemically and immunologically. These polypeptides have been identified by two-dimensional pH gradient gel electrophoresis as basic in charge, with apparent molecular weights of 20 and 23 kD, and they have been characterized immunologically and by amino acid analysis as non-keratin in nature. Monoclonal antibodies which react with both polypeptides have been used for immunohistochemical and immunogold electron-microscopic localization. Immunoreactivity was observed in suprabasal cells of reticulate scale epidermis, where it codistributed with bundles of -type cytokeratins in the -keratin-rich layers of epidermis known as the alpha stratum and in suprabasal cells of the outer epidermal surface of scutate scales, where it codistributed with -and -type keratin filament bundles in the -keratin-rich layers of epidermis known as the beta stratum.