scholarly journals Detailed analyses of the crucial functions of Zn transporter proteins in alkaline phosphatase activation

2020 ◽  
Vol 295 (17) ◽  
pp. 5669-5684 ◽  
Author(s):  
Eisuke Suzuki ◽  
Namino Ogawa ◽  
Taka-aki Takeda ◽  
Yukina Nishito ◽  
Yu-ki Tanaka ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Molecular Mechanisms ◽  
Genetically Engineered ◽  
Minor Role ◽  
Activation Process ◽  
Early Secretory Pathway ◽  
Zip Transporters ◽  
A Minor ◽  
Phosphatase Activation ◽  
Dt40 Cells

Numerous zinc ectoenzymes are metalated by zinc and activated in the compartments of the early secretory pathway before reaching their destination. Zn transporter (ZNT) proteins located in these compartments are essential for ectoenzyme activation. We have previously reported that ZNT proteins, specifically ZNT5–ZNT6 heterodimers and ZNT7 homodimers, play critical roles in the activation of zinc ectoenzymes, such as alkaline phosphatases (ALPs), by mobilizing cytosolic zinc into these compartments. However, this process remains incompletely understood. Here, using genetically-engineered chicken DT40 cells, we first determined that Zrt/Irt-like protein (ZIP) transporters that are localized to the compartments of the early secretory pathway play only a minor role in the ALP activation process. These transporters included ZIP7, ZIP9, and ZIP13, performing pivotal functions in maintaining cellular homeostasis by effluxing zinc out of the compartments. Next, using purified ALP proteins, we showed that zinc metalation on ALP produced in DT40 cells lacking ZNT5–ZNT6 heterodimers and ZNT7 homodimers is impaired. Finally, by genetically disrupting both ZNT5 and ZNT7 in human HAP1 cells, we directly demonstrated that the tissue-nonspecific ALP-activating functions of both ZNT complexes are conserved in human cells. Furthermore, using mutant HAP1 cells, we uncovered a previously-unrecognized and unique spatial regulation of ZNT5–ZNT6 heterodimer formation, wherein ZNT5 recruits ZNT6 to the Golgi apparatus to form the heterodimeric complex. These findings fill in major gaps in our understanding of the molecular mechanisms underlying zinc ectoenzyme activation in the compartments of the early secretory pathway.

scholarly journals Tissue Nonspecific Alkaline Phosphatase Is Activated via a Two-step Mechanism by Zinc Transport Complexes in the Early Secretory Pathway

2011 ◽  
Vol 286 (18) ◽  
pp. 16363-16373 ◽  
Author(s):  
Ayako Fukunaka ◽  
Yayoi Kurokawa ◽  
Fumie Teranishi ◽  
Israel Sekler ◽  
Kimimitsu Oda ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Secretory Pathway ◽  
Molecular Mechanisms ◽  
Zinc Content ◽  
Protein Stabilization ◽  
Zinc Transport ◽  
Early Secretory Pathway ◽  
Tissue Nonspecific Alkaline Phosphatase ◽  
Dt40 Cells ◽  
Step Mechanism

A number of enzymes become functional by binding to zinc during their journey through the early secretory pathway. The zinc transporters (ZnTs) located there play important roles in this step. We have previously shown that two zinc transport complexes, ZnT5/ZnT6 heterodimers and ZnT7 homo-oligomers, are required for the activation of alkaline phosphatases, by converting them from the apo- to the holo-form. Here, we investigated the molecular mechanisms of this activation. ZnT1 and ZnT4 expressed in chicken DT40 cells did not contribute to the activation of tissue nonspecific alkaline phosphatase (TNAP). The reduced activity of TNAP in DT40 cells deficient in both ZnT complexes was not restored by zinc supplementation nor by exogenous expression of other ZnTs that increase the zinc content in the secretory pathway. Moreover, we showed that expression of ZnT5/ZnT6 heterodimers reconstituted with zinc transport-incompetent ZnT5 mutant failed to restore TNAP activity but could stabilize the TNAP protein as the apo-form, regardless of zinc status. These findings demonstrate that TNAP is activated not simply by passive zinc binding but by an elaborate two-step mechanism via protein stabilization followed by enzyme conversion from the apo- to the holo-form with zinc loaded by ZnT complexes in the early secretory pathway.

The role of heredity in cancer.

1989 ◽  
Vol 7 (4) ◽  
pp. 527-540 ◽  
Author(s):  
E G Levine ◽  
R A King ◽  
C D Bloomfield
Keyword(s):  
Molecular Mechanisms ◽  
Familial Cancer ◽  
Tumor Development ◽  
Future Research ◽  
Minor Role ◽  
Research Activity ◽  
Environmental Interactions ◽  
Future Research Directions ◽  
A Minor

Heredity is generally felt to play a minor role in the development of cancer. This review critically examines this assumption. Topics discussed include evidence for heritable predisposition in animals and humans; the potential importance of genetic-environmental interactions; approaches that are being used to successfully locate genes responsible for heritable predisposition; comparability of genetic findings among heritable and corresponding sporadic malignancies; and future research directions. Breast, colon, and lung cancer are used to exemplify clinical and research activity in familial cancer; clinical phenotypes, segregation and linkage analyses, models for environmental interactions with inherited traits, and molecular mechanisms of tumor development are discussed. We conclude that the contribution of heredity to the cancer burden is greater than generally accepted, and that study of heritable predisposition will continue to reveal carcinogenic mechanisms important to the development of all cancers.

NgCAM and VAMP2 Reveal that Direct Delivery and Dendritic Degradation Maintain Axonal Polarity

2021 ◽  
Author(s):  
Alec T. Nabb ◽  
Marvin Bentley
Keyword(s):  
Membrane Proteins ◽  
Molecular Mechanisms ◽  
Minor Role ◽  
Axonal Membrane ◽  
Quantitative Analyses ◽  
Direct Delivery ◽  
Novel Strategy ◽  
Extreme Scale ◽  
A Minor

Neurons are polarized cells of extreme scale and compartmentalization. To fulfill their role in electrochemical signaling, axons must maintain a specific complement of membrane proteins. Despite being subject of considerable attention, the trafficking pathway of axonal membrane proteins is not well understood. Two pathways, direct delivery and transcytosis, have been proposed. Previous studies reached contradictory conclusions about which of these mediates delivery of axonal membrane proteins to their destination, in part because they evaluated long-term distribution changes and not vesicle transport. We developed a novel strategy to selectively label vesicles in different trafficking pathways and determined the trafficking of two canonical axonal membrane proteins, NgCAM and VAMP2. Results from detailed quantitative analyses of transporting vesicles differed substantially from previous studies and found that axonal membrane proteins overwhelmingly undergo direct delivery. Transcytosis plays only a minor role in axonal delivery of these proteins. In addition, we identified a novel pathway by which wayward axonal proteins that reach the dendritic plasma membrane are targeted to lysosomes. These results redefine how axonal proteins achieve their polarized distribution, a crucial requirement for elucidating the underlying molecular mechanisms. [Media: see text] [Media: see text] [Media: see text] [Media: see text]

scholarly journals A fluorimetry-based ssYFP secretion assay to monitor vasopressin-induced exocytosis in LLC-PK1 cells expressing aquaporin-2

2008 ◽  
Vol 295 (6) ◽  
pp. C1476-C1487 ◽  
Author(s):  
Paula Nunes ◽  
Udo Hasler ◽  
Mary McKee ◽  
Hua A. J. Lu ◽  
Richard Bouley ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Immunogold Electron Microscopy ◽  
Minor Role ◽  
Aquaporin 2 ◽  
Egg White Lysozyme ◽  
Amino Acid Signal Peptide ◽  
Immunofluorescence Labeling ◽  
A Minor

Vasopressin (VP)-induced exocytosis was dissected in native and aquaporin-2 (AQP2)-expressing renal LLC-PK1 cells by a fluorimetric exocytosis assay based on soluble secreted yellow fluorescent protein (ssYFP). YFP was targeted to the secretory pathway by addition of an 18-amino acid signal peptide from hen egg white lysozyme. Immunofluorescence labeling, together with analysis of Alexa 555-dextran internalization, revealed that ssYFP is exclusively located in the secretory pathway. Immunofluorescence and immunogold electron microscopy showed significant colocalization of ssYFP and AQP2. Fluorimetry and Western blot analysis demonstrated similar constitutive ssYFP secretion in native LLC-PK1 and AQP2-expressing cells. In AQP2-expressing cells, a twofold increase in ssYFP secretion was observed within 15 min of VP stimulation. This transient burst of ssYFP secretion was abolished by the PKA inhibitor H-89 and was not observed in native cells. The endocytotic inhibitor methyl-β-cyclodextrin, which also promotes membrane accumulation of AQP2, had no effect on ssYFP secretion. Although cells expressing phosphorylation-deficient AQP2-S256A showed significantly lower baseline levels of constitutive secretion, VP induced a significant increase in exocytosis. Our data indicate that 1) this assay can monitor exocytosis in cultured epithelial cells, 2) VP has an acute stimulatory effect on ssYFP secretion in AQP2-expressing, but not native, cells, and 3) phosphorylation of AQP2 at S256 may be involved in the regulation of constitutive AQP2 exocytosis and play only a minor role in the VP-induced burst. These results support the idea that, in addition to its role in reducing AQP2 endocytosis, VP increases AQP2 exocytosis.

Identification of Escherichia coli O157 : H7 genes influencing colonization of the bovine gastrointestinal tract using signature-tagged mutagenesis

Microbiology ◽  
2004 ◽  
Vol 150 (11) ◽  
pp. 3631-3645 ◽  
Author(s):  
Francis Dziva ◽  
Pauline M. van Diemen ◽  
Mark P. Stevens ◽  
Amanda J. Smith ◽  
Timothy S. Wallis
Keyword(s):  
Escherichia Coli ◽  
Molecular Mechanisms ◽  
Minor Role ◽  
Intestinal Colonization ◽  
E Coli ◽  
Type Iii ◽  
Fimbrial Subunit ◽  
Type Iii Protein Secretion ◽  
The Uk ◽  
A Minor

Enterohaemorrhagic Escherichia coli (EHEC) cause acute gastroenteritis in humans that may be complicated by life-threatening systemic sequelae. The predominant EHEC serotype affecting humans in the UK and North America is O157 : H7 and infections are frequently associated with contact with ruminant faeces. Strategies to reduce the carriage of EHEC in ruminants are expected to lower the incidence of human EHEC infections; however, the molecular mechanisms underlying persistence of EHEC in ruminants are poorly understood. This paper reports the first comprehensive survey for EHEC factors mediating colonization of the bovine intestines by using signature-tagged transposon mutagenesis. Seventy-nine E. coli O157 : H7 mutants impaired in their ability to colonize calves were isolated and 59 different genes required for intestinal colonization were identified by cloning and sequencing of the transposon insertion sites. Thirteen transposon insertions were clustered in the locus of enterocyte effacement (LEE), which encodes a type III protein secretion system required for the formation of attaching and effacing lesions on intestinal epithelia. A putative structural component of the apparatus (EscN) is essential for intestinal colonization; however, the type III secreted effector protein Map plays only a minor role. Other Type III secretion-associated genes were implicated in colonization of calves by E. coli O157 : H7, including z0990 (ecs0850), which encodes the non-LEE-encoded type III secreted effector NleD and the closely related z3023 (ecs2672) and z3026 (ecs2674) genes which encode homologues of Shigella IpaH proteins. We also identified a novel fimbrial locus required for intestinal colonization in calves by E. coli O157 : H7 (z2199-z2206; ecs2114-ecs2107/locus 8) and demonstrated that a mutant harbouring a deletion of the putative major fimbrial subunit gene is rapidly out-competed by the parent strain in co-infection studies. Our data provide valuable new information for the development of intervention strategies.

scholarly journals ER-Golgi Traffic Is a Prerequisite for Efficient ER Degradation

2002 ◽  
Vol 13 (6) ◽  
pp. 1806-1818 ◽  
Author(s):  
Christof Taxis ◽  
Frank Vogel ◽  
Dieter H. Wolf
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Protein Quality ◽  
Morphological Changes ◽  
Ubiquitin Proteasome System ◽  
Protein Traffic ◽  
Early Secretory Pathway ◽  
Maturation Rate ◽  
Ubiquitin Proteasome ◽  
A Minor

Protein quality control is an essential function of the endoplasmic reticulum. Misfolded proteins unable to acquire their native conformation are retained in the endoplasmic reticulum, retro-translocated back into the cytosol, and degraded via the ubiquitin-proteasome system. We show that efficient degradation of soluble malfolded proteins in yeast requires a fully competent early secretory pathway. Mutations in proteins essential for ER-Golgi protein traffic severely inhibit ER degradation of the model substrate CPY*. We found ER localization of CPY* in WT cells, but no other specific organelle for ER degradation could be identified by electron microscopy studies. Because CPY* is degraded in COPI coat mutants, only a minor fraction of CPY* or of a proteinaceous factor required for degradation seems to enter the recycling pathway between ER and Golgi. Therefore, we propose that the disorganized structure of the ER and/or the mislocalization of Kar2p, observed in early secretory mutants, is responsible for the reduction in CPY* degradation. Further, we observed that mutations in proteins directly involved in degradation of malfolded proteins (Der1p, Der3/Hrd1p, and Hrd3p) lead to morphological changes of the endoplasmic reticulum and the Golgi, escape of CPY* into the secretory pathway and a slower maturation rate of wild-type CPY.

scholarly journals Hog1 Mitogen-Activated Protein Kinase Plays Conserved and Distinct Roles in the Osmotolerant Yeast Torulaspora delbrueckii

2006 ◽  
Vol 5 (8) ◽  
pp. 1410-1419 ◽  
Author(s):  
María José Hernandez-Lopez ◽  
Francisca Randez-Gil ◽  
José Antonio Prieto
Keyword(s):  
Protein Kinase ◽  
Osmotic Stress ◽  
Molecular Mechanisms ◽  
Mitogen Activated Protein Kinase ◽  
Minor Role ◽  
Hog Pathway ◽  
Protection Mechanism ◽  
Torulaspora Delbrueckii ◽  
Mitogen Activated Protein ◽  
A Minor

ABSTRACT Torulaspora delbrueckii has emerged during evolution as one of the most osmotolerant yeasts. However, the molecular mechanisms underlying this unusual stress resistance are poorly understood. In this study, we have characterized the functional role of the high-osmolarity glycerol (HOG) mitogen-activated protein kinase pathway in mediating the osmotic stress response, among others, in T. delbrueckii. We show that the T. delbrueckii Hog1p homologue TdHog1p is phosphorylated after cell transfer to NaCl- or sorbitol-containing medium. However, TdHog1p plays a minor role in tolerance to conditions of moderate osmotic stress, a trait related mainly with the osmotic balance. In consonance with this, the absence of TdHog1p produced only a weak defect in the timing of the osmostress-induced glycerol and GPD1 mRNA overaccumulation. Tdhog1Δ mutants also failed to display aberrant morphology changes in response to osmotic stress. Furthermore, our data indicate that the T. delbrueckii HOG pathway has evolved to respond to specific environmental conditions and to play a pivotal role in the stress cross-protection mechanism.

scholarly journals IκB-Mediated Inhibition of Virus-Induced Beta Interferon Transcription

1999 ◽  
Vol 73 (4) ◽  
pp. 2694-2702 ◽  
Author(s):  
Michèle Algarté ◽  
Hannah Nguyen ◽  
Christophe Heylbroeck ◽  
Rongtuan Lin ◽  
John Hiscott
Keyword(s):  
Virus Infection ◽  
Sendai Virus ◽  
Gene Activation ◽  
Inhibitory Effect ◽  
Minor Role ◽  
Activation Process ◽  
High Concentration ◽  
Beta Interferon ◽  
Iκb Kinase ◽  
A Minor

ABSTRACT We have examined the consequences of overexpression of the IκBα and IκBβ inhibitory proteins on the regulation of NF-κB-dependent beta interferon (IFN-β) gene transcription in human cells after Sendai virus infection. In transient coexpression studies or in cell lines engineered to express different forms of IκB under tetracycline-inducible control, the IFN-β promoter (−281 to +19) linked to the chloramphenicol acetyltransferase reporter gene was differentially inhibited in response to virus infection. IκBα exhibited a strong inhibitory effect on virus-induced IFN-β expression, whereas IκBβ exerted an inhibitory effect only at a high concentration. Despite activation of the IκB kinase complex by Sendai virus infection, overexpression of the double-point-mutated (S32A/S36A) dominant repressors of IκBα (TD-IκBα) completely blocked IFN-β gene activation by Sendai virus. Endogenous IFN-β RNA production was also inhibited in Tet-inducible TD-IκBα-expressing cells. Inhibition of IFN-β expression directly correlated with a reduction in the binding of NF-κB (p50-RelA) complex to PRDII after Sendai virus infection in IκBα-expressing cells, whereas IFN-β expression and NF-κB binding were only slightly reduced in IκBβ-expressing cells. These experiments demonstrate a major role for IκBα in the regulation of NF-κB-induced IFN-β gene activation and a minor role for IκBβ in the activation process.

scholarly journals Evidence for Segregation of Sphingomyelin and Cholesterol during Formation of Copi-Coated Vesicles

2000 ◽  
Vol 151 (3) ◽  
pp. 507-518 ◽  
Author(s):  
Britta Brügger ◽  
Roger Sandhoff ◽  
Sabine Wegehingel ◽  
Karin Gorgas ◽  
Jörg Malsam ◽  
...  
Keyword(s):  
Cholesterol Synthesis ◽  
Secretory Pathway ◽  
Molecular Mechanisms ◽  
Lipid Analysis ◽  
Coated Vesicles ◽  
Early Secretory Pathway ◽  
Golgi Membranes ◽  
A Cell ◽  
Higher Eukaryotes ◽  
Specific Lipid

In higher eukaryotes, phospholipid and cholesterol synthesis occurs mainly in the endoplasmic reticulum, whereas sphingomyelin and higher glycosphingolipids are synthesized in the Golgi apparatus. Lipids like cholesterol and sphingomyelin are gradually enriched along the secretory pathway, with their highest concentration at the plasma membrane. How a cell succeeds in maintaining organelle-specific lipid compositions, despite a steady flow of incoming and outgoing transport carriers along the secretory pathway, is not yet clear. Transport and sorting along the secretory pathway of both proteins and most lipids are thought to be mediated by vesicular transport, with coat protein I (COPI) vesicles operating in the early secretory pathway. Although the protein constituents of these transport intermediates are characterized in great detail, much less is known about their lipid content. Using nano-electrospray ionization tandem mass spectrometry for quantitative lipid analysis of COPI-coated vesicles and their parental Golgi membranes, we find only low amounts of sphingomyelin and cholesterol in COPI-coated vesicles compared with their donor Golgi membranes, providing evidence for a significant segregation from COPI vesicles of these lipids. In addition, our data indicate a sorting of individual sphingomyelin molecular species. The possible molecular mechanisms underlying this segregation, as well as implications on COPI function, are discussed.

scholarly journals Neuroprogenitor Cells From Patients With TBCK Encephalopathy Suggest Deregulation of Early Secretory Vesicle Transport

2022 ◽  
Vol 15 ◽  
Author(s):  
Danielle de Paula Moreira ◽  
Angela May Suzuki ◽  
André Luiz Teles e Silva ◽  
Elisa Varella-Branco ◽  
Maria Cecília Zorél Meneghetti ◽  
...  
Keyword(s):  
Cell Cycle Progression ◽  
Secretory Pathway ◽  
Molecular Mechanisms ◽  
Secretory Vesicle ◽  
Psychomotor Retardation ◽  
Vesicle Transport ◽  
Early Secretory Pathway ◽  
Pathogenic Variants ◽  
Altered Cell ◽  
Induced Pluripotent

Biallelic pathogenic variants in TBCK cause encephaloneuropathy, infantile hypotonia with psychomotor retardation, and characteristic facies 3 (IHPRF3). The molecular mechanisms underlying its neuronal phenotype are largely unexplored. In this study, we reported two sisters, who harbored biallelic variants in TBCK and met diagnostic criteria for IHPRF3. We provided evidence that TBCK may play an important role in the early secretory pathway in neuroprogenitor cells (iNPC) differentiated from induced pluripotent stem cells (iPSC). Lack of functional TBCK protein in iNPC is associated with impaired endoplasmic reticulum-to-Golgi vesicle transport and autophagosome biogenesis, as well as altered cell cycle progression and severe impairment in the capacity of migration. Alteration in these processes, which are crucial for neurogenesis, neuronal migration, and cytoarchitecture organization, may represent an important causative mechanism of both neurodevelopmental and neurodegenerative phenotypes observed in IHPRF3. Whether reduced mechanistic target of rapamycin (mTOR) signaling is secondary to impaired TBCK function over other secretory transport regulators still needs further investigation.

Sign in / Sign up

Export Citation Format

Share Document