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Biomedical subjects

W A Maltese

Publications and source records attributed to W A Maltese.

At least 19 recordsLinked to original sources
PubMed2001

Different Rab GTPases associate preferentially with alpha or beta GDP-dissociation inhibitors.

GDIs (GDP-dissociation inhibitors) bind to Rab GTPases and mediate their membrane targeting and recycling. In vitro, most Rabs can bind to either of the major isoforms of GDI, leading to the assumption that the proportion of each specific Rab/GDI complex in vivo reflects the relative abundance of the alpha versus beta forms of GDI. Here we show that when human teratocarcinoma cells (Ntera2) are induced to differentiate into postmitotic neurons (NT2N), there is a major change in the proportion of GDIalpha relative to GDIbeta. Under these conditions, certain Rab GTPases associate preferentially with either GDIalpha or GDIbeta, irrespective of the relative abundance of the GDI isoform. These findings suggest that heretofore unrecognized functional specificity may exist between the two major forms of GDI.

Animals↗
PubMed2001

Membrane targeting of a Rab GTPase that fails to associate with Rab escort protein (REP) or guanine nucleotide dissociation inhibitor (GDI).

The targeting of various Rab proteins to different subcellular compartments appears to be determined by variable amino acid sequences located upstream from geranylgeranylated cysteine residues in the C-terminal tail. All nascent Rab proteins are prenylated by geranylgeranyltransferase II, which recognizes the Rab substrate only when it is bound to Rab escort protein (REP). After prenylation, REP remains associated with the modified Rab until it is delivered to the appropriate subcellular membrane. It remains unclear whether docking of the Rab with the correct membrane is solely a function of features contained within the prenylated Rab itself (with REP serving as a "passive" carrier) or whether REP actively participates in the targeting process. To address this issue, we took advantage of a mutation in the alpha2 helix of Rab1B (i.e. Y78D) that abolishes REP and GDI interaction without disrupting nucleotide binding or hydrolysis. These studies demonstrate that replacing the C-terminal GGCC residues of Rab1B(Y78D) with a CLLL motif permits this protein to be prenylated by geranylgeranyltransferase I but not II both in cell-free enzyme assays and in transfected cells. Subcellular fractionation and immunofluorescence studies reveal that the prenylated Rab1B(Y78D)CLLL, which remains deficient in REP and GDI association is, nonetheless, delivered to the Golgi and endoplasmic reticulum (ER) membranes. When the dominant-negative S22N mutation was inserted into Rab1B-CLLL, the resulting monoprenylated construct suppressed ER --> Golgi protein transport. However, when the Y78D mutation was added to the latter construct, its inhibitory effect on protein trafficking was lost despite the fact that it was localized to the ER/Golgi membrane. Therefore, protein interactions mediated by the alpha2 helical domain of Rab1B(S22N) appear to be essential for its functional interaction with components of the ER --> Golgi transport machinery.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Retention of the Alzheimer's amyloid precursor fragment C99 in the endoplasmic reticulum prevents formation of amyloid beta-peptide.

gamma-Secretase is a membrane-associated endoprotease that catalyzes the final step in the processing of Alzheimer's beta-amyloid precursor protein (APP), resulting in the release of amyloid beta-peptide (Abeta). The molecular identity of gamma-secretase remains in question, although recent studies have implicated the presenilins, which are membrane-spanning proteins localized predominantly in the endoplasmic reticulum (ER). Based on these observations, we have tested the hypothesis that gamma-secretase cleavage of the membrane-anchored C-terminal stump of APP (i.e. C99) occurs in the ER compartment. When recombinant C99 was expressed in 293 cells, it was localized mainly in the Golgi apparatus and gave rise to abundant amounts of Abeta. Co-expression of C99 with mutant forms of presenilin-1 (PS1) found in familial Alzheimer's disease resulted in a characteristic elevation of the Abeta(42)/Abeta(40) ratio, indicating that the N-terminal exodomain of APP is not required for mutant PS1 to influence the site of gamma-secretase cleavage. Biogenesis of both Abeta(40) and Abeta(42) was almost completely eliminated when C99 was prevented from leaving the ER by addition of a di-lysine retention motif (KKQN) or by co-expression with a dominant-negative mutant of the Rab1B GTPase. These findings indicate that the ER is not a major intracellular site for gamma-secretase cleavage of C99. Thus, by inference, PS1 localized in this compartment does not appear to be active as gamma-secretase. The results suggest that presenilins may acquire the characteristics of gamma-secretase after leaving the ER, possibly by assembling with other proteins in peripheral membranes.

Alzheimer Disease↗
PubMed2000

Rab24 is an atypical member of the Rab GTPase family. Deficient GTPase activity, GDP dissociation inhibitor interaction, and prenylation of Rab24 expressed in cultured cells.

The function of Rab24 is currently unknown, but other members of the Rab GTPase family are known to participate in various protein trafficking pathways. Rab proteins are thought to cycle on and off vesicle membranes in conjunction with changes in their guanine nucleotide state. The present studies indicate that Rab24 possesses several unusual characteristics that distinguish it from other Rab proteins. 1) Based on [(32)P]orthophosphate labeling of protein-bound nucleotide, Rab24 exists predominantly in the GTP state when expressed in cultured cells. The low GTPase activity is related to the presence of serine instead of glutamine at the position cognate to Ras Gln-61. 2) Posttranslational geranylgeranylation of Rab24, determined by metabolic labeling or detergent partitioning assays, is inefficient when compared with other Rabs ending with the common CXC and CC carboxyl-terminal motifs. This is partly due to the presence of two histidines distal to the target cysteines, but also involves other unidentified features. 3) Most of the Rab24 in the cytoplasmic compartment of cultured cells is not associated with Rab GDP dissociation inhibitors. These findings indicate that, if Rab24 functions in vesicular transport processes, it may operate through a novel mechanism that does not depend on GTP hydrolysis or GDP dissociation inhibitor-mediated recycling.

Amino Acid Sequence↗
PubMed1998

Prenylation of Rab8 GTPase by type I and type II geranylgeranyl transferases.

Rab GTPases are post-translationally modified by addition of geranylgeranyl moieties to carboxyl-terminal cysteine residues. For Rab proteins ending with xxCC xCxC and CCxx motifs this modification is catalysed by geranylgeranyltransferase type II (GGTaseII), and is entirely dependent on the Rab substrate being bound to Rab escort protein (REP). Several Rab proteins contain carboxyl-terminal CaaL prenylation motifs typical of members of the Rho family, which are modified in a REP-independent manner by geranylgeranyltransferase type I (GGTaseI). The present studies show that one such Rab protein (Rab8), which ends with a CVLL motif, is uniquely able to serve as a substrate for either REP/GGTaseII or GGTaseI in cell-free assays. The modification of Rab8 by GGTaseI did not require REP, indicating that a REP-induced conformational change is not essential for exposure of the Rab carboxyl-terminal cysteine prenylation site. To determine whether one enzyme plays a predominant role in Rab8 prenylation in vivo, the incorporation of [3H]mevalonate into Rab8 was measured in human embryonal kidney 293 cells under conditions where the activity of GGTaseI, but not GGTaseII, was blocked by the peptidomimetic inhibitor GGTI-298. The GGTaseI inhibitor did not prevent prenylation of either overexpressed Myc-tagged Rab8 or endogenous Rab8, whereas prenylation of a known GGTaseI substrate with the same carboxyl-terminal motif, Cdc42Hs, was completely blocked. To rule out the possibility that the apparent prenylation of Rab8 by GGTaseII occurs only when GGTaseI activity is eliminated, metabolic labelling studies were carried out in the absence of the GGTaseI inhibitor, using a REP-binding-deficient Rab8 construct (Y78D) that cannot serve as a substrate for GGTaseII, but is indistinguishable from wild-type Rab8 as a substrate for GGTaseI. Prenylation of the Y78D mutant was reduced by 60-70% in intact cells, consistent with the conclusion that the majority of Rab8 is prenylated by the REP/GGTaseII system in vivo.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Presenilin-1 mutations associated with familial Alzheimer's disease do not disrupt protein transport from the endoplasmic reticulum to the Golgi apparatus.

Mutations in genes encoding presenilin-1 (PS1) and presenilin-2 (PS2) have been linked to familial forms of Alzheimer's disease (AD). Cells expressing mutant presenilins produce elevated levels of Abeta42, the major amyloid peptide found in AD plaques. The mechanism whereby this occurs remains unknown, but the localization of presenilins to endoplasmic reticulum (ER) and Golgi compartments has suggested that they may function in intracellular trafficking pathways involved in processing beta-amyloid precursor proteins (APP). To test this possibility, we coexpressed PS1(wt), PS1(M146L), or PS1(L286V) in HEK293 cells together with the LDL receptor, a classic glycoprotein marker that undergoes post-translational O-glycosylation in the Golgi compartment. Pulse-chase analysis of the receptor indicated that mutant presenilins had no effect on ER-->Golgi transport. Similar results were obtained when the studies were carried out with cells expressing the Swedish variant of APP (SWAPP751) instead of the LDL receptor. Moreover, secretion of the soluble exodomain polypeptide fragments of SWAPP751 that arise from alpha-secretase and beta-secretase cleavage was not markedly affected by the PS1 mutants. Despite the lack of discernible effect of the PS1 mutants on trafficking of proteins through the Golgi apparatus, they caused a substantial increase in the proportion of Abeta42 relative to total Abeta in the culture medium. The results suggest that mutant forms of PS1 cause elevated production of Abeta42 by a mechanism that is independent of a major disruption of exocytic trafficking of APP.

Alzheimer Disease↗
PubMed1998

Expression of Rab3A GTPase and other synaptic proteins is induced in differentiated NT2N neurons.

Postmitotic NT2N cells, which are derived from human NT2 teratocarcinoma cells by treatment with retinoic acid (RA) and mitotic inhibitors, are viewed as a good in vitro model for mature neurons of the human central nervous system. Although NT2N cells exhibit many morphological and biochemical characteristics of neurons, the expression of key protein components involved in regulated exocytosis have not been firmly established. Here we show by immunoblot analysis that mature morphologically differentiated NT2N cells contain readily detectable quantities of the synaptic vesicle-associated proteins, synaptobrevin, synapsin, and synaptophysin. They also express the presynaptic plasma membrane protein, SNAP-25, and a Rab GTPase implicated in the control of Ca(2+)-dependent exocytosis, Rab3A. These proteins were not detected in untreated NT2 cells or cells exposed to RA for only 6 d. The induction of an array of proteins known to be involved in the docking and fusion of synaptic vesicles with the plasma membrane provides further support for the validity of NT2N cells as a model for human cortical neurons and suggests that these cells may be useful for in vitro molecular studies of the Ca(2+)-regulated exocytic pathway in nerve terminals.

Biomarkers, Tumor↗
PubMed1998

The putative "switch 2" domain of the Ras-related GTPase, Rab1B, plays an essential role in the interaction with Rab escort protein.

Posttranslational modification of Rab proteins by geranylgeranyltransferase type II requires that they first bind to Rab escort protein (REP). Following prenylation, REP is postulated to accompany the modified GTPase to its specific target membrane. REP binds preferentially to Rab proteins that are in the GDP state, but the specific structural domains involved in this interaction have not been defined. In p21 Ras, the alpha2 helix of the Switch 2 domain undergoes a major conformational change upon GTP hydrolysis. Therefore, we hypothesized that the corresponding region in Rab1B might play a key role in the interaction with REP. Introduction of amino acid substitutions (I73N, Y78D, and A81D) into the putative alpha2 helix of Myc-tagged Rab1B prevented prenylation of the recombinant protein in cell-free assays, whereas mutations in the alpha3 and alpha4 helices did not. Additionally, upon transient expression in transfected HEK-293 cells, the Myc-Rab1B alpha2 helix mutants were not efficiently prenylated as determined by incorporation of [3H]mevalonate. Metabolic labeling studies using [32P]orthophosphate indicated that the poor prenylation of the Rab1B alpha2 helix mutants was not directly correlated with major disruptions in guanine nucleotide binding or intrinsic GTPase activity. Finally, gel filtration analysis of cytosolic fractions from 293 cells that were coexpressing T7 epitope-tagged REP with various Myc-Rab1B constructs revealed that mutations in the alpha2 helix of Rab1B prevented the association of nascent (i.e., nonprenylated) Rab1B with REP. These data indicate that the Switch 2 domain of Rab1B is a key structural determinant for REP interaction and that nucleotide-dependent conformational changes in this region are largely responsible for the selective interaction of REP with the GDP-bound form of the Rab substrate.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Prenylation of a Rab1B mutant with altered GTPase activity is impaired in cell-free systems but not in intact mammalian cells.

Previous studies have reached differing conclusions as to whether or not guanine-nucleotide-dependent conformational changes affect the ability of Rab proteins to undergo post-translational modification by Rab:geranylgeranyltransferase (Rab-GGTase). We now show that the ability of a Rab1B mutant [Q67L (Gln-67-->Leu)] with reduced intrinsic GTPase activity to undergo geranylgeranylation in cell-free assays depends on the guanine nucleotide composition of the system. When GTP is the predominant nucleotide in the assay, Rab1BQ67L is a poor substrate. However, when GDP is present and GTP is omitted, prenylation of the Q67L mutant is comparable with that of the wild-type (WT) protein. These studies, coupled with the poor prenylation of Rab1BWT in the presence of the non-hydrolysable GTP analogue guanosine 5'-[gamma-thio]triphosphate, support the notion that Rab-GGTase prefers substrates in the GDP conformation. When the abilities of Rab1BQ67L and Rab1BWT to undergo prenylation were compared by metabolic labelling of transiently expressed proteins in cultured human 293 cells, we did not observe a decline in prenylation of the mutant protein as predicted on the basis of the cell-free assays. Moreover, the Q67L mutant was comparable with the wild-type Rab1B in its ability to associate with co-expressed Rab GDP dissociation inhibitors in 293 cells. These findings raise the possibility that unidentified proteins present in intact cells may compensate for the reduced intrinsic GTPase activity of the Q67L mutant, allowing a significant proportion of the nascent Rab1BQ67L to assume a GDP conformation. The differential prenylation of Rab1BQ67L in cell-free systems versus intact cells underscores the importance of evaluating the post-translational modification of specific Rab mutants in vivo, where poorly characterized regulatory proteins may have a significant effect on GTPase activity or nucleotide exchange rates.

Amino Acid Sequence↗
PubMed1996

Association of Rab1B with GDP-dissociation inhibitor (GDI) is required for recycling but not initial membrane targeting of the Rab protein.

We have identified the Rab1B effector-domain mutant (D44N) that, when geranylgeranylated by Rab:geranylgeranyltransferase (GGTase II) in cell-free systems or intact cells, fails to form detectable complexes with GDP-dissociation inhibitors (GDIs). GDI-Rab complexes were collected on anti-FLAG affinity beads after incubating recombinant geranylgeranylated Rab1B with FLAG epitope-tagged GDI in vitro, or transiently coexpressing Myc-tagged Rab1B with FLAG-GDI-alpha or FLAG-GDI-2 in human embryonal kidney 293 cells. [3H]Mevalonate labeling and immunoprecipitation studies confirmed that the inability of Myc-Rab1BD44N to associate with GDI in vivo was not due to failure of the mutant to undergo geranylgeranylation. Immunofluorescence localization and immunoblot analysis of subcellular fractions indicated that expressed Myc-Rab1BD44N was efficiently delivered to intracellular membranes in 293 cells. This was confirmed when the fate of the prenylated pool of Rab1BD44N in 293 cells was traced by labeling the geranylgeranyl groups attached to the nascent protein with [3H]meval onate. However, in contrast to the prenylated Rab1BWT, which was distributed in both the membrane and soluble fractions, the prenylated Rab1BD44N was completely absent from the cytosol. Overexpression of Myc-Rab1BD44N did not impair ER --> Golgi glycoprotein trafficking in 293 cells, which was assessed by monitoring the Golgi-dependent processing of coexpressed beta-amyloid precursor protein. The current findings suggest that nascent prenylated Rab1B can be delivered to intracellular membranes in intact cells without forming a stable complex with GDI, but that recycling of prenylated Rab1B to the cytosolic compartment is absolutely dependent on GDI interaction.

Amino Acid Sequence↗
PubMed1996

Differential effects of a Rab6 mutant on secretory versus amyloidogenic processing of Alzheimer's beta-amyloid precursor protein.

The Ras-related GTP-binding protein, Rab6, is localized in late Golgi compartments where it mediates intra-Golgi vesicular trafficking. Herein we report that coexpression of Alzheimer's beta-amyloid precursor protein (beta APP751) with a dominant-negative Rab6 mutant (Rab6N126I) in human embryonal kidney 293 cells causes an increase in secretion of the soluble amino-terminal exodomain (s-APP alpha) derived from non-amyloidogenic processing of beta-APP751 by alpha-secretase. The effect was specific to Rab6N126I, since the corresponding mutation in Rab8 (i.e. Rab8N121I), which has been implicated in protein transport to the plasma membrane, caused a modest reduction in s-APP alpha secretion. While Rab6N126I stimulated secretion of APP alpha, the accumulation of amyloid beta peptide (A beta) in the medium was either moderately reduced or unaffected. Similar differential effects of Rab6N126I on secretion of s-APP alpha versus A beta were observed in cell cultures that were overproducing A beta after transfection with a plasmid encoding Swedish variant of beta APP751. Moreover, assays of medium from the latter cultures revealed a marked increase in secretion of s-APP alpha relative to s-APP beta (the immediate product derived from cleavage of beta APP by beta-secretase). The results indicate that vesicular transport events controlled by Rab6 occur at or near a critical juncture in the trans-Golgi network where beta APP is sorted into either the constitutive alpha-secretase pathway or the amyloidogenic beta-secretase pathway.

Amyloid↗
PubMed1995

Regions outside of the CAAX motif influence the specificity of prenylation of G protein gamma subunits.

A family of GTP-binding regulatory proteins (G proteins) transduces signals across the plasma membrane from a large number of receptors to a smaller number of effectors. Recent studies indicate that a series of post-translational modifications are required for their association with the plasma membrane and for their function. In the case of the G protein gamma subunits, the post-translational modifications include the prenylation of a cysteine residue within a carboxyl-terminal CAAX motif. Although prenylation has been shown to involve the addition of either a C15 farnesyl or a C20 geranylgeranyl group to proteins, the structural requirements and functional consequences of adding different types of prenyl groups to various members of the gamma subunit family have not been examined. In the present study, we have employed the baculovirus expression system to study the structural requirements for attaching different types of prenyl groups to various members of the gamma subunit family. We show that the gamma 2 subunit is modified by a C20 geranylgeranyl group, consistent with the presence of a geranylgeranylation target sequence in this protein. However, we found that the gamma 1 and mutant gamma 2(Ser-71) subunits are modified by both C15 farnesyl and C20 geranylgeranyl groups, despite the presence of an accepted farnesylation target sequence in both of these proteins. Using chimeras of the gamma 1 and gamma 2 subunits, we provide evidence indicating that structural elements upstream of the carboxyl-terminal CAAX motif play a role in the recognition of members of the gamma subunit family by the appropriate insect and mammalian prenyltransferases.

Alkyl and Aryl Transferases↗
PubMed1995

The Ras-related GTP-binding protein, Rab1B, regulates early steps in exocytic transport and processing of beta-amyloid precursor protein.

The role of the Ras-related GTP-binding protein, Rab1B, in intracellular trafficking of beta-amyloid precursor protein (beta APP) was studied in cultured 293 cells. beta APP is processed via one of two alternative routes. In the major secretory pathway, beta APP is cleaved by alpha-secretase within the region comprising the beta-amyloid peptide (A beta), resulting in release of a soluble NH2-terminal exodomain (APP alpha) and a 3-kDa peptide (p3) derived from the carboxyl-terminal tail. In the alternative amyloidogenic pathway, beta APP is cleaved by beta-secretase, with the release of a truncated exodomain (APP beta) and an intact A beta peptide. When beta APP751 was coexpressed with Rab1B(wt) or dominant-negative Rab1B mutants (Rab1BN121I or Rab1BS22N) there was a marked decrease in conversion of the immature Endo-H sensitive form of beta APP751 (108 kDa) to the mature O-glycosylated form of beta APP751 (130 kDa) in cells expressing the mutant forms of Rab1B. The block in Golgi-dependent processing of beta APP was accompanied by inhibition of secretion of APPS (APP alpha). A similar decrease in secretion of APPS (APP alpha+APP beta) was observed in cells that were coexpressing Rab1BN121I with the "Swedish" variant of beta APP751 (i.e. beta APPSW751), which undergoes increased amyloidogenic processing. Coincident with the decline in APPS secretion, the cells coexpressing beta APPSW751 with Rab1BN121I showed a 90% decrease in A beta secretion. The data indicate that Rab1B plays a key role in endoplasmic reticulum-->Golgi transport of beta APP, and that beta APP must pass through a late Golgi compartment before entering either the alpha-secretase or the amyloidogenic beta-secretase pathway. The results also suggest that mutant versions of other Rab proteins that function in different parts of the exocytic and endocytic pathways may be useful in defining the specific routes of beta APP transport involved in the biogenesis of A beta.

Amyloid beta-Protein Precursor↗
PubMed1993

Isoprenylation of Rab1B is impaired by mutations in its effector domain.

Low molecular mass GTP-binding proteins encoded by the Rab gene family are posttranslationally modified by a specific geranylgeranyltransferase (GGTase II), which catalyzes the thioether linkage of geranylgeranyl isoprenoids to cysteines within one of the following carboxyl-terminal sequence motifs: GGCC, CXC, CCSN. Short peptides containing these sequences are poor substrates for isoprenylation in vitro, suggesting that structural domains remote from the carboxyl terminus are required for interactions between Rab proteins and GGTase II. To begin to define these domains, deletions and point mutations were created within the Rab1B gene, and the ability of the mutant translation products to undergo isoprenylation was evaluated in reticulocyte lysates. Deletion of amino acids 2-9 diminished but did not eliminate isoprenylation of Rab1B, suggesting that the extreme amino-terminal region is not absolutely required for interaction with GGTase II. Longer deletions in the amino-terminal region, which probably disrupt the overall conformation of Rab1B, completely prevented isoprenylation. Site-directed mutations predicted to lie in the amino-terminal variable region (Y5N), the beta 3 strand (Q60E), and Loop 7 (A110D) of the Rab1B structure did not reduce isoprenylation. However, two mutations (I41N, D44N) in the effector domain, which appears to mediate interactions with proteins that stimulate GTP hydrolysis or GDP dissociation, essentially abolished the ability of Rab1B to undergo isoprenylation. These findings imply that the effector domain plays a key role in the isoprenylation of Rab proteins, either by serving as a prenyltransferase binding site or by facilitating interactions with accessory proteins that allow Rab1B to assume a specific guanine nucleotide-dependent conformation that is recognized by GGTase II.

Amino Acids↗