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#25984893   2015/05/29 Save this To Up

The GST-BHMT assay reveals a distinct mechanism underlying proteasome inhibition-induced macroautophagy in mammalian cells.

By monitoring the fragmentation of a GST-BHMT (a protein fusion of glutathionine S-transferase N-terminal to betaine-homocysteine S-methyltransferase) reporter in lysosomes, the GST-BHMT assay has previously been established as an endpoint, cargo-based assay for starvation-induced autophagy that is largely nonselective. Here, we demonstrate that under nutrient-rich conditions, proteasome inhibition by either pharmaceutical or genetic manipulations induces similar autophagy-dependent GST-BHMT processing. However, mechanistically this proteasome inhibition-induced autophagy is different from that induced by starvation as it does not rely on regulation by MTOR (mechanistic target of rapamycin [serine/threonine kinase]) and PRKAA/AMPK (protein kinase, AMP-activated, α catalytic subunit), the upstream central sensors of cellular nutrition and energy status, but requires the presence of the cargo receptors SQSTM1/p62 (sequestosome 1) and NBR1 (neighbor of BRCA1 gene 1) that are normally involved in the selective autophagy pathway. Further, it depends on ER (endoplasmic reticulum) stress signaling, in particular ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1) and its main downstream effector MAPK8/JNK1 (mitogen-activated protein kinase 8), but not XBP1 (X-box binding protein 1), by regulating the phosphorylation-dependent disassociation of BCL2 (B-cell CLL/lymphoma 2) from BECN1 (Beclin 1, autophagy related). Moreover, the multimerization domain of GST-BHMT is required for its processing in response to proteasome inhibition, in contrast to its dispensable role in starvation-induced processing. Together, these findings support a model in which under nutrient-rich conditions, proteasome inactivation induces autophagy-dependent processing of the GST-BHMT reporter through a distinct mechanism that bears notable similarity with the yeast Cvt (cytoplasm-to-vacuole targeting) pathway, and suggest the GST-BHMT reporter might be employed as a convenient assay to study selective macroautophagy in mammalian cells.

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#25286230   2014/10/07 Save this To Up

Hyperhomocysteinemia is a result, rather than a cause, of depression under chronic stress.

Although the accumulation of homocysteine (Hcy) has been implicated in the pathogenesis of depression, whether Hcy is directly involved and acts as the primary cause of depressive symptoms remains unclear. The present study was designed to clarify whether increased Hcy plays an important role in stress-induced depression.

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#24895213   2014/09/26 Save this To Up

Specific potassium ion interactions facilitate homocysteine binding to betaine-homocysteine S-methyltransferase.

Betaine-homocysteine S-methyltransferase (BHMT) is a zinc-dependent methyltransferase that uses betaine as the methyl donor for the remethylation of homocysteine to form methionine. This reaction supports S-adenosylmethionine biosynthesis, which is required for hundreds of methylation reactions in humans. Herein we report that BHMT is activated by potassium ions with an apparent K(M) for K⁺ of about 100 µM. The presence of potassium ions lowers the apparent K(M) of the enzyme for homocysteine, but it does not affect the apparent K(M) for betaine or the apparent k(cat) for either substrate. We employed molecular dynamics (MD) simulations to theoretically predict and protein crystallography to experimentally localize the binding site(s) for potassium ion(s). Simulations predicted that K⁺ ion would interact with residues Asp26 and/or Glu159. Our crystal structure of BHMT bound to homocysteine confirms these sites of interaction and reveals further contacts between K⁺ ion and BHMT residues Gly27, Gln72, Gln247, and Gly298. The potassium binding residues in BHMT partially overlap with the previously identified DGG (Asp26-Gly27-Gly28) fingerprint in the Pfam 02574 group of methyltransferases. Subsequent biochemical characterization of several site-specific BHMT mutants confirmed the results obtained by the MD simulations and crystallographic data. Together, the data herein indicate that the role of potassium ions in BHMT is structural and that potassium ion facilitates the specific binding of homocysteine to the active site of the enzyme.

1162 related Products with: Specific potassium ion interactions facilitate homocysteine binding to betaine-homocysteine S-methyltransferase.

betaine-homocysteine meth betaine-homocysteine meth N Acetyl DL Homocysteine S-(5'-Adenosyl)-L-homocys S-(5'-Adenosyl)-L-homocys SYNTHETIC S ADENOSYL L HO Nycodenz, non ionic, non S-2-Benzothiazolyl-L-homo S-Benzyl-L-homocysteine C Potassium Ion Assay (Enzy CARDIOVASCULAR DISEASES E Rabbit Anti-S-Adenosyl-Ho

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#22775318   2012/08/09 Save this To Up

Double-headed sulfur-linked amino acids as first inhibitors for betaine-homocysteine S-methyltransferase 2.

Betaine-homocysteine S-methyltransferase 2 (BHMT-2) catalyzes the transfer of a methyl group from S-methylmethionine to l-homocysteine, yielding two molecules of l-methionine. It is one of three homocysteine methyltransferases in mammals, but its overall contribution to homocysteine remethylation and sulfur amino acid homeostasis is not known. Moreover, recombinant BHMT-2 is highly unstable, which has slowed research on its structural and catalytic properties. In this study, we have prepared the first series of BHMT-2 inhibitors to be described, and we have tested them with human recombinant BHMT-2 that has been stabilized by copurification with human recombinant BHMT. Among the compounds synthesized, (2S,8RS,11RS)-5-thia-2,11-diamino-8-methyldodecanedioic acid (11) was the most potent (K(i)(app) ∼77 nM) and selective inhibitor of BHMT-2. Compound 11 only weakly inhibited human BHMT (IC(50) about 77 μM). This compound (11) may be useful in future in vivo studies to probe the physiological significance of BHMT-2 in sulfur amino acid metabolism.

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#19200878   2009/02/09 Save this To Up

The GST-BHMT assay and related assays for autophagy.

The endpoint of the autophagic process is the breakdown of delivered cytoplasmic cargo in lysosomes. Therefore, assays based on degradation of cargo are of particular interest in that they can measure regulation of the entire autophagic process, including changes in cargo delivery and breakdown in the lytic compartment. Betaine homocysteine methyltransferase (BHMT) is one of many cytosolic proteins found in the mammalian autophagosome, and delivery of BHMT to the lysosome results in its proteolysis to discrete fragments under certain conditions. Making use of these observations, the GST-BHMT assay was developed as an endpoint, cargo-based autophagy assay. Using this assay as a starting point, additional cargo-based assays have been developed with the potential to measure autophagic degradation of specific subcellular compartments. Here we describe the development and validation of these assays.

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#18498552   2008/05/23 Save this To Up

Effect of transgenic extrahepatic expression of betaine-homocysteine methyltransferase on alcohol or homocysteine-induced fatty liver.

Chronic alcohol feeding induces hyperhomocysteinemia (HHcy). Previously, we reported a protective role of betaine-homocysteine methyltransferase (BHMT) in homocysteine-induced injury in cultured hepatocytes. In this study, we investigated the direct role of BHMT in alcohol or homocysteine-induced liver injury.

2015 related Products with: Effect of transgenic extrahepatic expression of betaine-homocysteine methyltransferase on alcohol or homocysteine-induced fatty liver.

betaine-homocysteine meth betaine-homocysteine meth DNA (cytosine 5) methyltr N Acetyl DL Homocysteine S-(5'-Adenosyl)-L-homocys S-(5'-Adenosyl)-L-homocys SYNTHETIC S ADENOSYL L HO S-2-Benzothiazolyl-L-homo S-Benzyl-L-homocysteine C CARDIOVASCULAR DISEASES E Rabbit Anti-S-Adenosyl-Ho pCAMBIA1381Xa Vector (Fus

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#18230605   2008/03/31 Save this To Up

Betaine-homocysteine S-methyltransferase-2 is an S-methylmethionine-homocysteine methyltransferase.

We demonstrate that purified recombinant human betainehomocysteine methyltransferase-2 (BHMT-2) is a zinc metalloenzyme that uses S-methylmethionine (SMM) as a methyl donor for the methylation of homocysteine. Unlike the highly homologous betaine-homocysteine methyltransferase (BHMT), BHMT-2 cannot use betaine. The K(m) of BHMT-2 for SMM was determined to be 0.94 mm, and it has a turnover number similar to BHMT. Several compounds were tested as inhibitors of recombinant human BHMT and BHMT-2. The SMM-specific methyltransferase activity of BHMT-2 is not inhibited by dimethylglycine and betaine, whereas the former is a potent inhibitor of BHMT. Methionine is a stronger inhibitor of BHMT-2 than BHMT, and S-adenosylmethionine does not inhibit BHMT but is a weak inhibitor of BHMT-2. BHMT can use SMM as a methyl donor with a k(cat)/K(m) that is 5-fold lower than the k(cat)/K(m) for betaine. However, SMM does not inhibit BHMT activity when it is presented to the enzyme at concentrations that are 10-fold greater than the subsaturating amounts of betaine used in the assay. Based on these data, it is our current hypothesis that in vivo most if not all of the SMM-dependent methylation of homocysteine occurs via BHMT-2.

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#18059170   2008/01/21 Save this To Up

Macroautophagy-dependent, intralysosomal cleavage of a betaine homocysteine methyltransferase fusion protein requires stable multimerization.

Cargo-based assays have proven invaluable in the study of macroautophagy in yeast and mammalian cells. Proteomic analysis of autolysosomes identified the metabolic enzyme, betaine homocysteine methyltransferase (BHMT), as a potential cargo-based, end-point marker for mammalian macroautophagy. To test whether degradation of BHMT can be used to measure macroautophagic flux in mammalian cells, we created a BHMT fusion protein (GST-BHMT) that demonstrates starvation-induced, site-specific fragmentation in a variety of cell lines. Subcellular fractionation studies show that the GST-BHMT fragment co-fractionates with vesicles containing lysosomal and autolysosomal markers. Furthermore, both pharmacological inhibitors of macroautophagy and depletion of macroautophagy-specific proteins reduce accumulation of the fragment. In the course of these studies, we observed that fragmentation of GST-BHMT did not occur in forms of the reporter with truncation or point mutations that destabilize oligomerization. Since stable oligomerization of BHMT is essential for its catalytic activity, a point mutation known to ablate BHMT activity was tested. We show that accumulation of the GST-BHMT fragment is not impaired in a catalytically inactive mutant, indicating that selective proteolysis of GST-BHMT requires stable quaternary structure independent of effects on activity. Also, the loss of fragmentation observed in the oligomerization deficient mutants does not seem to be due to a defect of sequestration and lysosomal loading, suggesting that disruption of stable quaternary structure affects the ability of a lysosomal protease to cleave the newly-delivered cargo. Finally, we propose that the cargo-based GST-BHMT assay will be a valuable addition to existing macroautophagy assays in mammalian cells.

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#16953798   2006/12/07 Save this To Up

Inhibition of human betaine-homocysteine methyltransferase expression by S-adenosylmethionine and methylthioadenosine.

BHMT (betaine-homocysteine methyltransferase) remethylates homocysteine to form methionine. SAM (S-adenosylmethionine) inhibits BHMT activity, but whether SAM modulates BHMT gene expression is unknown. Transcriptional regulation of the human BHMT is also unknown. The present study examined regulation of the human BHMT gene by SAM and its metabolite, MTA (5'-methylthioadenosine). To facilitate these studies, we cloned the 2.7 kb 5'-flanking region of the human BHMT gene (GenBank accession number AY325901). Both SAM and MTA treatment of HepG2 cells resulted in a dose- and time-dependent decrease in BHMT mRNA levels, which paralleled their effects on the BHMT promoter activity. Maximal suppression was observed with the BHMT promoter construct -347/+33, which contains a number of NF-kappaB (nuclear factor kappaB) binding sites. SAM and MTA treatment increased NF-kappaB nuclear binding and NF-kappaB-driven luciferase activities, and increased nuclear binding activity of multiple histone deacetylase co-repressors to the NF-kappaB sites. Overexpression of p50 and p65 decreased BHMT promoter activity, while blocking NF-kappaB activation increased BHMT expression and promoter activity, and prevented SAM but not MTA's ability to inhibit BHMT expression. The NF-kappaB binding site at -301 is responsible, at least in part, for this effect. Lower BHMT expression can impair homocysteine metabolism, which can induce ER (endoplasmic reticulum) stress. Indeed, MTA treatment resulted in increased expression ER stress markers. In conclusion, SAM and MTA down-regulate BHMT expression in HepG2 cells in part by inducing NF-kappaB, which acts as a repressor for the human BHMT gene. While SAM's mechanism is NF-kappaB-dependent, MTA has both NF-kappaB-dependent and -independent mechanisms.

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#15130780   2004/05/07 Save this To Up

Oligomerization is required for betaine-homocysteine S-methyltransferase function.

Betaine-homocysteine methyltransferase (BHMT) is a member of a family (Pfam 02574) of zinc- and thiol/selenol-dependent methyltransferases. All family members purified to date are monomers, except BHMT, which is an oligomer. We have studied how C-terminal truncation or mutagenic replacement of residues within or associated with the unique dimerization arm of this enzyme affects oligomerization and function. Two C-terminal truncation mutants, S325 and D371, do not express well in Escherichia coli and are inactive. Residues within the dimerization arm (H338, R346, W352, R361, P362, Y363, N364, and P365) and one that forms a hydrogen bond to the arm (E266) were changed to alanine. All mutants maintained a normal or near-normal ability to bind zinc. E266A, R361A, P362A, Y363A, N364A, and P365A displayed near-normal catalytic activity, but H338A had only 10% of the wild-type enzyme activity. Like the wild-type enzyme, most mutants eluted as tetramers from gel filtration columns and formed discrete bands on SDS-PAGE gels following glutaraldehyde crosslinking. Mutants R346A and W352A had negligible activity, eluted as dimers, and displayed aberrant crosslinking properties. These data indicate that unlike other Pfam 02574 members, oligomerization of BHMT is required for function.

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