Base-catalyzed reactivation of glucogen phosphorylase reconstituted with a coenzyme-substrate conjugate and its analogues

AbstractGlycogen phosphorylase reconstituted with pyridoxal (5′)diphospho(1)-α-D-glucose (PLDP-Glc) is catalytically inactive but slowly converted to the active enzyme through the cleavage of the pyrophosphate linkage. A similar reaction occurs more rapidly on PLDP-Gal and -Xyl but not on PLDP-Man. Values of pKa for all the reactions are about 8.3, suggesting the participation of a common basic residue in these reactions. Based on the present and other results, it is presumed that Tyr-573 or Lys-574 acts as the base abstracting the proton from 2-hydroxyl group of the glucosyl moiety of PLDP-Glc

SNARE-associated proteins and receptor trafficking

A wide variety of receptors that function on the cell surface are regulated, at least in part, through intracellular membrane trafficking including endocytosis, recycling and subsequent degradation. Soluble N-ethylmaleimide sensitive factor (NSF) attachment protein (SNAP) receptors (SNAREs) are essential molecules for the final step of intracellular membrane trafficking, i.e. fusion of transport vesicles with the target membrane. SNAREs on two opposing membranes form a trans-SNARE complex consisting of a four-helical bundle and drive a membrane fusion. The resultant cis-SNARE complex is disassembled through a process mediated by NSF and SNAPs. Cells contain families of SNAREs, and the interaction of cognate SNAREs at least contributes to the specificity of membrane fusion. The SNARE complex formation and dissociation are modulated by many SNARE-associated proteins at multiple steps including tethering, assembly and disassembly. Diverse molecular mechanisms, such as scaffolding, phosphorylation and ubiquitylation of SNARE proteins, and phosphoinositide production, are utilized for the modulation. In this review, we summarize recent progress in understanding the role of SNARE-associated proteins required for the endocytic recycling and degradation of epidermal growth factor receptor, transferrin receptor and integrins. We also discuss the physiological and pathological relevance of SNAREs and SNARE-associated proteins in the receptor trafficking

TorsinA participates in endoplasmic reticulum-associated degradation

TorsinA is an $AAA^+$ ATPase located within the lumen of the endoplasmic reticulum and nuclear envelope, with a mutant form causing early onset torsion dystonia (DYT1). Here we report a new function for torsinA in endoplasmic reticulum-associated degradation (ERAD). Retro-translocation and proteosomal degradation of a mutant cystic fibrosis transmembrane conductance regulator $(CFTR\Delta F508)$ was inhibited by downregulation of torsinA or overexpression of mutant torsinA, and facilitated by increased torsinA. Retro-translocation of cholera toxin was also decreased by downregulation of torsinA. TorsinA associates with proteins implicated in ERAD, including Derlin-1, VIMP, and p97. Further, torsinA reduces endoplasmic reticulum stress in nematodes overexpressing $CFTR\Delta F508$, and fibroblasts from DYT1 dystonia patients are more sensitive than controls to endoplasmic reticulum stress and less able to degrade mutant CFTR. Therefore, compromised ERAD function in the cells of DYT1 patients may increase sensitivity to endoplasmic reticulum stress with consequent alterations in neuronal function contributing to the disease state

Identification of cell cycle–arrested quiescent osteoclast precursors in vivo

Osteoclasts are multinucleated cells that resorb bone. Although osteoclasts originate from the monocyte/macrophage lineage, osteoclast precursors are not well characterized in vivo. The relationship between proliferation and differentiation of osteoclast precursors is examined in this study using murine macrophage cultures treated with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB (RANK) ligand (RANKL). Cell cycle–arrested quiescent osteoclast precursors (QuOPs) were identified as the committed osteoclast precursors in vitro. In vivo experiments show that QuOPs survive for several weeks and differentiate into osteoclasts in response to M-CSF and RANKL. Administration of 5-fluorouracil to mice induces myelosuppression, but QuOPs survive and differentiate into osteoclasts in response to an active vitamin D3 analogue given to those mice. Mononuclear cells expressing c-Fms and RANK but not Ki67 are detected along bone surfaces in the vicinity of osteoblasts in RANKL-deficient mice. These results suggest that QuOPs preexist at the site of osteoclastogenesis and that osteoblasts are important for maintenance of QuOPs

SNARE-associated proteins and receptor trafficking

A wide variety of receptors that function on the cell surface are regulated, at least in part, through intracellular membrane trafficking including endocytosis, recycling and subsequent degradation. Soluble N-ethylmaleimide sensitive factor (NSF) attachment protein (SNAP) receptors (SNAREs) are essential molecules for the final step of intracellular membrane trafficking, i.e. fusion of transport vesicles with the target membrane. SNAREs on two opposing membranes form a trans-SNARE complex consisting of a four-helical bundle and drive a membrane fusion. The resultant cis-SNARE complex is disassembled through a process mediated by NSF and SNAPs. Cells contain families of SNAREs, and the interaction of cognate SNAREs at least contributes to the specificity of membrane fusion. The SNARE complex formation and dissociation are modulated by many SNARE-associated proteins at multiple steps including tethering, assembly and disassembly. Diverse molecular mechanisms, such as scaffolding, phosphorylation and ubiquitylation of SNARE proteins, and phosphoinositide production, are utilized for the modulation. In this review, we summarize recent progress in understanding the role of SNARE-associated proteins required for the endocytic recycling and degradation of epidermal growth factor receptor, transferrin receptor and integrins. We also discuss the physiological and pathological relevance of SNAREs and SNARE-associated proteins in the receptor trafficking

STX17: an ancient SNARE protein whose roles have not been conserved

Mammalian STX17 (syntaxin 17) plays different cellular roles, including in mitochondrial fission, lipid droplet expansion and macroautophagy/autophagy, depending on the nutritional status. STX17 has a long C-terminal hydrophobic domain (CHD) with a hairpin-like structure, flanked by a basic amino acid-enriched C-terminal tail (C-tail). STX17 is present in a wide variety of eukaryotes, but has been lost in several lineages during evolution. Moreover, the structure of its C-tail remarkably varies, although the CHD is highly conserved. Recently, we compared the localization and function of fly and nematode Syx17/SYX-17 proteins expressed in mammalian cells with that of human STX17. Fly Syx17 expressed in mammalian cells localizes almost exclusively to the cytosol and translocates to autophagosomes upon starvation, whereas nematode SYX-17 is mainly distributed to mitochondria and promotes mitochondrial fission, but does not participate in autophagy. In vivo experiments showed that fly and nematode STX17 homologs are not involved in mitochondrial fission and autophagy, respectively. These results provide important insights into the localization and function of STX17, which acts as a molecular sensor for different nutritional conditions