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Ora A Weisz

Publications and source records attributed to Ora A Weisz.

At least 19 recordsLinked to original sources
PubMed2007

Epithelial Na+ channels are fully activated by furin- and prostasin-dependent release of an inhibitory peptide from the gamma-subunit.

Epithelial sodium channels (ENaC) are expressed in the apical membrane of high resistance Na(+) transporting epithelia and have a key role in regulating extracellular fluid volume and the volume of airway surface liquids. Maturation and activation of ENaC subunits involves furin-dependent cleavage of the ectodomain at two sites in the alpha subunit and at a single site within the gamma subunit. We now report that the serine protease prostasin further activates ENaC by inducing cleavage of the gamma subunit at a site distal to the furin cleavage site. Dual cleavage of the gamma subunit is predicted to release a 43-amino acid peptide. Channels with a gamma subunit lacking this 43-residue tract have increased activity due to a high open probability. A synthetic peptide corresponding to the fragment cleaved from the gamma subunit is a reversible inhibitor of endogenous ENaCs in mouse cortical-collecting duct cells and in primary cultures of human airway epithelial cells. Our results suggest that multiple proteases cleave ENaC gamma subunits to fully activate the channel.

Animals↗
PubMed2006

N-WASP inhibitor wiskostatin nonselectively perturbs membrane transport by decreasing cellular ATP levels.

Wiskott-Aldrich syndrome protein (WASP) and WAVE stimulate actin-related protein (Arp)2/3-mediated actin polymerization, leading to diverse downstream effects, including the formation and remodeling of cell surface protrusions, modulation of cell migration, and intracytoplasmic propulsion of organelles and pathogens. Selective inhibitors of individual Arp2/3 activators would enable more exact dissection of WASP- and WAVE-dependent cellular pathways and are potential therapeutic targets for viral pathogenesis. Wiskostatin is a recently described chemical inhibitor that selectively inhibits neuronal WASP (N-WASP)-mediated actin polymerization in vitro. A growing number of recent studies have utilized this drug in vivo to uncover novel cellular functions for N-WASP; however, the selectivity of wiskostatin in intact cells has not been carefully explored. In our studies with this drug, we observed rapid and dose-dependent inhibition of N-WASP-dependent membrane trafficking steps. Additionally, however, we found that addition of wiskostatin inhibited numerous other cellular functions that are not believed to be N-WASP dependent. Further studies revealed that wiskostatin treatment caused a rapid, profound, and irreversible decrease in cellular ATP levels, consistent with its global effects on cell function. Our data caution against the use of this drug as a selective perturbant of N-WASP-dependent actin dynamics in vivo.

Actin-Related Protein 2-3 Complex↗
PubMed2006

Insulin-like peptide 6: characterization of secretory status and posttranslational modifications.

Insulin-like peptide 6 (Insl6) is a member of the insulin/relaxin superfamily with unknown biological function(s). In the current report, we establish that meiotic and postmeiotic germ cells of the testis are the principal sites of expression of Insl6. Analysis of stably or transiently transfected cells revealed that Insl6 is a secreted protein localized to the endoplasmic reticulum and Golgi. Secretion could be detected in both CHO and GC2 germ cells and was sensitive to brefeldin A treatment. In cell lysates, the predominant Insl6 band was approximately 28 kDa in size. In contrast, the predominant Insl6 species in the supernatant was 8 kDa in size, suggesting posttranslational processing of the precursor protein. Ectopically expressed Insl6 is processed and secreted in furin-deficient LoVo cells and in CHO cells treated with a furin inhibitor, although the size profile of the secreted protein is altered suggesting that Insl6 is a substrate for furin action. Furthermore, mutation of a putative furin cleavage site in the Insl6 peptide resulted in aberrant processing of the Insl6 peptide. Additional investigations of the structure of Insl6 protein provided evidence for posttranslational modifications of Insl6, including the presence of disulfide bonds, glycosylation, and ubiquitination. On the basis of the demonstrated secretory status of Insl6, we speculate that the physical proximity of the germ cell to the Sertoli cell renders the Sertoli cell a likely candidate for Insl6 action.

Amino Acid Sequence↗
PubMed2006

Polarized biosynthetic traffic in renal epithelial cells: sorting, sorting, everywhere.

The maintenance of apical and basolateral membrane domains with distinct protein and lipid compositions is necessary for the proper function of polarized epithelial cells. Delivery of cargo to the basolateral surface is thought to be mediated by the interaction of cytoplasmically disposed sorting signals with sorting receptors, whereas apically destined cargoes are sorted via mechanisms dependent on cytoplasmic, glycan-mediated, or lipid-interacting sorting signals. Apical and basolateral cargo are delivered to the surface in discrete tubular and vesicular carriers that bud from the trans-Golgi network (TGN). While it has long been thought that the TGN is the primary compartment in which apical and basolateral cargoes are segregated, recent studies suggest that sorting may begin earlier along the biosynthetic pathway. Moreover, rather than being delivered directly from the TGN to the cell surface, at least a subset of biosynthetic cargo appears to transit recycling endosomes en route to the plasma membrane. The implications and limitations of these challenges to the conventional model for how proteins are sorted and trafficked along the biosynthetic pathway are discussed.

Animals↗
PubMed2006

Phosphatidylinositol 5-kinase stimulates apical biosynthetic delivery via an Arp2/3-dependent mechanism.

The mechanisms by which polarized epithelial cells target distinct carriers enriched in newly synthesized proteins to the apical or basolateral membrane remain largely unknown. Here we investigated the effect of phosphatidylinositol metabolism and modulation of the actin cytoskeleton, two regulatory mechanisms that have individually been suggested to function in biosynthetic traffic, on polarized traffic in Madin-Darby canine kidney cells. Overexpression of phosphatidylinositol 5-kinase (PI5K) increased actin comet frequency in Madin-Darby canine kidney cells and concomitantly stimulated trans-Golgi network (TGN) to apical membrane delivery of the raft-associated protein influenza hemagglutinin (HA), but did not affect delivery of a non-raft-associated apical protein or a basolateral marker. Modulation of actin comet formation by pharmacologic means, by overexpression of the TGN-localized inositol polyphosphate 5-phosphatase Ocrl, or by blockade of Arp2/3 function had parallel effects on the rate of apical delivery of HA. Moreover, HA released from a TGN block was colocalized in transport carriers in association with PI5K and actin comets. Inhibition of Arp2/3 function in combination with microtubule depolymerization led to a virtual block in HA delivery, suggesting synergistic coordination of these cytoskeletal assemblies in membrane transport. Our results suggest a previously unidentified role for actin comet-mediated propulsion in the biosynthetic delivery of a subset of apical proteins.

Actin-Related Protein 2-3 Complex↗
PubMed2006

Posttranslational cleavage and adaptor protein complex-dependent trafficking of mucolipin-1.

Mucolipin-1 (ML1) is a member of the transient receptor potential ion channel superfamily that is thought to function in the biogenesis of lysosomes. Mutations in ML1 result in mucolipidosis type IV, a lysosomal storage disease characterized by the intracellular accumulation of enlarged vacuolar structures containing phospholipids, sphingolipids, and mucopolysaccharides. Little is known about how ML1 trafficking or activity is regulated. Here we have examined the processing and trafficking of ML1 in a variety of cell types. We find that a significant fraction of ML1 undergoes cell type-independent cleavage within the first extracellular loop of the protein during a late step in its biosynthetic delivery. To determine the trafficking route of ML1, we systematically examined the effect of ablating adaptor protein complexes on the localization of this protein. Whereas ML1 trafficking was not apparently affected in fibroblasts from mocha mice that lack functional adaptor protein complex (AP)-3, small interfering RNA-mediated knockdown revealed a requirement for AP-1 in Golgi export of ML1. Knockdown of functional AP-2 had no effect on ML1 localization. Interestingly, cleavage of ML1 was not compromised in AP-1-deficient cells, suggesting that proteolysis occurs in a prelysosomal compartment, possibly the trans-Golgi network. Our results suggest that posttranslational processing of ML1 is more complex than previously described and that this protein is delivered to lysosomes primarily via an AP-1-dependent route that does not involve passage via the cell surface.

Animals↗
PubMed2006

Recycling of MUC1 is dependent on its palmitoylation.

MUC1 is a mucin-like transmembrane protein expressed on the apical surface of epithelia, where it protects the cell surface. The cytoplasmic domain has numerous sites for phosphorylation and docking of proteins involved in signal transduction. In a previous study, we showed that the cytoplasmic YXXphi motif Y20HPM and the tyrosine-phosphorylated Y60TNP motif are required for MUC1 clathrin-mediated endocytosis through binding AP-2 and Grb2, respectively (Kinlough, C. L., Poland, P. A., Bruns, J. B., Harkleroad, K. L., and Hughey, R. P. (2004) J. Biol. Chem. 279, 53071-53077). Palmitoylation of transmembrane proteins can affect their membrane trafficking, and the MUC1 sequence CQC3RRK at the boundary of the transmembrane and cytoplasmic domains mimics reported site(s) of S-palmitoylation. [3H]Palmitate labeling of Chinese hamster ovary cells expressing MUC1 with mutations in CQC3RRK revealed that MUC1 is dually palmitoylated at the CQC motif independent of RRK. Lack of palmitoylation did not affect the cold detergent solubility profile of a chimera (Tac ectodomain and MUC1 transmembrane and cytoplasmic domains), the rate of chimera delivery to the cell surface, or its half-life. Calculation of rate constants for membrane trafficking of wild-type and mutant Tac-MUC1 indicated that the lack of palmitoylation blocked recycling, but not endocytosis, and caused the chimera to accumulate in a EGFP-Rab11-positive endosomal compartment. Mutations CQC/AQA and Y20N inhibited Tac-MUC1 co-immunoprecipitation with AP-1, although mutant Y20N had reduced rates of both endocytosis and recycling, but a normal subcellular distribution. The double mutant chimera AQA+Y20N had reduced endocytosis and recycling rates and accumulated in EGFP-Rab11-positive endosomes, indicating that palmitoylation is the dominant feature modulating MUC1 recycling from endosomes back to the plasma membrane.

Animals↗
PubMed2006

Phosphatidylinositol 4-phosphate formation at ER exit sites regulates ER export.

The mechanisms that regulate endoplasmic reticulum (ER) exit-site (ERES) assembly and COPII-mediated ER export are currently unknown. We analyzed the role of phosphatidylinositols (PtdIns) in regulating ER export. Utilizing pleckstrin homology domains and a PtdIns phosphatase to specifically sequester or reduce phosphorylated PtdIns levels, we found that PtdIns 4-phosphate (PtsIns4P) is required to promote COPII-mediated ER export. Biochemical and morphological in vitro analysis revealed dynamic and localized PtsIns4P formation at ERES. PtdIns4P was utilized to support Sar1-induced proliferation and constriction of ERES membranes. PtdIns4P also assisted in Sar1-induced COPII nucleation at ERES. Therefore, localized dynamic remodeling of PtdIns marks ERES membranes to regulate COPII-mediated ER export.

Animals↗
PubMed2006

N-glycans mediate apical recycling of the sialomucin endolyn in polarized MDCK cells.

Apical and basolateral proteins are maintained within distinct membrane subdomains in polarized epithelial cells by biosynthetic and postendocytic sorting processes. Sorting of basolateral proteins in these processes has been well studied; however, the sorting signals and mechanisms that direct proteins to the apical surface are less well understood. We previously demonstrated that an N-glycan-dependent sorting signal directs the sialomucin endolyn to the apical surface in polarized Madin-Darby canine kidney cells. Terminal processing of a subset of endolyn's N-glycans is key for polarized biosynthetic delivery to the apical membrane. Endolyn is subsequently internalized, and via a cytoplasmic tyrosine-based sorting motif is targeted to lysosomes from where it constitutively cycles to the cell surface. Here, we examine the polarized sorting of endolyn along the postendocytic pathway in polarized cells. Our results suggest that similar N-glycan sorting determinants are required for apical delivery of endolyn along both the biosynthetic and the postendocytic pathways.

Animals↗
PubMed2006

Role of N- and O-glycans in polarized biosynthetic sorting.

The maintenance of proper epithelial function requires efficient sorting of newly synthesized and recycling proteins to the apical and basolateral surfaces of differentiated cells. Whereas basolateral protein sorting signals are generally confined to their cytoplasmic regions, apical targeting signals have been identified that localize to luminal, transmembrane, and cytoplasmic aspects of proteins. In the past few years, both N- and O-linked glycans have been identified as apical sorting determinants. Glycan structures are extraordinarily diverse and have tremendous information potential. Moreover, because the oligosaccharides added to a given protein can change depending on cell type and developmental stage, the potential exists for altering sorting pathways by modulation of the expression pattern of enzymes involved in glycan synthesis. In this review, we discuss the evidence for glycan-mediated apical sorting along the biosynthetic pathway and present possible mechanisms by which these common and heterogeneous posttranslational modifications might function as specific sorting signals.

Animals↗
PubMed2005

Functions of early (AP-2) and late (AIP1/ALIX) endocytic proteins in equine infectious anemia virus budding.

The proline-rich L domains of human immunodeficiency virus 1 (HIV-1) and other retroviruses interact with late endocytic proteins during virion assembly and budding. In contrast, the YPDL L domain of equine infectious anemia virus (EIAV) is apparently unique in its reported ability to interact both with the mu2 subunit of the AP-2 adaptor protein complex and with ALG-2-interacting protein 1 (AIP1/Alix) protein factors involved in early and late endosome formation, respectively. To define further the mechanisms by which EIAV adapts vesicle trafficking machinery to facilitate virion production, we have examined the specificity of EIAV p9 binding to endocytic factors and the effects on virion production of alterations in early and late endocytic protein expression. The results of these studies demonstrated that (i) an approximately 300-residue region of AIP1/Alix-(409-715) was sufficient for binding to the EIAV YPDL motif; (ii) overexpression of AIP1/Alix or AP-2 mu2 subunit specifically inhibited YPDL-mediated EIAV budding; (iii) virion budding from a replication-competent EIAV variant with its L domain replaced by the HIV PTAP sequence was inhibited by wild type or mutant mu2 to a level similar to that observed when a dominant-negative mutant of Tsg101 was expressed; and (iv) overexpression or siRNA silencing of AIP1/Alix and AP-2 revealed additive suppression of YPDL-mediated EIAV budding. Taken together, these results indicated that both early and late endocytic proteins facilitate EIAV production mediated by either YPDL or PTAP L domains, suggesting a comprehensive involvement of endocytic factors in retroviral assembly and budding that can be accessed by distinct L domain specificities.

Adaptor Protein Complex 2↗
PubMed2005

Distinct Golgi populations of phosphatidylinositol 4-phosphate regulated by phosphatidylinositol 4-kinases.

Phosphatidylinositol 4-phosphate (PI4P) regulates biosynthetic membrane traffic at multiple steps and differentially affects the surface delivery of apically and basolaterally destined proteins in polarized cells. Two phosphatidylinositol 4-kinases (PI4Ks) have been localized to the Golgi complex in mammalian cells, type III PI4Kbeta (PI4KIIIbeta) and type II PI4Kalpha (PI4KIIalpha). Here we report that PI4KIIIbeta and PI4KIIalpha localize to discrete subcompartments of the Golgi complex in Madin-Darby canine kidney (MDCK) cells. PI4KIIIbeta was enriched in early Golgi compartments, whereas PI4KIIalpha colocalized with markers of the trans-Golgi network (TGN). To understand the temporal and spatial control of PI4P generation across the Golgi complex, we quantitated the steady state distribution of a fluorescent PI4P-binding domain relative to cis/medial Golgi and TGN markers in transiently transfected MDCK cells. The density of the signal from this PI4P reporter was roughly 2-fold greater in the early Golgi compartments compared with that of the TGN. Furthermore, this ratio could be modulated in vivo by overexpression of catalytically inactive PI4KIIIbeta and PI4KIIalpha or in vitro by the PI4KIIIbeta inhibitor wortmannin. Our data suggest that both PI4KIIIbeta and PI4KIIalpha contribute to the compartmental regulation of PI4P synthesis within the Golgi complex. We discuss our results with respect to the kinetic effects of modulating PI4K activity on polarized biosynthetic traffic in MDCK cells.

1-Phosphatidylinositol 4-Kinase↗
PubMed2005

Receptor-mediated entry by equine infectious anemia virus utilizes a pH-dependent endocytic pathway.

Previous studies of human and nonhuman primate lentiviral entry mechanisms indicate a predominant use of pH-independent pathways, although more recent studies of human immunodeficiency virus type 1 entry appear to reveal the use of a low-pH-dependent entry pathway in certain target cells. To expand the characterization of the specificity of lentiviral entry mechanisms, we have in the current study examined the entry pathway of equine infectious anemia virus (EIAV) during infection of its natural target, equine macrophages, permissive equine fibroblastic cell lines, and an engineered mouse cell line expressing the recently defined equine lentivirus receptor-1. The specificity of EIAV entry into these various cells was determined by assaying the effects of specific drug treatments on the level of virus entry as measured by quantitative real-time PCR assay of early reverse transcripts or by measurements of virion production. The results of these studies demonstrated that EIAV entry into all cell types was substantially inhibited in a dose-dependent manner by treatment with the vacuolar H+-ATPase inhibitors concanamycin A and bafilomycin A1 or the lysosomotropic weak base ammonium chloride. In contrast, treatments with sucrose to block clathrin-mediated endocytosis or with chloroquine to block organelle acidification failed to inhibit EIAV entry into the same target cells. The observed inhibition of EIAV entry was shown not to be related to cytotoxicity. Taken together, these experiments reveal for the first time that EIAV receptor-mediated entry into target cells is via a low-pH-dependent endocytic pathway.

Ammonium Chloride↗
PubMed2004

ADP-ribosylation factor 1-independent protein sorting and export from the trans-Golgi network.

Polarized epithelial cells efficiently sort newly synthesized apical and basolateral proteins into distinct transport carriers that emerge from the trans-Golgi network (TGN), and this sorting is recapitulated in nonpolarized cells. While the targeting signals of basolaterally destined proteins are generally cytoplasmically disposed, apical sorting signals are not typically accessible to the cytosol, and the transport machinery required for segregation and export of apical cargo remains largely unknown. Here we investigated the molecular requirements for TGN export of the apical marker influenza hemagglutinin (HA) in HeLa cells using an in vitro reconstitution assay. HA was released from the TGN in intact membrane-bound compartments, and export was dependent on addition of an ATP-regenerating system and exogenous cytosol. HA release was inhibited by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) as well as under conditions known to negatively regulate apical transport in vivo, including expression of the acid-activated proton channel influenza M2. Interestingly, release of HA was unaffected by depletion of ADP-ribosylation factor 1, a small GTPase that has been implicated in the recruitment of all known adaptors and coat proteins to the Golgi complex. Furthermore, regulation of HA release by GTPgammaS or M2 expression was unaffected by cytosolic depletion of ADP-ribosylation factor 1, suggesting that HA sorting remains functionally intact in the absence of the small GTPase. These data suggest that TGN sorting and export of influenza HA does not require classical adaptors involved in the formation of other classes of exocytic carriers and thus appears to proceed via a novel mechanism.

ADP-Ribosylation Factor 1↗
PubMed2004

Differential current decay profiles of epithelial sodium channel subunit combinations in polarized renal epithelial cells.

In many epithelial tissues in the body, the rate of Na(+) reabsorption is governed by the activity of the epithelial sodium channel (ENaC). The assembly, trafficking, and turnover of the three ENaC subunits (alpha, beta, and gamma) is complex and not well understood. Recent experiments suggest that ENaC must be proteolytically cleaved for maximal activity and may explain the discrepancies reported in prior biochemical approaches focused on quantitating the trafficking and half-life of full-length subunits. As an alternative approach to examining the dynamics of ENaC subunits, we have generated doxycycline-repressible replication-defective recombinant adenoviruses encoding individual epitope-tagged mouse ENaC subunits and expressed these in polarized MDCK I cells. Co-infection with these viruses encoding all three subunits generates robust amiloride-sensitive currents in polarized MDCK cells. Significant current was also observed in cells expressing alpha- and gamma-mENaC in the absence of beta-mENaC. These currents did not appear to result from association with endogenous canine beta-ENaC. Treatment of alpha beta gamma-expressing cells with cycloheximide (CHX) resulted in the rapid inhibition (within 3 h) of approximately 50-80% of the initial current; however, a sizable fraction of the initial current remained even after 6 h of CHX. By contrast, CHX addition to cells expressing only alpha- and gamma-mENaC resulted in rapid decay in current with no residual fraction. Our data suggest that ENaC channels of differing stoichiometries are differentially trafficked and degraded and provide support for the possibility that noncoordinate trafficking of ENaC subunits may function in vivo as a mechanism to modulate ENaC activity.

Adenoviridae↗
PubMed2004

Differential effects of actin cytoskeleton dynamics on equine infectious anemia virus particle production.

Retrovirus assembly and budding involve a highly dynamic and concerted interaction of viral and cellular proteins. Previous studies have shown that retroviral Gag proteins interact with actin filaments, but the significance of these interactions remains to be defined. Using equine infectious anemia virus (EIAV), we now demonstrate differential effects of cellular actin dynamics at distinct stages of retrovirus assembly and budding. First, virion production was reduced when EIAV-infected cells were treated with phallacidin, a cell-permeable reagent that stabilizes actin filaments by slowing down their depolymerization. Confocal microscopy confirmed that the inhibition of EIAV production correlated temporally over several days with the incorporation dynamics of phallacidin into the actin cytoskeleton. Although the overall structure of the actin cytoskeleton and expression of viral protein appeared to be unaffected, phallacidin treatment dramatically reduced the amount of full-length Gag protein associated with the actin cytoskeleton. These data suggest that an association of full-length Gag proteins with de novo actin filaments might contribute to Gag assembly and budding. On the other hand, virion production was enhanced when EIAV-infected cells were incubated briefly (2 h) with the actin-depolymerizing drugs cytochalasin D and latrunculin B. Interestingly, the enhanced virion production induced by cytochalasin D required a functional late (L) domain, either the EIAV YPDL L-domain or the proline-rich L domains derived from human immunodeficiency virus type 1 or Rous sarcoma virus, respectively. Thus, depolymerization of actin filaments may be a common function mediated by retrovirus L domains during late stages of viral budding. Taken together, these observations indicate that dynamic actin polymerization and depolymerization may be associated with different stages of viral production.

Actins↗
PubMed2003

Specific N-glycans direct apical delivery of transmembrane, but not soluble or glycosylphosphatidylinositol-anchored forms of endolyn in Madin-Darby canine kidney cells.

The sialomucin endolyn is a transmembrane protein with a unique trafficking pattern in polarized Madin-Darby canine kidney cells. Despite the presence of a cytoplasmic tyrosine motif that, in isolation, is sufficient to mediate basolateral sorting of a reporter protein, endolyn predominantly traverses the apical surface en route to lysosomes. Apical delivery of endolyn is disrupted in tunicamycin-treated cells, implicating a role for N-glycosylation in apical sorting. Site-directed mutagenesis of endolyn's eight N-glycosylation sites was used to identify two N-glycans that seem to be the major determinants for efficient apical sorting of the protein. In addition, apical delivery of endolyn was disrupted when terminal processing of N-glycans was blocked using glycosidase inhibitors. Missorting of endolyn occurred independently of the presence or absence of the basolateral sorting signal, because apical delivery was also inhibited by tunicamycin when the cytoplasmic tyrosine motif was mutated. However, we found that apical secretion of a soluble mutant of endolyn was N-glycan independent, as was delivery of glycosylphosphatidylinositol-anchored endolyn. Thus, specific N-glycans are only essential for the apical sorting of transmembrane endolyn, suggesting fundamental differences in the mechanisms by which soluble, glycosylphosphatidylinositol-anchored, and transmembrane proteins are sorted.

Animals↗