Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.23.5 (cathepsin D)
 4,130 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Net changes in the concentrations of 18 amino acids in perfusate and skeletal muscle were followed during perfusion of hemicorpus preparations from fed rat. Perfusate levels of 16 amino acids showed little change from their initial concentrations during the 1st h, but increased dramatically during the 2nd and 3rd h. Aspartate and glutamate levels decreased continuously throughout the perfusion. Release of alanine and glutamine accounted for approximately 50% of the total change in perfusate amino acids. The increase in perfusate amino acids was derived from net breakdown of muscle proteins and not from leakage from the intracellular pool as evidenced by elevated concentrations of intracellular amino acids in perfused muscle. Addition of insulin to the perfusate did not change the pattern of amino acid release during the 1st h of perfusion. However, during the 2nd and 3rd h the hormone completely prevented the net release of most amino acids and maintained intracellular concentrations of most amino acids at levels found in upperfused tissue. Effects of time of perfusion and insulin on amino acid release were accounted for by changes in the rate of protein turnover. Protein synthesis in gastrocemius and psoas muscles in control perfusions decreased after 1 h to approximately 50% of the initial rate. This decrease was accompanied by a 2-fold increase in the level of ribosomal subunits, indicating development of a block in peptide chain initiation. Addition of insulin maintained the initial rate of synthesis and the in vivo level of ribosomal subunits, demonstrating that the hormone prevented the block in peptide chain initiation from forming. Addition of insulin after 2 h reversed the perfusion-induced block in initiation. Synthesis of the specific muscle protein myosin was increased 45% over the control rate in the presence of insulin. Insulin also produced a 50% decrease in the rate of protein degradation during the 2nd and 3rd h of perfusion. A similar effect was noted when protein synthesis was inhibited by addition of cycloheximide. Higher concentrations of insulin were required to maximally inhibit protein degradation than to increase protein synthesis. Involvement of lysosomal proteases in the effect of insulin on protein degradation was evaluated by measuring cathepsin D activity in psoas muscle homogenates. "Free" enzyme activity increased as a result of perfusion while addition of insulin maintained this activity at the unperfused level. Neither perfusion nor insulin had any effect on total cathepsin D activity. Alterations in protein degradation and lysosomal enzyme activity were not due to changes in levels of adenine nucleotides, GTP, or creatine phosphate.
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PMID:Regulation by insulin of amino acid release and protein turnover in the perfused rat hemicorpus. 83 25

The proteolytic maturation of cathepsin D polypeptides was studied in lysosomes isolated from metabolically labeled fibroblasts. In lysosomes isolated from fibroblasts labeled with [35S]methionine, 70-95% of labeled cathepsin D polypeptides were represented by a Mr = 47,000 polypeptide after a 20-min pulse and 75-min chase. When these lysosomes were incubated in vitro, up to 70% of the Mr = 47,000 polypeptide was processed to mature cathepsin D polypeptides. The processing was dependent on the integrity of the lysosomes, had an optimum between pH 6 and 7, and could be stimulated by dithiothreitol and ATP. The noncleavable ATP analogue, adenosine 5'-(beta, gamma-imido)triphosphate, and GTP, CTP, and UTP could not substitute for ATP. The ATP-dependent stimulation was associated with an acidification of lysosomes. It was inhibited by agents that dissipate the lysosomal pH gradient (carbonyl cyanide p-trifluoromethoxyphenylhydrazone, N,N'-dicyclohexylcarbodiimide, nigericin, NH4Cl). A stimulatory effect of ATP was observed also at pH 5.5. The stimulation at pH 5.5 was not associated with acidification of lysosomes and was resistant to protonophores. Inhibitors of lysosomal cysteine proteinases and N-ethylmaleimide inhibited the processing. In the presence of ATP the processing activity was partially protected from inhibition by N-ethylmaleimide. In conclusion, the maturation of cathepsin D in lysosomes depends on cysteine proteinases and is stimulated by the ATP-driven acidification of lysosomes. In addition, ATP stimulates maturation at pH 5.5 by a mechanism not involving the proton pump.
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PMID:Processing of human cathepsin D in lysosomes in vitro. 397 22

Cathepsin L was capable of destroying rabbit muscle aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) activity towards the substrate fructose 1,6-bisphosphate. The rate of loss of activity towards this substrate was stimulated (approx. 2-fold) by physiological concentrations of ATP and to a lesser degree by GTP, CTP, UTP, ADP and cyclic AMP, while PPi and Pi decreased the rate of inactivation. Other proteinases (cathepsin B, cathepsin D, trypsin and chymotrypsin) also decreased aldolase activity toward fructose 1,6-bisphosphate more rapidly in the presence of ATP and more slowly in the presence of Pi. Cathepsin L, at higher concentrations, was capable of inactivating aldolase activity towards fructose 1-phosphate and extensively degrading the enzyme; these reactions were not affected by ATP and Pi. The thermostability of aldolase was also unaffected by these ligands. ATP and Pi had no effect on the rates of hydrolysis of other proteins (hemoglobin, bovine serum albumin, casein and azocasein) by cathepsin L. These data indicate that the effects of ATP and Pi are due to interactions of these ligands with aldolase that make the enzyme more vulnerable to limited but not extensive proteolysis; these ligands do not directly affect cathepsin L activity.
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PMID:Inactivation of fructose-1,6-bisphosphate aldolase by cathepsin L. Stimulation by ATP. 669 88

Dynamin is the mammalian homologue to the Drosophila shibire gene product. Mutations in this 100-kD GTPase cause a pleiotropic defect in endocytosis. To further investigate its role, we generated stable HeLa cell lines expressing either wild-type dynamin or a mutant defective in GTP binding and hydrolysis driven by a tightly controlled, tetracycline-inducible promoter. Overexpression of wild-type dynamin had no effect. In contrast, coated pits failed to become constricted and coated vesicles failed to bud in cells overexpressing mutant dynamin so that endocytosis via both transferrin (Tfn) and EGF receptors was potently inhibited. Coated pit assembly, invagination, and the recruitment of receptors into coated pits were unaffected. Other vesicular transport pathways, including Tfn receptor recycling, Tfn receptor biosynthesis, and cathepsin D transport to lysosomes via Golgi-derived coated vesicles, were unaffected. Bulk fluid-phase uptake also continued at the same initial rates as wild type. EM immunolocalization showed that membrane-bound dynamin was specifically associated with clathrin-coated pits on the plasma membrane. Dynamin was also associated with isolated coated vesicles, suggesting that it plays a role in vesicle budding. Like the Drosophila shibire mutant, HeLa cells overexpressing mutant dynamin accumulated long tubules, many of which remained connected to the plasma membrane. We conclude that dynamin is specifically required for endocytic coated vesicle formation, and that its GTP binding and hydrolysis activities are required to form constricted coated pits and, subsequently, for coated vesicle budding.
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PMID:Induction of mutant dynamin specifically blocks endocytic coated vesicle formation. 796 76

RAP1A protein is a small Ras-like GTPase that accumulates during muscle differentiation. In this study, we observed variable intracellular location of the endogenous RAP1A protein and concomitant relocation of the late endocytic compartments in differentiating myogenic cells. By monitoring the nucleotide-bound form of RAP1A protein, we established that the various protein localizations were related to the GTP/GDP-bound state. To carry on our study, we raised stable myogenic cell lines overexpressing wild-type or mutated forms of RAP1A. Myoblasts overexpressing the GTP-bound mutant did not display specific changes of RAP1A and of late endocytic compartments locations. In contrast, the GDP-bound mutant clustered with acidic structures in the perinuclear region of myoblasts. In addition, we observed that overexpression of GDP-bound RAP1A protein induces disturbances in the maturation process of the lysosomal enzyme cathepsin D. Whereas ectopic expression of wild-type or GTP-bound RAP1A proteins inhibited myogenic differentiation, the GDP-bound mutant favors myotubes formation. From our results, we propose that RAP1A protein may regulate the morphological organization of the late endocytic compartments and therefore affect the intracellular degradations occurring during myogenic differentiation.
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PMID:RAP1A GTP/GDP cycles determine the intracellular location of the late endocytic compartments and contribute to myogenic differentiation. 988 15

The transport and sorting of soluble and membrane-associated macromolecules arriving at endosomal compartments require a complex set of Rab proteins. Rab22a has been localized to the endocytic compartment; however, very little is known about the function of Rab22a and inconsistent results have been reported in studies performed in different cell lines. To characterize the function of Rab22a in endocytic transport, the wild-type protein (Rab22a WT), a hydrolysis-deficient mutant (Rab22a Q64L), and a mutant with reduced affinity for GTP (Rab22a S19N) were expressed in CHO cells. None of the three Rab22a constructs affected the transport of rhodamine-dextran to lysosomes, the digestion of internalized proteins, or the lysosomal localization of cathepsin D. In contrast with the mild effect of Rab22a on the endosome-lysosome route, cells expressing Rab22a WT and Rab22a Q64L presented a strong delay in the retrograde transport of cholera toxin from endosomes to the Golgi apparatus. Moreover, these cells accumulated the cation independent mannose 6-phosphate receptor in endosomes. These observations indicate that Rab22a can affect the trafficking from endosomes to the Golgi apparatus probably by promoting fusion among endosomes and impairing the proper segregation of membrane domains required for targeting to the trans-Golgi network (TGN).
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PMID:Overexpression of Rab22a hampers the transport between endosomes and the Golgi apparatus. 1574 82

CVAK104 is a novel coated vesicle-associated protein with a serine/threonine kinase homology domain that was recently shown to phosphorylate the beta2-subunit of the adaptor protein (AP) complex AP2 in vitro. Here, we demonstrate that a C-terminal segment of CVAK104 interacts with the N-terminal domain of clathrin and with the alpha-appendage of AP2. CVAK104 localizes predominantly to the perinuclear region of HeLa and COS-7 cells, but it is also present on peripheral vesicular structures that are accessible to endocytosed transferrin. The distribution of CVAK104 overlaps extensively with that of AP1, AP3, the mannose 6-phosphate receptor, and clathrin but not at all with its putative phosphorylation target AP2. RNA interference-mediated clathrin knockdown reduced the membrane association of CVAK104. Recruitment of CVAK104 to perinuclear membranes of permeabilized cells is enhanced by guanosine 5'-O-(3-thio)triphosphate, and brefeldin A redistributes CVAK104 in cells. Both observations suggest a direct or indirect requirement for GTP-binding proteins in the membrane association of CVAK104. Live-cell imaging showed colocalization of green fluorescent protein-CVAK104 with endocytosed transferrin and with red fluorescent protein-clathrin on rapidly moving endosomes. Like AP1-depleted COS-7 cells, CVAK104-depleted cells missort the lysosomal hydrolase cathepsin D. Together, our data suggest a function for CVAK104 in clathrin-dependent pathways between the trans-Golgi network and the endosomal system.
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PMID:Clathrin-dependent association of CVAK104 with endosomes and the trans-Golgi network. 1691 21

Regulated secretory vesicles produce, store, and secrete active peptide hormones and neurotransmitters that function in cell-cell communication. To gain knowledge of the protein systems involved in such secretory vesicle functions, we analyzed proteins in the soluble and membrane fractions of dense core secretory vesicles purified from neuroendocrine chromaffin cells. Soluble and membrane fractions of these vesicles were subjected to SDS-PAGE separation, and proteins from systematically sectioned gel lanes were identified by microcapillary LC-MS/MS (microLC-MS/MS) of tryptic peptides. The identified proteins revealed functional categories of prohormones, proteases, catecholamine neurotransmitter metabolism, protein folding, redox regulation, ATPases, calcium regulation, signaling components, exocytotic mechanisms, and related functions. Several novel secretory vesicle components involved in proteolysis were identified consisting of cathepsin B, cathepsin D, cystatin C, ubiquitin, and TIMP, as well carboxypeptidase E/H and proprotein convertases that are known to participate in prohormone processing. Significantly, the membrane fraction exclusively contained an extensive number of GTP nucleotide-binding proteins related to Rab, Rho, and Ras signaling molecules, together with SNARE-related proteins and annexins that are involved in trafficking and exocytosis of secretory vesicle components. Membranes also preferentially contained ATPases that regulate proton translocation. These results implicate membrane-specific functions for signaling and exocytosis that allow these secretory vesicles to produce, store, and secrete active peptide hormones and neurotransmitters released from adrenal medulla for the control of physiological functions in health and disease. In summary, this proteomic study illustrates secretory vesicle protein systems utilized for the production and secretion of regulatory factors that control neuroendocrine functions.
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PMID:Proteomics of neuroendocrine secretory vesicles reveal distinct functional systems for biosynthesis and exocytosis of peptide hormones and neurotransmitters. 1740 50

Rab7b is a recently identified member of the Rab GTPase protein family and has high similarity to Rab7. It has been reported that Rab7b is lysosome associated, that it is involved in monocytic differentiation and that it promotes lysosomal degradation of TLR4 and TLR9. Here we investigated further the localization and function of this GTPase. We found that wild-type Rab7b is lysosome associated whereas an activated, GTP-bound form of Rab7b localizes to the Golgi apparatus. In contrast to Rab7, Rab7b is not involved in EGF and EGFR degradation. Depletion of Rab7b or expression of Rab7b T22N, a Rab7b dominant-negative mutant, impairs cathepsin-D maturation and causes increased secretion of hexosaminidase. Moreover, expression of Rab7b T22N or depletion of Rab7b alters TGN46 distribution, cation-independent mannose-6-phosphate receptor (CI-MPR) trafficking, and causes an increase in the levels of the late endosomal markers CI-MPR and cathepsin D. Vesicular stomatitis virus G protein (VSV-G) trafficking, by contrast, is normal in Rab7b-depleted or Rab7b-T22N-expressing cells. In addition, depletion of Rab7b prevents cholera toxin B-subunit from reaching the Golgi. Altogether, these data indicate that Rab7b is required for normal lysosome function, and, in particular, that it is an essential factor for retrograde transport from endosomes to the trans-Golgi network (TGN).
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PMID:Rab7b controls trafficking from endosomes to the TGN. 2037 62

Burkholderia pseudomallei is a gram-negative, facultative intracellular bacterium, which causes a disease known as melioidosis. Professional phagocytes represent a crucial first line of innate defense against invading pathogens. Uptake of pathogens by these cells involves the formation of a phagosome that matures by fusing with early and late endocytic vesicles, resulting in killing of ingested microbes. Host Rab GTPases are central regulators of vesicular trafficking following pathogen phagocytosis. However, it is unclear how Rab GTPases interact with B. pseudomallei to regulate the transport and maturation of bacterial-containing phagosomes. Here, we showed that the host Rab32 plays an important role in mediating antimicrobial activity by promoting phagosome maturation at an early phase of infection with B. pseudomallei. And we demonstrated that the expression level of Rab32 is increased through the downregulation of the synthesis of miR-30b/30c in B. pseudomallei infected macrophages. Subsequently, we showed that B. pseudomallei resides temporarily in Rab32-positive compartments with late endocytic features. And Rab32 enhances phagosome acidification and promotes the fusion of B. pseudomallei-containing phagosomes with lysosomes to activate cathepsin D, resulting in restricted intracellular growth of B. pseudomallei. Additionally, Rab32 mediates phagosome maturation depending on its guanosine triphosphate/guanosine diphosphate (GTP/GDP) binding state. Finally, we report the previously unrecognized role of miR-30b/30c in regulating B. pseudomallei-containing phagosome maturation by targeting Rab32 in macrophages. Altogether, we provide a novel insight into the host immune-regulated cellular pathway against B. pseudomallei infection is partially dependent on Rab32 trafficking pathway, which regulates phagosome maturation and enhances the killing of this bacterium in macrophages.
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PMID:Rab32 GTPase, as a direct target of miR-30b/c, controls the intracellular survival of Burkholderia pseudomallei by regulating phagosome maturation. 3119 52


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