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J P Hendrick

Publications and source records attributed to J P Hendrick.

At least 19 recordsLinked to original sources
PubMed2004

Dynamics of {beta}-amyloid reductions in brain, cerebrospinal fluid, and plasma of {beta}-amyloid precursor protein transgenic mice treated with a {gamma}-secretase inhibitor.

gamma-Secretase inhibitors are one promising approach to the development of a therapeutic for Alzheimer's disease (AD). gamma-Secretase inhibitors reduce brain beta-amyloid peptide (Abeta), which is believed to be a major contributor in the etiology of AD. Transgenic mice overexpressing the human beta-amyloid precursor protein (APP) are valuable models to examine the dynamics of Abeta changes with gamma-secretase inhibitors in plaque-free and plaque-bearing animals. BMS-299897 2-[(1R)-1-[[(4-chlorophenyl)sulfony](2,5-difluorophenyl)amino]ethyl]-5-fluorobenzenepropanoic acid, a gamma-secretase inhibitor, showed dose- and time dependent reductions of Abeta in brain, cerebrospinal fluid (CSF), and plasma in young transgenic mice, with a significant correlation between brain and CSF Abeta levels. Because CSF and brain interstitial fluid are distinct compartments in composition and location, this correlation could not be assumed. In contrast, aged transgenic mice with large accumulations of Abeta in plaques showed reductions in CSF Abeta in the absence of measurable changes in plaque Abeta in the brain after up to 2 weeks of treatment. Hence, CSF Abeta levels were a valuable measure of gamma-secretase activity in the central nervous system in either the presence or absence of plaques. Transgenic mice were also used to examine potential side effects due to Notch inhibition. BMS-299897 was 15-fold more effective at preventing the cleavage of APP than of Notch in vitro. No changes in the maturation of CD8(+) thymocytes or of intestinal goblet cells were observed in mice treated with BMS-299897, showing that it is possible for gamma-secretase inhibitors to reduce brain Abeta without causing Notch-mediated toxicity.

Aging↗
PubMed1998

Mass spectrometry of ribosomes and ribosomal subunits.

Nanoflow electrospray ionization has been used to introduce intact Escherichia coli ribosomes into the ion source of a mass spectrometer. Mass spectra of remarkable quality result from a partial, but selective, dissociation of the particles within the mass spectrometer. Peaks in the spectra have been assigned to individual ribosomal proteins and to noncovalent complexes of up to five component proteins. The pattern of dissociation correlates strongly with predicted features of ribosomal protein-protein and protein-RNA interactions. The spectra allow the dynamics and state of folding of specific proteins to be investigated in the context of the intact ribosome. This study demonstrates a potentially general strategy to probe interactions within complex biological assemblies.

Escherichia coli↗
PubMed1997

In vivo observation of polypeptide flux through the bacterial chaperonin system.

The quantitative contribution of chaperonin GroEL to protein folding in E. coli was analyzed. A diverse set of newly synthesized polypeptides, predominantly between 10-55 kDa, interacts with GroEL, accounting for 10%-15% of all cytoplasmic protein under normal growth conditions, and for 30% or more upon exposure to heat stress. Most proteins leave GroEL rapidly within 10-30 s. We distinguish three classes of substrate proteins: (I) proteins with a chaperonin-independent folding pathway; (II) proteins, more than 50% of total, with an intermediate chaperonin dependence for which normally only a small fraction transits GroEL; and (III) a set of highly chaperonin-dependent proteins, many of which dissociate slowly from GroEL and probably require sequestration of aggregation-sensitive intermediates within the GroEL cavity for successful folding.

Bacterial Proteins↗
PubMed1995

The role of molecular chaperones in protein folding.

Folding of newly synthesized polypeptides in the crowded cellular environment requires the assistance of so-called molecular chaperone proteins. Chaperones of the Hsp70 class and their partner proteins interact with nascent polypeptide chains on ribosomes and prevent their premature (mis)folding at least until a domain capable of forming a stable structure is synthesized. For many proteins, completion of folding requires the subsequent interaction with one of the large oligomeric ring-shaped proteins of the chaperonin family, which is composed of the GroEL-like proteins in eubacteria, mitochondria, and chloroplasts, and the TRiC family in eukaryotic cytosol and archaea. These proteins bind partially folded polypeptide in their central cavity and promote folding by ATP-dependent cycles of release and rebinding. In these reactions, molecular chaperones interact predominantly with the hydrophobic surfaces exposed by nonnative polypeptides, thereby preventing incorrect folding and aggregation.

Animals↗
PubMed1993

Control of folding and membrane translocation by binding of the chaperone DnaJ to nascent polypeptides.

Recent evidence supports the view that cellular protein folding may be mediated by molecular chaperones. A fundamental question concerns the stage in its biogenesis at which the folding protein makes first contact with these components. We show here by crosslinking that the chaperone DnaJ binds nascent ribosome-bound polypeptide chains as short as 55 residues. Cotranslational binding of DnaJ to firefly luciferase and chloramphenicol acetyltransferase resulted in an arrest of folding as long as the functional partners of DnaJ in Escherichia coli, DnaK and GrpE, were missing. Protein uptake into microsomes and mitochondria was also interrupted by DnaJ. Both folding and post-translational translocation recommenced upon addition of DnaK and GrpE. We propose that DnaJ protects nascent polypeptide chains against aggregation and, in cooperation with Hsp70, controls their productive folding once a complete polypeptide or a polypeptide domain has been synthesized.

Animals↗
PubMed1991

SecA protein needs both acidic phospholipids and SecY/E protein for functional high-affinity binding to the Escherichia coli plasma membrane.

Translocation of preproteins across the Escherichia coli inner membrane requires acidic phospholipids. We have studied the translocation of the precursor protein proOmpA across inverted inner membrane vesicles prepared from cells depleted of phosphatidylglycerol and cardiolipin. These membranes support neither translocation nor the translocation ATPase activity of the SecA subunit of preprotein translocase. We now report that inner membrane vesicles which are depleted of acidic phospholipids are unable to bind SecA protein with high affinity. These membranes can be restored to translocation competence by fusion with liposomes containing phosphatidylglycerol, suggesting that the defect in SecA binding is a direct effect of phospholipid depletion rather than a general derangement of inner membrane structure. Reconstitution of SecY/E, the membrane-embedded domain of translocase, into proteoliposomes containing predominantly a single synthetic acidic lipid, dioleoylphosphatidylglycerol, allows efficient catalysis of preprotein translocation.

Adenosine Triphosphatases↗
PubMed1990

The binding cascade of SecB to SecA to SecY/E mediates preprotein targeting to the E. coli plasma membrane.

The export of many E. coli proteins such as proOmpA requires the cytosolic chaperone SecB and the membrane-bound preprotein translocase. Translocase is a multisubunit enzyme with the SecA protein as its peripheral membrane domain and the SecY/E protein as its integral domain. SecB, by binding to proOmpA in the cytosol, prevents its aggregation or association with membranes at nonproductive sites. The SecA receptor binds the proOmpA-SecB complex (Kd approximately 6 x 10(-8) M) through direct recognition of both the SecB (Kd approximately 2 x 10(-7) M) as well as the leader and mature domains of the precursor protein. SecB has a dual function in stabilizing the precursor and in passing it on to membrane-bound SecA, the next step in the pathway. SecA itself is bound to the membrane by its affinity (Kd approximately 4 x 10(-8) M) for SecY/E and for acidic lipids. The functions of SecB and SecA as a two-stage receptor system are linked by their affinity for each other.

Bacterial Outer Membrane Proteins↗
PubMed1990

The purified E. coli integral membrane protein SecY/E is sufficient for reconstitution of SecA-dependent precursor protein translocation.

We have previously reconstituted the soluble phase of precursor protein translocation in vitro using purified proteins (the precursor proOmpA, the chaperone SecB, and the ATPase SecA) in addition to isolated inner membrane vesicles. We now report the isolation of the SecY/E protein, the integral membrane protein component of the E. coli preprotein translocase. The SecY/E protein, reconstituted into proteoliposomes, acts together with SecA protein to support translocation of proOmpA, the precursor form of outer membrane protein A. This translocation requires ATP and is strongly stimulated by the protonmotive force. The initial rates and the extents of translocation into either native membrane vesicles or proteoliposomes with pure SecY/E are comparable. The SecY/E protein consists of SecY, SecE, and an additional polypeptide. Antiserum against SecY immunoprecipitates all three components of the SecY/E protein.

Adenosine Triphosphatases↗
PubMed1989

Survey of amino-terminal proteolytic cleavage sites in mitochondrial precursor proteins: leader peptides cleaved by two matrix proteases share a three-amino acid motif.

We have compiled sequences of precursor proteins for 50 mitochondrial proteins for which the mature amino terminus has been determined by amino acid sequence analysis. Included in this set are 8 precursors that have leader peptides that are cleaved in two places by mitochondrial matrix proteases. When these eight leader peptides are aligned and compared, a highly conserved three-amino acid motif is identified as being common to this class of leader peptides. This motif includes an arginine at position -10, a hydrophobic residue at position -8, and serine, threonine, or glycine at position -5 relative to the mature amino terminus. The initial cleavage of these peptides by matrix processing protease occurs within the motif, between residues at -9 and -8, such that arginine at position -10 is at position -2 relative to the cleaved bond. The rest of the motif is within the octapeptide removed by subsequent cleavage catalyzed by intermediate-specific protease. An additional 14 leader peptides in this collection (all of those that contain an arginine at -10) conform to this motif. Assuming that these 14 precursors are matured in two steps, we compared the internal cleavage sites at position -8 with the ends of the other 30 leader peptides in the collection. We find that 74% of matrix processing protease cleavage sites follow an arginine at position -2 relative to cleavage.

Amino Acid Sequence↗
PubMed1988

Two mitochondrial matrix proteases act sequentially in the processing of mammalian matrix enzymes.

The imported precursors of the mammalian matrix enzymes malate dehydrogenase [(S)-malate:NAD+ oxidoreductase, EC 1.1.1.37] and ornithine transcarbamylase (carbamoyl-phosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) are cleaved to their mature subunits in two steps, each catalyzed by matrix-localized processing proteases. The number and properties of these proteases are the subjects of this report. We have identified and characterized two distinct protease activities in a crude matrix fraction from rat liver: processing protease I, which cleaves these precursors to the corresponding intermediate form; and processing protease II, which cleaves the intermediate forms to mature subunits. Protease I is insensitive to chelation by EDTA and to inactivation with N-ethylmaleimide; protease II is inhibited by 5 mM EDTA and is inactivated by treatment with N-ethylmaleimide. We have prepared from mitochondrial matrix an 800-fold-enriched protease I fraction free of protease II activity by using the following steps: ion exchange, hydroxyapatite, molecular sieving, and hydrophobic chromatography. Using similar procedures, we also have prepared an approximately 2000-fold-enriched protease II fraction, which has a trace amount of contaminating protease I. This enriched protease II fraction has little or no cleavage activity toward mitochondrial precursors but rapidly and efficiently converts intermediate forms to mature size. Finally, we show that protease I alone is sufficient to cleave the precursor of a third nuclear-encoded mitochondrial protein subunit--the beta subunit of propionyl-CoA carboxylase [propanoyl-CoA:carbon dioxide ligase (ADP-forming), EC 6.4.1.3]--to its mature size.

Animals↗
PubMed1988

A 5S rRNA/L5 complex is a precursor to ribosome assembly in mammalian cells.

A novel 5S RNA-protein (RNP) complex in human and mouse cells has been analyzed using patient autoantibodies. The RNP is small (approximately 7S) and contains most of the nonribosome-associated 5S RNA molecules in HeLa cells. The 5S RNA in the particle is matured at its 3' end, consistent with the results of in vivo pulse-chase experiments which indicate that this RNP represents a later step in 5S biogenesis than a previously described 5S*/La protein complex. The protein moiety of the 5S RNP has been identified as ribosomal protein L5, which is known to be released from ribosomes in a complex with 5S after various treatments of the 60S subunit. Indirect immunofluorescence indicates that the L5/5S complex is concentrated in the nucleolus. L5 may therefore play a role in delivering 5S rRNA to the nucleolus for assembly into ribosomes.

Animals↗
PubMed1988

Mitochondrial import and processing of mutant human ornithine transcarbamylase precursors in cultured cells.

We have investigated mitochondrial import and processing of the precursor for human ornithine transcarbamylase (OTC; carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) in HeLa cells stably transformed with cDNA sequences encoding OTC precursors carrying mutations in their leader peptides. The mutant precursors studied included two with amino acid substitutions in the 32-amino-acid leader peptide (glycine for arginine at position 23, designated gly23; glycines for arginines at positions 15, 23, and 26, designated gly15,23,26) and two with deletions (deletion of residues 8 to 22, designated d8-22; deletion of residues 17 to 32, designated N16). Specific immunoprecipitation with anti-OTC antiserum of extracts of L-[35S]methionine-labeled cells expressing these mutations yielded only precursor species; neither mature nor intermediate-size OTC subunits were observed. Fractionation of radiolabeled cells, however, revealed important differences among the various mutants: the gly23 precursor was associated with mitochondria and was not detected in the cytosol; the d8-22 and N16 precursors were found with both the mitochondrial fraction and the cytosol; only the gly15,23,26 precursor was detected exclusively in the cytosol. A large fraction of each of the mitochondrially associated OTC species was in a trypsin-protected compartment. In particular, the gly23 precursor behaved in trypsin protection and mitochondrial fractionation studies in a manner consistent with its translocation into the mitochondrial matrix. On the other hand, the lack of binding of the gly23 protein to a delta-N-phosphonoacetyl-L-ornithine affinity column, which specifically recognizes active OTC enzyme, indicated that, despite its intramitochondrial location, the mutant protein did not assemble into the normal, active trimer. Further, the gly23 mutant precursor was unstable within the mitochondria and was degraded with a t1/2 of less further than 4 h. Thus, we have shown that, in intact HeLa cells, cleavage of the OTC leader peptide is not required for translocation into mitochondria, but is required for assembly into active enzyme.

Cytosol↗
PubMed1987

Import and processing of human ornithine transcarbamoylase precursor by mitochondria from Saccharomyces cerevisiae.

Expression of the subunit precursor of the human mitochondrial matrix enzyme ornithine transcarbamoylase (OTCase; EC 2.1.3.3) was programmed in Saccharomyces cerevisiae from a 2-micron plasmid by using an inducible galactose operon promoter. In the presence of the inducing sugar (galactose), two polypeptides were specifically precipitable with anti-OTCase antiserum: the human OTCase precursor (40 kDa); and the mature OTCase subunit (36 kDa). When yeast cells containing these species were lysed and fractionated, the OTCase precursor was found to be associated with mitochondrial membranes, while the mature subunit was found partly with mitochondrial membranes and partly in the soluble mitochondrial matrix-containing fraction. When OTCase enzymatic activity was assayed in fractions similarly derived from an S. cerevisiae strain devoid of yeast OTCase activity (an arg3 mutant) but expressing human OTCase, activity was detected specifically in the mitochondrial matrix fraction. A mutant human OTCase precursor containing an artificial mutation in the NH2-terminal leader peptide (arginine-23 to glycine) was similarly examined. As was previously observed with mammalian mitochondria, this precursor failed both to reach the matrix compartment and to be proteolytically processed; it also failed to exhibit OTCase enzymatic activity. Presence of OTCase enzymatic activity in an arg3 strain expressing wild-type precursor was utilized to obtain selective growth in a medium devoid of arginine but supplemented with the OTCase substrate ornithine. We conclude that, during evolution, the pathway of mitochondrial import utilized by the human OTCase precursor is conserved between yeast and humans, and that, by using selective growth conditions, it may be possible to examine genetically this pathway in S. cerevisiae.

Enzyme Precursors↗
PubMed1987

Import of rat ornithine transcarbamylase precursor into mitochondria: two-step processing of the leader peptide.

The mitochondrial matrix enzyme ornithine transcarbamylase (OTC) is synthesized on cytoplasmic polyribosomes as a precursor (pOTC) with an NH2-terminal extension of 32 amino acids. We report here that rat pOTC synthesized in vitro is internalized and cleaved by isolated rat liver mitochondria in two, temporally separate steps. In the first step, which is dependent upon an intact mitochondrial membrane potential, pOTC is translocated into mitochondria and cleaved by a matrix protease to a product designated iOTC, intermediate in size between pOTC and mature OTC. This product is in a trypsin-protected mitochondrial location. The same intermediate-sized OTC is produced in vivo in frog oocytes injected with in vitro-synthesized pOTC. The proteolytic processing of pOTC to iOTC involves the removal of 24 amino acids from the NH2 terminus of the precursor and utilizes a cleavage site two residues away from a critical arginine residue at position 23. In a second cleavage step, also catalyzed by a matrix protease, iOTC is converted to mature OTC by removal of the remaining eight residues of leader sequence. To define the critical regions in the OTC leader peptide required for these events, we have synthesized OTC precursors with alterations in the leader. Substitution of either an acidic (aspartate) or a "helix-breaking" (glycine) amino acid residue for arginine 23 of the leader inhibits formation of both iOTC and OTC, without affecting translocation. These mutant precursors are cleaved at an otherwise cryptic cleavage site between residues 16 and 17 of the leader. Interestingly, this cleavage occurs at a site two residues away from an arginine at position 15. The data indicate that conversion of pOTC to mature OTC proceeds via the formation of a third discrete species: an intermediate-sized OTC. The data suggest further that, in the rat pOTC leader, the essential elements required for translocation differ from those necessary for correct cleavage to either iOTC or mature OTC.

Animals↗
PubMed1984

RNA required for import of precursor proteins into mitochondria.

A cytoplasmic RNA moiety is necessary for posttranslational uptake of nuclear-encoded mammalian proteins destined for the mitochondrial matrix. Post-translational addition of ribonuclease to a reticulocyte lysate-programmed cell-free translation mixture inhibited subsequent import of six different mitochondrial matrix enzyme precursors into rat liver mitochondria. The required RNA is highly protected, as indicated by the high concentrations of ribonuclease necessary to produce this inhibition. The dependence of the inhibitory effect on temperature, duration of exposure to ribonuclease, and availability of divalent cations is characteristic of the nuclease susceptibility of ribonucleoproteins. The ribonuclease-sensitive component was found in a 400-kilodalton fraction which contains the mitochondrial protein precursors.

Animals↗
PubMed1983

A mammalian tRNAHis-containing antigen is recognized by the polymyositis-specific antibody anti-Jo-1.

The mammalian cell antigen reactive with the autoantibody anti-Jo-1 has been shown to contain tRNAHis. The RNA sequence of this human and mouse cell tRNA was determined in a search for unusual features that might be related to antigenicity. The 5' terminal nucleotide is unique among other sequenced tRNAs in that it is a methylated guanine. The presence of the hypermodified base queuine, which occurs in the wobble position of the anticodon of tRNAHis from several species, was not detected in the tRNAHis immunoprecipitated by anti-Jo-1 from either human HeLa or mouse Friend erytholeukemia cell extracts. The binding of protein(s) appears to confer antigenicity on tRNAHis since either proteinase K treatment or phenol extraction resulted in the loss of immunoprecipitability. However, we have not succeeded in identifying an antigenic protein, and we find that the antigenic complex is not resolved from purified tRNAHis by Sephacryl S-200 column chromatography. Immunofluorescence studies indicate that the antigenic form of tRNAHis is located preferentially in the mammalian cell cytoplasm. The results presented here are discussed in light of an earlier report (1) on the nature of the Jo-1 antigen.

Animals↗