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Biomedical subjects

Louis B Hersh

Publications and source records attributed to Louis B Hersh.

At least 19 recordsLinked to original sources
PubMed2007

The vesicular monoamine transporter 2 contains trafficking signals in both its N-glycosylation and C-terminal domains.

The vesicular acetylcholine transporter (VAChT) and the vesicular monoamine transporter (VMAT) belong to the same transporter family that packages acetylcholine into synaptic vesicles (SVs) and biogenic amines into large dense core vesicles (LDCVs) and/or SVs, respectively. These transporters share similarities in sequence and structure with their N- and C-terminal domains located in the cytoplasm. When expressed in PC12 cells, VMAT2 localizes to LDCV, whereas VAChT is found mainly on synaptic-like microvesicles. Previous studies have shown that the cytoplasmic C-terminal domain of VAChT contains signals targeting this transporter to SVs. However, the targeting signals for VMAT have not been completely elucidated. To identify signals targeting VMAT2 to LDCV, the subcellular localization of VMAT2-VAChT chimeras was analyzed in PC12 cells. Chimeras having either the N-terminal region through transmembrane domain 2 of VMAT2 or the C-terminal domain of VMAT2 do not traffic to LDCV efficiently. In contrast, chimeras having both of these regions, or the luminal glycosylated loop in conjunction with transmembrane domains 1 and 2 and the C-terminal domain of VMAT2, traffic to LDCV. Treatment of PC12 cells with 1-deoxymannojirimycin, a specific alpha-mannosidase I inhibitor, causes VMAT2 to localize to synaptic-like microvesicles. The results indicate that both mature N-linked glycosylation and the C-terminus are important for proper trafficking of VMAT2 and that the locations of trafficking signals in VMAT2 and VAChT are surprisingly different.

Animals↗
PubMed2006

Proteolytic fragments of insulysin (IDE) retain substrate binding but lose allosteric regulation.

Treatment of an N-terminal-containing His6-tagged insulysin (His6-IDE) with proteinase K led to the initial cleavage of the His tag and linker region. This was followed by C-terminal cleavages resulting in intermediate fragments of approximately 95 and approximately 76 kDa and finally a relatively stable approximately 56 kDa fragment. The approximately 76 and approximately 56 kDa fragments exhibited a low level of catalytic activity but retained the ability to bind the substrate with a similar affinity as the native enzyme. The kinetics of the reaction of the IDE approximately 76 and approximately 56 kDa proteolytic fragments with a synthetic fluorogenic substrate produced hyperbolic substrate versus velocity curves, rather than the sigmoidal curve obtained with His6-IDE. The approximately 76 and approximately 56 kDa IDE proteolytic fragments were active toward the physiological peptides beta-endorphin, insulin, and amyloid beta peptide 1-40. Although activity was reduced by a factor of approximately 103-104 with these substrates, the relative activity and the cleavage sites were unchanged. Both the approximately 76 and approximately 56 kDa fragments retained the regulatory cationic binding site that binds ATP. Thus, the two proteinase K cleavage fragments of IDE retain the substrate- and ATP-binding sites but have low catalytic activity and lose the allosteric kinetic behavior of IDE. These data suggest a role of the C-terminal region of IDE in allosteric regulation.

Allosteric Regulation↗
PubMed2006

Characterization of the binding of the fluorescent ATP analog TNP-ATP to insulysin.

It has recently been reported that insulin-degrading enzyme (IDE) contains an allosteric site which binds polyanions such as ATP and PPPi. This site is distinct from the catalytic site where homotrophic allosteric effects are produced. In this study, we have characterized the binding of ATP to this anion binding site using the fluorescent ATP analog 2',3'-O-(2,4,6-trinitrophenyl)-adenosine triphosphate (TNP-ATP), which exhibits a higher affinity to the enzyme than ATP itself. TNP-ATP binding to IDE was accompanied by a more than 4-fold increase in fluorescence. The dissociation constant (K(D)) of TNP-ATP was determined as 1.15 microM, while the activation constant (K(A)) was determined to be 1.6 microM. Competition experiments were used to show that ATP (Ki = 1.3 mM) and PPPi (Ki = 0.9mM) bind with a higher affinity than ADP (2.2 mM) and AMP (4.0 mM). Adenosine did not bind to the anion binding site.

Adenosine Triphosphate↗
PubMed2006

Characterization of the REST/NRSF-interacting LIM domain protein (RILP): localization and interaction with REST/NRSF.

We previously identified a nuclear envelope protein repressor element-1 silencing transcription factor (REST)/neuron-restrictive silencer factor (NRSF)-interacting Lin-11, Isl-1 and Mec-3 (LIM) domain protein (RILP) that we proposed functions in the nuclear translocation of the transcriptional repressor REST/NRSF. In this study we assessed the functionality of the prenylation motif, protein kinase A (PKA) phosphorylation sites and nuclear localization sequences (NLSs) of RILP. [(3)H]-mevalonolactone labeled endogenous RILP, showing that RILP is indeed prenylated, while phosphorylation analysis showed that the two PKA sites are phosphorylated. Blocking RILP prenylation, mutating the NLSs or mutating the PKA phosphorylation sites caused RILP to mislocalize to the cytosol. Concurrent with this mislocalization of RILP, REST/NRSF and REST4, which are normally found in the nucleus, co-localized in the cytosol with the RILP mutants. This provides additional evidence that RILP interacts with REST/NRSF and REST4 in vivo, and is involved in the nuclear localization of REST/NRSF and REST4. Reporter gene analysis using the promoter region of the human cholinergic gene locus revealed that these RILP mutants prevented repression of the reporter gene. By trapping REST/NRSF in the cytosol, the RILP mutants prevented translocation to the nucleus where REST/NRSF binds to an RE-1/NRSE element to repress gene transcription. These results show that RILP is required for REST/NRSF nuclear targeting and function.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Tat peptides inhibit neprilysin.

Dementia associated with human immunodeficiency virus (HIV) infection occurs commonly in the aging population and amyloid depositions are noted in the brains of patients with HIV infection in younger age groups. This suggests a dysregulation of amyloid processing in the setting of HIV infection. The Tat protein of HIV has been implicated in the neuropathogenesis of HIV infection due to its neurotoxic and glial activation properties. However, Tat protein and Tat-derived peptides were recently also shown to inhibit neprilysin, the major amyloid beta peptide degrading enzyme in brain, in a cell aggregate system. This effect could contribute to the observed accumulation of amyloid in the brain of HIV-infected patients. The authors report here that peptides derived from the Tat protein, but not Tat protein itself, inhibit homogeneous recombinant neprilysin. This inhibition was found to be competitive and reversible and therefore does not involve covalent bond formation. Tat peptides and Tat protein were slowly hydrolyzed by neprilysin. Thus the accumulation of Tat-derived proteolytic fragments may serve to inhibit neprilysin and increase amyloid beta peptide levels.

AIDS Dementia Complex↗
PubMed2006

Interaction of the brain-specific protein p42IP4/centaurin-alpha1 with the peptidase nardilysin is regulated by the cognate ligands of p42IP4, PtdIns(3,4,5)P3 and Ins(1,3,4,5)P4, with stereospecificity.

The brain-specific protein p42IP4, also called centaurin-alpha1, specifically binds phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. Here, we investigate the interaction of p42IP4/centaurin-alpha1 with nardilysin (NRDc), a member of the M16 family of zinc metalloendopeptidases. Members of this peptidase family exhibit enzymatic activity and also act as receptors for other proteins. We found that p42IP4/centaurin-alpha1 binds specifically to NRDc from rat brain. We further detected that centaurin-alpha2, a protein that is highly homologous to p42IP4/centaurin-alpha1 and expressed ubiquitously, also binds to NRDc. In vivo interaction was demonstrated by co-immunoprecipitation of p42IP4/centaurin-alpha1 with NRDc from rat brain. The acidic domain of NRDc (NRDc-AD), which does not participate in catalysis, is sufficient for the protein interaction with p42IP4. Interestingly, preincubation of p42IP4 with its cognate ligands D-Ins(1,3,4,5)P4 and the lipid diC8PtdIns(3,4,5)P3 negatively modulates the interaction between the two proteins. D-Ins(1,3,4,5)P4 and diC8PtdIns(3,4,5)P3 suppress the interaction with virtually identical concentration dependencies. This inhibition is highly ligand specific. The enantiomer L-Ins(1,3,4,5)P4 is not effective. Similarly, the phosphoinositides diC8PtdIns(3,4)P2, diC8PtdIns(3,5)P2 and diC8PtdIns(4,5)P2 all have no influence on the interaction. Further experiments revealed that endogenous p42IP4 from rat brain binds to glutathione-S-transferase (GST)-NRDc-AD. The proteins dissociate from each other when incubated with D-Ins(1,3,4,5)P4, but not with inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. In summary, we demonstrate that p42IP4 binds to NRDc via the NRDc-AD, and that this interaction is controlled by the cognate cellular ligands of p42IP4/centaurin-alpha1. Thus, specific ligands of p42IP4 can modulate the recruitment of proteins, which are docked to p42IP4, to specific cellular compartments.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Susceptibility of amyloid beta peptide degrading enzymes to oxidative damage: a potential Alzheimer's disease spiral.

Insulysin (IDE) and neprilysin (NEP) were found to be inactivated by oxidation with hydrogen peroxide, an iron-ascorbate oxidation system, and by treatment with 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). In each case reaction led to the introduction of protein carbonyl groups as judged by reaction with 2,4-dintrophenylhydrazine. IDE was inactivated by reaction with 4-hydroxy-2-nonenal (HNE) with the concomitant formation of protein adducts. NEP was not inactivated to a significant extent by HNE, but some HNE-adduct formation did occur. Prior reaction with hydrogen peroxide or AAPH led to enhanced formation of HNE adducts. Treatment of IDE with AAHP or hydrogen peroxide increased its susceptibility to proteolysis, while treatment of NEP with iron/ascorbate or hydrogen peroxide increased its susceptibility to proteolysis. Since IDE and NEP play a prominent role in the clearance of amyloid beta peptides, their oxidative inactivation and enhanced proteolysis can contribute to the onset and/or progression of Alzheimer's disease.

Aldehydes↗
PubMed2005

Environmental enrichment reduces Abeta levels and amyloid deposition in transgenic mice.

Cerebral deposition of beta-amyloid (Abeta) peptides is an invariant pathological hallmark in brains of patients with Alzheimer's disease (AD) and transgenic mice coexpressing familial AD-linked APP and PS1 variants. We now report that exposure of transgenic mice to an "enriched environment" results in pronounced reductions in cerebral Abeta levels and amyloid deposits, compared to animals raised under "standard housing" conditions. The enzymatic activity of an Abeta-degrading endopeptidase, neprilysin, is elevated in the brains of "enriched" mice and inversely correlated with amyloid burden. Moreover, DNA microarray analysis revealed selective upregulation in levels of transcripts encoded by genes associated with learning and memory, vasculogenesis, neurogenesis, cell survival pathways, Abeta sequestration, and prostaglandin synthesis. These studies provide evidence that environmental enrichment leads to reductions in steady-state levels of cerebral Abeta peptides and amyloid deposition and selective upregulation in levels of specific transcripts in brains of transgenic mice.

Alzheimer Disease↗
PubMed2005

Nardilysin facilitates complex formation between mitochondrial malate dehydrogenase and citrate synthase.

Gel filtration chromatography showed that nardilysin activity in a rat testis or rat brain extract exhibited an apparent molecular weight of approximately 300 kDa compared to approximately 187 kDa for the purified enzyme. The addition of purified nardilysin to a rat brain extract, but not to an E. coli extract, produced the higher molecular species. The addition of a GST fusion protein containing the acidic domain of nardilysin eliminated the higher molecular weight nardilysin forms, suggesting that oligomerization involves the acidic domain of nardilysin. Using an immobilized nardilysin column, mitochondrial malate dehydrogenase (mMDH) and citrate synthase (CS) were isolated from a fractionated rat brain extract. Porcine mMDH, but not porcine cytosolic MDH, was shown to form a heterodimer with nardilysin. Mitochondrial MDH increased nardilysin activity about 50%, while nardilysin stabilized mMDH towards heat inactivation. CS was co-immunoprecipitated with mMDH only in the presence of nardilysin showing that nardilysin facilitates complex formation.

Animals↗
PubMed2005

Mutation of active site residues of insulin-degrading enzyme alters allosteric interactions.

The active site glutamate (Glu(111)) and the active site histidine (His(112)) of insulin-degrading enzyme (IDE) were mutated. These mutant enzymes exhibit, in addition to a large decrease in catalytic activity, a change in the substrate-velocity response from a sigmoidal one seen with the native enzyme (Hill coefficient > 2), to a hyperbolic response. With 2-aminobenzoyl-GGFLRKHGQ-N-(2,4-dinitrophenyl)ethylenediamine as substrate, ATP and triphosphate increase the reaction rate of the wild type enzyme some 50-80-fold. This effect is dampened with glutamate mutants to no effect or less than a 3-fold increase in activity and changed to inhibition with the histidine mutants. Sedimentation equilibrium shows the IDE mutants exhibit a similar oligomeric distribution as the wild type enzyme, being predominantly monomeric, with triphosphate having little if any effect on the oligomeric state. Triphosphate did induce aggregation of many of the IDE mutants. Thus, the oligomeric state of IDE does not correlate with kinetic properties. The His(112) mutants were shown to bind zinc, but with a lower affinity than the wild type enzyme. The glutamate mutants displayed an altered cleavage profile for the peptide beta-endorphin. Wild type IDE cleaved beta-endorphin at Leu(17)-Phe(18) and Phe(18)-Lys(19), whereas the glutamate mutants cleaved at these sites, but in addition at Lys(19)-Asn(20) and at Met(5)-Thr(6). Thus, active site mutations of IDE are suggested to not only reduce catalytic activity but also cause local conformational changes that affect the allosteric properties of the enzyme.

Allosteric Regulation↗
PubMed2005

Subcellular localization of nardilysin during mouse oocyte maturation.

We have previously shown that the peptidase, nardilysin, contains a bipartite nuclear localization signal that permits the enzyme to cycle between the nucleus and cytoplasm. In the present study, we report that nardilysin accumulates in the nucleus of an oocyte as a function of its maturation. Nardilysin is predominantly localized in the cytoplasm of an oocyte when initially placed into culture. The enzyme starts to accumulate in the nucleus within 30 min of in vitro culture. After 3 h, nardilysin is found as a spherical structure surrounded by condensed chromosomal DNA. After 18 h of in vitro culture, it co-localizes with beta-tubulin at the spindle apparatus. Cilostamide, a phosphodiesterase 3A inhibitor that inhibits meiosis, blocks accumulation of nuclear nardilysin. This finding demonstrates that the nuclear entry of nardilysin is tightly controlled in the oocyte. Taken together, these experiments strongly suggest a role for nardilysin in meiosis through its dynamic translocation from cytosol to nucleus, and then to the spindle apparatus.

Active Transport, Cell Nucleus↗
PubMed2005

Insulysin: an allosteric enzyme as a target for Alzheimer's disease.

That the zinc metalloendopeptidase insulysin (insulin-degrading enzyme IDE) is a major b-amyloid (A(beta)) peptide-degrading enzyme in vivo is shown by the higher A(beta) peptide levels in the brain of an insulysin-deficient mouse. Insulysin was shown to initially cleave A(beta)1-40and A(beta)1-42 at His13-Gln14, His14-Gln15, and Phe19-Phe20. The insulysin-dependent cleavage of A(beta) prevents both the neurotoxic effects of the peptide as well as the ability of A(beta) to deposit onto synthetic amyloid plaques. The kinetics of the reaction of insulysin with the synthetic peptide substrate Abz-G-G-F-L-R-K-H-G-Q-EDDnp displays allosteric properties indicative of a regulated enzyme. Small peptide substrates increase the activity of insulysin toward the hydrolysis of A(beta)1-40 without affecting the activity of the enzyme toward insulin. These studies indicate that insulysin is a target for drug development in which small-molecule peptide analogs can be used to increase the rate of A(beta) clearance without affecting insulin levels.

Allosteric Regulation↗
PubMed2004

ATP effects on insulin-degrading enzyme are mediated primarily through its triphosphate moiety.

It has been reported previously that ATP inhibits the insulysin reaction (Camberos, M. C., Perez, A. A., Udrisar, D. P., Wanderley, M. I., and Cresto, J. C. (2001) Exp. Biol. Med. 226, 334-341). We report here that with 2-aminobenzoyl-GGFLRKHGQ-ethylenediamine-2,4-dinitrophenyl as substrate, ATP and other nucleotides increase the rate >20-fold in Tris buffer. There is no specificity with respect to the nucleotide; however, ATP is more effective than ADP, which is more effective than AMP. Triphosphate itself was as effective as ATP, indicating it is this moiety that is responsible for activation. The binding of triphosphate was shown to be at a site distinct from the active site, thus acting as a noncompetitive activator. With the physiological substrates insulin and amyloid beta peptide, nucleotides and triphosphate were without effect. However, with small physiological peptides such as bradykinin and dynorphin B-9, ATP and triphosphate increased the rate of hydrolysis approximately 10-fold. Triphosphate and ATP shifted the oligomeric state of the enzyme from primarily dimer-tetramers to a monomer. These data suggest the presence of an allosteric regulatory site on insulysin that may shift its specificity toward small peptide substrates.

Adenosine Triphosphate↗
PubMed2004

Estrogen regulates neprilysin activity in rat brain.

Neprilysin is a zinc metalloendopeptidase that regulates the activity of a number of physiological peptides through hydrolytic inactivation. Most recently, evidence has accumulated that neprilysin is involved in the clearance of amyloid beta peptides in the brain. Previous studies have shown that the neprilysin gene responds to progesterone, androgen, and glucocorticoids. We now show that estrogen regulates neprilysin activity in rat brain. Ovariectomy leads to a 30% decrease in neprilysin activity at 45 or 85 days, but not 21 days, post surgery. Estrogen replacement restores neprilysin levels back to control values. These changes in neprilysin activity suggest that in women estrogen is required to maintain basal levels of neprilysin.

Analysis of Variance↗
PubMed2004

Choline acetyltransferase structure reveals distribution of mutations that cause motor disorders.

Choline acetyltransferase (ChAT) synthesizes acetylcholine in neurons and other cell types. Decreases in ChAT activity are associated with a number of disease states, and mutations in ChAT cause congenital neuromuscular disorders. The crystal structure of ChAT reported here shows the enzyme divided into two domains with the active site in a solvent accessible tunnel at the domain interface. A low-resolution view of the complex with one substrate, coenzyme A, defines its binding site and suggests an additional interaction not found in the related carnitine acetyltransferase. Also, the preference for choline over carnitine as an acetyl acceptor is seen to result from both electrostatic and steric blocks to carnitine binding at the active site. While half of the mutations that cause motor disorders are positioned to affect enzyme activity directly, the remaining changes are surprisingly distant from the active site and must exert indirect effects. The structure indicates how ChAT is regulated by phosphorylation and reveals an unusual pattern of basic surface patches that may mediate membrane association or macromolecular interactions.

Amino Acid Sequence↗
PubMed2004

Regulation of the cholinergic gene locus by the repressor element-1 silencing transcription factor/neuron restrictive silencer factor (REST/NRSF).

The cholinergic gene locus is comprised of two genes, the choline acetyltransferase gene and the vesicular acetylcholine transporter gene. The vesicular acetylcholine transporter gene is located within the first intron of the choline acetyltransferase gene. This arrangement permits coordinate regulation of the locus. Protein kinase A regulates expression of the cholinergic gene locus in PC12 cells. This regulation was found to be dependent on the presence of a 21-bp DNA sequence known as the repressor element- (RE- 1)/neuron-restrictive silencer element(NRSE). Repressor element-I silencing transcription factor (REST)/ neuron-restrictive silencer factor (NRSF), which binds to the RE-I/NRSE, is a zinc finger containing transcriptional repressor that blocks the expression of many neuronal RE-I/NRSE containing genes in nonneuronal cells. However, REST/NRSF expression has also been observed in neurons as well as the PC 12 cell line used in these studies. REST/NRSF truncated isoforms were expressed in neuronal cells, suggesting they also function in regulating neuronal gene expression. A study of REST4, one of the REST/NRSF isoforms, suggests that it regulates transcription of the cholinergic gene locus by blocking the repressor activity of REST/NRSF. Protein kinase A regulation of the cholinergic gene locus in PC 12 cells can thus be attributed, at least in part, to increased synthesis of REST4, which in turn derepresses the repressor activity of REST/NRSF.

Animals↗
PubMed2004

Nuclear shuttling of the peptidase nardilysin.

The metalloendopeptidase nardilysin contains a putative N-terminal nuclear localization signal. The functionality of this sequence was tested with nardilysin-GFP fusion constructs. Expression in NIH3T3 cells showed approximately 90-95% of nardilysin-GFP as cytoplasmic. However, 3-6% of transfected cells showed both cytosolic and nuclear staining, while 2-4% showed predominantly nuclear staining. A nuclear localization signal mutant and an N-terminally truncated nardilysin-GFP with the nuclear localization signal deleted were completely cytoplasmic. Although endogenous nardilysin was barely detectable in the nucleus, after treatment with leptomycin B, nuclear nardilysin rose to approximately 15% and to over 25% after addition of spermine. The ability of a methionine 49 to act as the sole initiator methionine, as previously proposed, was tested by inserting a c-myc epitope between leucine28 and glycine29. Expression in HEK293 cells showed the presence of the c-myc tag, demonstrating that the enzyme can be translated from the first methionine and contains the nuclear localization signal.

Amino Acid Sequence↗