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  1. Article ; Online: Identification of residues involved in allosteric signal transmission from amino acid binding site of pyruvate kinase muscle isoform 2.

    Nandi, Suparno / Dey, Mishtu

    PloS one

    2023  Volume 18, Issue 3, Page(s) e0282508

    Abstract: PKM2 is a rate-limiting enzyme in the glycolytic process and is involved in regulating tumor proliferation. Several amino acids (AAs) such as Asn, Asp, Val, and Cys have been shown to bind to the AA binding pocket of PKM2 and modulate its oligomeric ... ...

    Abstract PKM2 is a rate-limiting enzyme in the glycolytic process and is involved in regulating tumor proliferation. Several amino acids (AAs) such as Asn, Asp, Val, and Cys have been shown to bind to the AA binding pocket of PKM2 and modulate its oligomeric state, substrate binding affinity, and activity. Although previous studies have attributed that the main chain and side chain of bound AAs are responsible for initiating signal to regulate PKM2, the signal transduction pathway remains elusive. To identify the residues involved in signal transfer process, N70 and N75 located at two ends of a β strand connecting the active site and AA binding pocket were altered. Biochemical studies of these variants with various AA ligands (Asn, Asp, Val, and Cys), illustrate that N70 and N75, along with β1 connecting these residues are part of the signal transduction pathway between the AA binding pocket and the active site. The results demonstrate that mutation of N70 to D prevents the transfer of the inhibitory signal mediated by Val and Cys, whereas N75 to L alteration blocks the activating signal initiated by Asn and Asp. Taken together, this study confirms that N70 is one of the residues responsible for transmitting the inhibitory signal and N75 is involved in the activation signal flow.
    MeSH term(s) Amino Acids/metabolism ; Catalytic Domain ; Protein Isoforms/metabolism ; Pyruvate Kinase/chemistry ; Pyruvate Kinase/metabolism ; Humans ; Signal Transduction ; Thyroid Hormone-Binding Proteins
    Chemical Substances Amino Acids ; Protein Isoforms ; Pyruvate Kinase (EC 2.7.1.40)
    Language English
    Publishing date 2023-03-10
    Publishing country United States
    Document type Journal Article ; Research Support, U.S. Gov't, Non-P.H.S.
    ZDB-ID 2267670-3
    ISSN 1932-6203 ; 1932-6203
    ISSN (online) 1932-6203
    ISSN 1932-6203
    DOI 10.1371/journal.pone.0282508
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: Biochemical and structural insights into how amino acids regulate pyruvate kinase muscle isoform 2.

    Nandi, Suparno / Dey, Mishtu

    The Journal of biological chemistry

    2020  Volume 295, Issue 16, Page(s) 5390–5403

    Abstract: Pyruvate kinase muscle isoform 2 (PKM2) is a key glycolytic enzyme involved in ATP generation and critical for cancer metabolism. PKM2 is expressed in many human cancers and is regulated by complex mechanisms that promote tumor growth and proliferation. ... ...

    Abstract Pyruvate kinase muscle isoform 2 (PKM2) is a key glycolytic enzyme involved in ATP generation and critical for cancer metabolism. PKM2 is expressed in many human cancers and is regulated by complex mechanisms that promote tumor growth and proliferation. Therefore, it is considered an attractive therapeutic target for modulating tumor metabolism. Various stimuli allosterically regulate PKM2 by cycling it between highly active and less active states. Several small molecules activate PKM2 by binding to its intersubunit interface. Serine and cysteine serve as an activator and inhibitor of PKM2, respectively, by binding to its amino acid (AA)-binding pocket, which therefore represents a potential druggable site. Despite binding similarly to PKM2, how cysteine and serine differentially regulate this enzyme remains elusive. Using kinetic analyses, fluorescence binding, X-ray crystallography, and gel filtration experiments with asparagine, aspartate, and valine as PKM2 ligands, we examined whether the differences in the side-chain polarity of these AAs trigger distinct allosteric responses in PKM2. We found that Asn (polar) and Asp (charged) activate PKM2 and that Val (hydrophobic) inhibits it. The results also indicate that both Asn and Asp can restore the activity of Val-inhibited PKM2. AA-bound crystal structures of PKM2 displayed distinctive interactions within the binding pocket, causing unique allosteric effects in the enzyme. These structure-function analyses of AA-mediated PKM2 regulation shed light on the chemical requirements in the development of mechanism-based small-molecule modulators targeting the AA-binding pocket of PKM2 and provide broader insights into the regulatory mechanisms of complex allosteric enzymes.
    MeSH term(s) Allosteric Regulation ; Allosteric Site ; Amino Acids/chemistry ; Amino Acids/metabolism ; Carrier Proteins/antagonists & inhibitors ; Carrier Proteins/chemistry ; Carrier Proteins/metabolism ; Enzyme Inhibitors/chemistry ; Enzyme Inhibitors/pharmacology ; Humans ; Membrane Proteins/antagonists & inhibitors ; Membrane Proteins/chemistry ; Membrane Proteins/metabolism ; Protein Binding ; Protein Multimerization ; Thyroid Hormones/chemistry ; Thyroid Hormones/metabolism ; Thyroid Hormone-Binding Proteins
    Chemical Substances Amino Acids ; Carrier Proteins ; Enzyme Inhibitors ; Membrane Proteins ; Thyroid Hormones
    Language English
    Publishing date 2020-03-06
    Publishing country United States
    Document type Journal Article ; Research Support, U.S. Gov't, Non-P.H.S.
    ZDB-ID 2997-x
    ISSN 1083-351X ; 0021-9258
    ISSN (online) 1083-351X
    ISSN 0021-9258
    DOI 10.1074/jbc.RA120.013030
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    Uc I Zs.108: Show issues Location:
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  3. Article ; Online: Structural basis for allosteric regulation of pyruvate kinase M2 by phosphorylation and acetylation.

    Nandi, Suparno / Razzaghi, Mortezaali / Srivastava, Dhiraj / Dey, Mishtu

    The Journal of biological chemistry

    2020  Volume 295, Issue 51, Page(s) 17425–17440

    Abstract: Pyruvate kinase muscle isoform 2 (PKM2) is a key glycolytic enzyme and transcriptional coactivator and is critical for tumor metabolism. In cancer cells, native tetrameric PKM2 is phosphorylated or acetylated, which initiates a switch to a dimeric/ ... ...

    Abstract Pyruvate kinase muscle isoform 2 (PKM2) is a key glycolytic enzyme and transcriptional coactivator and is critical for tumor metabolism. In cancer cells, native tetrameric PKM2 is phosphorylated or acetylated, which initiates a switch to a dimeric/monomeric form that translocates into the nucleus, causing oncogene transcription. However, it is not known how these post-translational modifications (PTMs) disrupt the oligomeric state of PKM2. We explored this question via crystallographic and biophysical analyses of PKM2 mutants containing residues that mimic phosphorylation and acetylation. We find that the PTMs elicit major structural reorganization of the fructose 1,6-bisphosphate (FBP), an allosteric activator, binding site, impacting the interaction with FBP and causing a disruption in oligomerization. To gain insight into how these modifications might cause unique outcomes in cancer cells, we examined the impact of increasing the intracellular pH (pH
    MeSH term(s) Acetylation ; Allosteric Regulation ; Glycolysis ; Humans ; Hydrogen-Ion Concentration ; Lysine/metabolism ; Molecular Mimicry ; Phosphorylation ; Phosphoserine/metabolism ; Protein Binding ; Pyruvate Kinase/metabolism
    Chemical Substances Phosphoserine (17885-08-4) ; Pyruvate Kinase (EC 2.7.1.40) ; Lysine (K3Z4F929H6)
    Language English
    Publishing date 2020-12-18
    Publishing country United States
    Document type Journal Article ; Research Support, U.S. Gov't, Non-P.H.S.
    ZDB-ID 2997-x
    ISSN 1083-351X ; 0021-9258
    ISSN (online) 1083-351X
    ISSN 0021-9258
    DOI 10.1074/jbc.RA120.015800
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: Mechanistic and Structural Insights into Cysteine-Mediated Inhibition of Pyruvate Kinase Muscle Isoform 2.

    Srivastava, Dhiraj / Nandi, Suparno / Dey, Mishtu

    Biochemistry

    2019  Volume 58, Issue 35, Page(s) 3669–3682

    Abstract: Cancer cells regulate key enzymes in the glycolytic pathway to control the glycolytic flux, which is necessary for their growth and proliferation. One of the enzymes is pyruvate kinase muscle isoform 2 (PKM2), which is allosterically regulated by various ...

    Abstract Cancer cells regulate key enzymes in the glycolytic pathway to control the glycolytic flux, which is necessary for their growth and proliferation. One of the enzymes is pyruvate kinase muscle isoform 2 (PKM2), which is allosterically regulated by various small molecules. Using detailed biochemical and kinetic studies, we demonstrate that cysteine inhibits wild-type (wt) PKM2 by shifting from an active tetramer to a mixture of a tetramer and a less active dimer/monomer equilibrium and that the inhibition is dependent on cysteine concentration. The cysteine-mediated PKM2 inhibition is reversed by fructose 1,6-bisphosphate, an allosteric activator of PKM2. Furthermore, kinetic studies using two dimeric PKM2 variants, S437Y PKM2 and G415R PKM2, show that the reversal is caused by the tetramerization of wtPKM2. The crystal structure of the wtPKM2-Cys complex was determined at 2.25 Å, which showed that cysteine is held to the amino acid binding site via its main chain groups, similar to that observed for phenylalanine, alanine, serine, and tryptophan. Notably, ligand binding studies using fluorescence and isothermal titration calorimetry show that the presence of phosphoenolpyruvate alters the binding affinities of amino acids for wtPKM2 and vice versa, thereby unravelling the existence of a functionally bidirectional coupling between the amino acid binding site and the active site of wtPKM2.
    MeSH term(s) Amino Acid Substitution/genetics ; Amino Acids/chemistry ; Amino Acids/metabolism ; Carrier Proteins/antagonists & inhibitors ; Carrier Proteins/chemistry ; Carrier Proteins/genetics ; Carrier Proteins/metabolism ; Catalysis ; Catalytic Domain/genetics ; Crystallography, X-Ray ; Cysteine/pharmacology ; Enzyme Inhibitors/pharmacology ; Humans ; Isoenzymes/antagonists & inhibitors ; Isoenzymes/chemistry ; Isoenzymes/genetics ; Isoenzymes/metabolism ; Ligands ; Membrane Proteins/antagonists & inhibitors ; Membrane Proteins/chemistry ; Membrane Proteins/genetics ; Membrane Proteins/metabolism ; Models, Molecular ; Mutant Proteins/antagonists & inhibitors ; Mutant Proteins/chemistry ; Mutant Proteins/metabolism ; Phosphoenolpyruvate/chemistry ; Phosphoenolpyruvate/metabolism ; Protein Binding ; Protein Conformation ; Serine/genetics ; Thyroid Hormones/chemistry ; Thyroid Hormones/genetics ; Thyroid Hormones/metabolism ; Tyrosine/genetics ; Thyroid Hormone-Binding Proteins
    Chemical Substances Amino Acids ; Carrier Proteins ; Enzyme Inhibitors ; Isoenzymes ; Ligands ; Membrane Proteins ; Mutant Proteins ; Thyroid Hormones ; Tyrosine (42HK56048U) ; Serine (452VLY9402) ; Phosphoenolpyruvate (73-89-2) ; Cysteine (K848JZ4886)
    Language English
    Publishing date 2019-08-20
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
    ZDB-ID 1108-3
    ISSN 1520-4995 ; 0006-2960
    ISSN (online) 1520-4995
    ISSN 0006-2960
    DOI 10.1021/acs.biochem.9b00349
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Zs.A 217: Show issues Location:
    Je nach Verfügbarkeit (siehe Angabe bei Bestand)
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    Jg. 1995 - 2021: Lesesall (1.OG)
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  5. Article: Mechanistic and Structural Insights into Cysteine-Mediated Inhibition of Pyruvate Kinase Muscle Isoform 2

    Srivastava, Dhiraj / Nandi, Suparno / Dey, Mishtu

    Biochemistry. 2019 Aug. 06, v. 58, no. 35

    2019  

    Abstract: Cancer cells regulate key enzymes in the glycolytic pathway to control the glycolytic flux, which is necessary for their growth and proliferation. One of the enzymes is pyruvate kinase muscle isoform 2 (PKM2), which is allosterically regulated by various ...

    Abstract Cancer cells regulate key enzymes in the glycolytic pathway to control the glycolytic flux, which is necessary for their growth and proliferation. One of the enzymes is pyruvate kinase muscle isoform 2 (PKM2), which is allosterically regulated by various small molecules. Using detailed biochemical and kinetic studies, we demonstrate that cysteine inhibits wild-type (wt) PKM2 by shifting from an active tetramer to a mixture of a tetramer and a less active dimer/monomer equilibrium and that the inhibition is dependent on cysteine concentration. The cysteine-mediated PKM2 inhibition is reversed by fructose 1,6-bisphosphate, an allosteric activator of PKM2. Furthermore, kinetic studies using two dimeric PKM2 variants, S437Y PKM2 and G415R PKM2, show that the reversal is caused by the tetramerization of wtPKM2. The crystal structure of the wtPKM2–Cys complex was determined at 2.25 Å, which showed that cysteine is held to the amino acid binding site via its main chain groups, similar to that observed for phenylalanine, alanine, serine, and tryptophan. Notably, ligand binding studies using fluorescence and isothermal titration calorimetry show that the presence of phosphoenolpyruvate alters the binding affinities of amino acids for wtPKM2 and vice versa, thereby unravelling the existence of a functionally bidirectional coupling between the amino acid binding site and the active site of wtPKM2.
    Keywords active sites ; alanine ; binding capacity ; binding sites ; calorimetry ; crystal structure ; cysteine ; fluorescence ; fructose ; glycolysis ; ligands ; muscles ; neoplasm cells ; phenylalanine ; pyruvate kinase ; serine ; titration ; tryptophan
    Language English
    Dates of publication 2019-0806
    Size p. 3669-3682.
    Publishing place American Chemical Society
    Document type Article
    Note NAL-light
    ZDB-ID 1108-3
    ISSN 1520-4995 ; 0006-2960
    ISSN (online) 1520-4995
    ISSN 0006-2960
    DOI 10.1021/acs.biochem.9b00349
    Database NAL-Catalogue (AGRICOLA)

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    Zs.A 217: Show issues Location:
    Je nach Verfügbarkeit (siehe Angabe bei Bestand)
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  6. Article ; Online: 50S subunit recognition and modification by the

    Laughlin, Zane T / Nandi, Suparno / Dey, Debayan / Zelinskaya, Natalia / Witek, Marta A / Srinivas, Pooja / Nguyen, Ha An / Kuiper, Emily G / Comstock, Lindsay R / Dunham, Christine M / Conn, Graeme L

    Proceedings of the National Academy of Sciences of the United States of America

    2022  Volume 119, Issue 14, Page(s) e2120352119

    Abstract: Changes in bacterial ribosomal RNA (rRNA) methylation status can alter the activity of diverse groups of ribosome-targeting antibiotics. These modifications are typically incorporated by a single methyltransferase that acts on one nucleotide target and ... ...

    Abstract Changes in bacterial ribosomal RNA (rRNA) methylation status can alter the activity of diverse groups of ribosome-targeting antibiotics. These modifications are typically incorporated by a single methyltransferase that acts on one nucleotide target and rRNA methylation directly prevents drug binding, thereby conferring drug resistance. Loss of intrinsic methylation can also result in antibiotic resistance. For example, Mycobacterium tuberculosis becomes sensitized to tuberactinomycin antibiotics, such as capreomycin and viomycin, due to the action of the intrinsic methyltransferase TlyA. TlyA is unique among antibiotic resistance-associated methyltransferases as it has dual 16S and 23S rRNA substrate specificity and can incorporate cytidine-2′-O-methylations within two structurally distinct contexts. Here, we report the structure of a mycobacterial 50S subunit-TlyA complex trapped in a postcatalytic state with a S-adenosyl-L-methionine analog using single-particle cryogenic electron microscopy. Together with complementary functional analyses, this structure reveals critical roles in 23S rRNA substrate recognition for conserved residues across an interaction surface that spans both TlyA domains. These interactions position the TlyA active site over the target nucleotide C2144, which is flipped from 23S Helix 69 in a process stabilized by stacking of TlyA residue Phe157 on the adjacent A2143. Base flipping may thus be a common strategy among rRNA methyltransferase enzymes, even in cases where the target site is accessible without such structural reorganization. Finally, functional studies with 30S subunit suggest that the same TlyA interaction surface is employed to recognize this second substrate, but with distinct dependencies on essential conserved residues.
    MeSH term(s) Bacterial Proteins/chemistry ; Catalytic Domain ; Drug Resistance, Bacterial/genetics ; Methyltransferases/chemistry ; Mycobacterium tuberculosis/enzymology ; Mycobacterium tuberculosis/genetics ; Protein Conformation, alpha-Helical ; RNA, Ribosomal, 16S/chemistry ; RNA, Ribosomal, 23S/chemistry ; Ribosome Subunits, Large, Bacterial/chemistry
    Chemical Substances Bacterial Proteins ; RNA, Ribosomal, 16S ; RNA, Ribosomal, 23S ; TlyA protein, Mycobacterium tuberculosis ; Methyltransferases (EC 2.1.1.-) ; rRNA (adenosine-O-2'-)methyltransferase (EC 2.1.1.230)
    Language English
    Publishing date 2022-03-31
    Publishing country United States
    Document type Journal Article
    ZDB-ID 209104-5
    ISSN 1091-6490 ; 0027-8424
    ISSN (online) 1091-6490
    ISSN 0027-8424
    DOI 10.1073/pnas.2120352119
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    Zs.A 1148: Show issues Location:
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