WO2013175015A1 - Identification and functional expression of the mitochondrial pyruvate carrier, mpc1 - Google Patents

Identification and functional expression of the mitochondrial pyruvate carrier, mpc1 Download PDF

Info

Publication number
WO2013175015A1
WO2013175015A1 PCT/EP2013/060815 EP2013060815W WO2013175015A1 WO 2013175015 A1 WO2013175015 A1 WO 2013175015A1 EP 2013060815 W EP2013060815 W EP 2013060815W WO 2013175015 A1 WO2013175015 A1 WO 2013175015A1
Authority
WO
WIPO (PCT)
Prior art keywords
pyruvate
isolated
modulator
family
import
Prior art date
Application number
PCT/EP2013/060815
Other languages
French (fr)
Inventor
Sébastien HERZIG
Jean-Claude Martinou
Original Assignee
University Of Geneva
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University Of Geneva filed Critical University Of Geneva
Priority to EP13727081.5A priority Critical patent/EP2855532A1/en
Publication of WO2013175015A1 publication Critical patent/WO2013175015A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present invention relates to the characterization of a novel isolated and purified mitochondrial Pyruvate carrier (MFC) family capable of transporting Pyruvate into mitochondria. The invention also relates to modulators of the Pyruvate import activity for use in therapy of disorders related to pyruvate uptake. The invention further relates to the screening of drugs for disorders related to pyruvate uptake.

Description

IDENTIFICATION AND FUNCTIONAL EXPRESSION OF THE MITOCHONDRIAL PYRUVATE CARRIER , MPC1
FIELD OF THE INVENTION
The present invention relates to the characterization of a novel isolated and purified mitochondrial
Pyruvate carrier (MPC) family capable of transporting Pyruvate into mitochondria. The invention also relates to modulators of the Pyruvate import activity for use in therapy of disorders related to pyruvate uptake. The invention further relates to the screening of drugs for disorders related to pyruvate uptake.
BACKGROUND OF THE INVENTION
In the mitochondrion, pyruvate is converted into acetyl-CoA by the pyruvate dehydrogenase (PDH) complex and participates in the synthesis of branched-chain amino acids (BCAA) in yeast. Acetyl - CoA donates carbon atoms to the citric acid cycle and participates in the synthesis of octanoic acid, the precursor of lipoic acid. Lipoic acid is an essential cofactor of several miilti-subunit complexes in the mitochondrial matrix, including PDH, a-keloglutarate dehydrogenase (a -K.DH) and the branched-chain keto acid dehydrogenase (BCKDH).
Acetyl-CoA, which feeds the TCA cycle, generates reductive power that is used for energy production through the respiratory chain. Import of pyruvate across the inner mitochondrial membrane (IMM) requires a specific carrier whose molecular identity has not been established.
However, the existence of a specific carrier for pyruvate in the mitochondrial matrix was firmly established by Andrew Halestrap in 1975 by the use of a specific inhibitor, a-eyano-4- hydroxycinnamate (CHC) (1), confirming earlier studies by Papa et al. (2).
Nevertheless, the molecular identity of this carrier was unknown.
Therefore, there is a need for new activators (agonists) and inhibitors (antagonists) capable of modulating Pyruvate import to treat disorders associated with the pyruvate transport and uptake. SUMMARY OF THE INVENTION
The transport of pyruvate, the end product of glycolysis, into mitochondria is an essential process that provides the organelle with a major oxidative fuel. Although the existence of a specific mitochondrial pyruvate carrier (MPC) has been anticipated, its molecular identity remained unknown. Applicants report that MPC is a heterocomplex formed by two members of a family of previously uncharacterized membrane proteins that are conserved from yeast to mammals. Members of the MPC family were found in the inner mitochondrial membrane, and yeast mutants lacking MPC proteins showed severe defects in mitochondrial pyruvate uptake. Coexpression of mouse MPC1 and MPC2 in Lactococcm lactis was sufficient to allow transport of pyruvate across the membrane. This finding firmly establishes these proteins as essential components of the MPC.
One object of the present invention is to provide an isolated and purified mitochondrial Pyruvate carrier (MPC) family comprising the heterocomplex association of two members of a family of membrane proteins consisting of an isolated and purified MPC1 member (Brp44L) together with an isolated and purified MPC2 member (Brp44), wherein said isolated and purified MPC family is capable of transporting Pyruvate into mitochondria.
Other object of the invention will be described in the detailed description.
BRIEF DESCRIPTION OF THE FIGURES Figure 1. Plienotypes of yeast deleted for MPC genes.
A. Spot assays of strains (as indicated), on media differing in amino acid composition or carbon source. The plates are representative of at least three independent experiments. B. Spot assays of mpclA cells transformed with p2U vector containing the indicated gene. C. Spot assays of
Figure imgf000003_0001
cells transformed with p2U vector containing the indicated gene. D. Valine or leucine decarboxylation in WT (black bars) or mpclA (white bars) cells. The means of 6 independent experiments are indicated above the bars +/- S.D. E. Valine decarboxylation in the indicated strains. The mean of at least 3 independent experiments +/- S.D. is shown. F. PDH and a -KDH activities in WT (black bars) or mpclA (white bars) mitochondria, mean +/- S.D. of three independent experiments for PDH and two independent experiments for a -KDH are shown. Figure 2. Lipoic acid production defects in mpc mutant strains
A-B. Protein immunoblot analysis of cell extracts using antibodies raised against lipoic acid in strains and media as indicated. Ponceau staining is shown as loading control. The blots are representative of at least three independent experiments. C. PDH activity of mitochondria isolated from the indicated strains grown in lactate medium. N.D: not detected. Data is expressed as the mean +/- SD of three independent experiments.
Figure 3. Impaired import of pyruvate in mpc mutant strains.
A. Kinetics of pyruvate import into isolated mitochondria of WT (closed circles), mpc I A (open circles), mpc2A (closed triangles), mpc3A (open triangles) or mpc2Ampc3A (squares) cells. Data are representative of at least three independent experiments B. Rate of pyruvate import calculated from the first two minutes of import. Data are mean of at least three independent experiments +/- S.E.M. N.D. not detected. C. Protein immunoblot analysis of protein levels using genomically 3x11A tagged proteins in the indicated medium, probed with anti-HA antibodies. Ponceau staining is shown as a loading control. The blot is representative of three independent experiments.
Figure 4. mMPCl and mMPC2 functionally reconstitute the pyruvate carrier in Lactococcus lactis,
A. Spot assay in amino acid-free medium of the indicated yeast strains transformed with an empty vector or expressing murine MPCl (mMPCl) or MPC2 (mMPC2). B. Protein immunoblot analysis of expression of the different proteins from strains shown in A. C. Protein immunoblotting of Lactococcus lactis strains showing expression of mMPC proteins. D. Time course of pyruvate import in Lactococcus lactis strains with empty vector (diamonds), mMPCl (circles), mMPC2 (triangles) or both (squares). The mean of three independent experiment +/- SEM is shown. E. Quantification of the import rate from the first 20 minutes of import in L. lactis strains expressing the indicated proteins. 2DG: 2-deoxyglucose. *: p<0.05, two-tailed T-test.
Figure 5, MPCl and MPC2 are conserved inner mitochondrial membrane proteins.
A. Mitochondria isolated from 293T cells expressing MPC2-HA were treated as indicated, resolved on SDS-PAGE, transferred to nitrocellulose and blotted with antibodies raised against endogenous MPCl or the HA epitope to detect overexpressed MPC2-HA. B. Sodium carbonate extract of carbonate extract of mitochondria from cells described in A were ultracentrifiiged and the pellet (P) and supernatant (S) were resolved on SDS-PAGE, transferred to nitrocellulose, and blotted with antibodies against the indicated proteins or HA epitope. Blots are representative of three independent experiments.
C-D. Protein sequence alignment of MPCl (C) and MPC2(D) homologs in the indicated species. Highlighted are the amino acids according to their hydropathy and the predicted transmembrane domains (PsiPred). Accession numbers are: Arabidopsis thaliana MPCl: NP_ 001078606.1, MPC2: NP_567439.1; Oryza sativa MPCl: NPJX) 1061794.1; Chlamydomonas reinhardti MPCl: A8I311; Magnaporthe oryzae MPCl: XP 368396.1, MPC2: XP 366592.1; Neurospora crassa MPCl: XP_956237.1, MPC2: XP 964561.1; Schizosaccharomyces pombe MPCl: NP_587737.1, MPC2: NP 592846.1; Saccharomyces cerevisiae MPCl: NP 011435.1, MPC2: NP_012032.1, MPC3: NP 011759.1; Homo sapiens MPCl: NP 057! 82.1, MPC2: NP 001137146.1; Pan troglodytes MPCl: XP_0()1137474.1, MPC2: XP 001174837.1; Bos taurus MPCl: NP_001070510.1, MPC2: Xl\_001174837.1; Mus musculus MPCl: NP_061289.1. MPC2: NP_081706.1: Rattus norvegicus MPCl: NP 598245.1; Gallus gallus MPCl: NP 001026695.1, MPC2: XP 001231387.2; Nematostella vectensis MPCl: A7SC70, MPC2: A7SQZ7; Anopheles gambiae MPCl: XP_321716.4, MPC2: XP_314997.3; Drosophila me!anogaster MPCl: NP_650762.1, MPC2:
NP 649912.1; Caenorhabditis elegaiis MPCl: NP_491234.1, MPC2: NP_497894.1; Dictyostelium discoideum MPCl: Q55GU4; Canis lupus MPC2: XP 537209.1; Danio rerio MPC2: NP_997757.1; Ashbya gossypii MPC2: NP 984651.1; Kluyveromyces lactis MPC2: XP_451243.1.
Figure 6. Mpcl is an inner mitochondrial membrane protein in yeast.
A. WT cells expressing Mpcl-GFP fusion and mitochondria targeted mClierry (mito-mCherry) from p2U and pYXl 22 vectors respectively were imaged by fluorescence microscopy. B. Mitochondria isolated from yeast expressing Mpcl-HA were treated as indicated, resolved on SDS- PAGE, transferred to nitrocellulose and blotted with antibodies raised against the indicated proteins. C. Sodium carbonate extract of mitochondria expressing Mpcl-HA were ultracentrifiiged and the pellet (P) and supernatant (S) were resolved on SDS-PAGE, transferred to nitrocellulose, and blotted with antibodies against the indicated proteins. Blots are representative of three independent experiments. Figure 7. Phenoty pes of MFC deletion strains.
A. Growth curves of WT (black symbols) or mpclA (white symbols) cells grown overnight in SD, diluted to an OD60o of 0, 1 in SD medium containing the indicated supplement at 100 pg/ml and grown for 48 h at 30° in 96 -we 11 plates, ODgoo was measured every 30 minutes. The data is the average of three independent experiments +/- S.D. B. Protein immunoblot analysis of co- immunoprecipitation (IP) in mitochondria from Mpcl-3xHA or Mpcl -3xHA Mpc2-13xmyc strains. Tim50 is shown as a control for the inner mitochondrial membrane. The lysate before IP (input), the anti-MYC IP (MYC IP) or anti-ΗΛ IP (HA IP) are shown. Mpc2-Myc appeared as two bands, likely due to proteolytic cleavage of part of the tag. The blots are representative of three independent experiments. C, Western blot analysis of lipoylation in mpclA cells transformed with an empty vector (p2U) or with p2U containing yeast MPC1. I). Lipoic acid was quantified in WT (black bars) or mpclA (white bars) cells grown overnight in YPD or SD. Values are the mean of three independent experiments +/- S.E.M. E. Autoradiogram of Reverse-Phase TLC analysis of the fatty acids synthesized in mitochondria isolated from indicated strain grown in SD medium.
Figure 8. Model of the fate of pyruvate inside the mitochondria.
Pyruvate serves as a precursor for synthesis of BCAA or as a source of acetyl-CoA via PDH for the Krebs cycle and lipoic acid synthesis. Lipoic acid is required for PDH and BCKDH activity, which bypasses PDH and allows generation of acetyl-CoA from leucine. Hfal is the mitochondrial acetyl- CoA carboxylase, which converts acetyl-CoA to malonyl-CoA. Lip5 is the lipoic acid synthase essential for the conversion of octanoic to lipoic acid.
Figure 9. MFC mutants have normal mitochondrial membrane potential.
Mitochondrial membrane potential was assessed by flow cytometry in the indicated strains grown in lactate medium and stained with 50nM rliodamine 123 (Rh 123) in the absence or presence of 20mM azide (Rhl23 + azide) by flow cytometry. Data are mean +/- SD of three independent experiments.
Figure 10: Downregulation of MPC1 slows down proliferation of 143B osteosarcoma cells
A. Cells were seeded at 20' 000 cells per well in 6 -we 11 plates and grown for 6 days with the indicated concentration of doxycycline (ng/nil). Cells were then detached with trypsin and counted with a Casy counter. The average of three wells in shown +/- SD. B. Total protein extract were prepared from cells treated as in A. Protein were resolved by SDS- PAGE followed by protein immunoblotting using antibodies against MPC1. The band corresponding to the size of MFC 1 is shown with an arrow.
SEQUENCE LISTING
SEQ ID NO: 1 represents the amino acid sequence of M PCI in Human (Homo sapiens)
SEQ ID NO: 2 represents the amino acid sequence of MPC2 in Human (Homo sapiens)
SEQ ID NO: 3 represents the amino acid sequence of MPC3 in Saccharomyces cerevisiae
SEQ ID NO: 4 represents the DNA sequence of hMPCl in Human (Homo sapiens)
SEQ ID NO: 5 represents the DNA sequence of hMPC2 in Human (Homo sapiens)
SEQ ID NO: 6 represents the artificial shRNA sequence against MPC! .
DETAILED DESCRIPTION OF THE INVENTION
Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The publications and applications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.
In the case of conflict, the present specification, including definitions, will control. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in art to which the subject matter herein belongs. As used herein, the following definitions are supplied in order to facilitate the understanding of the present invention.
As used herein, the term "comprise" is generally used in the sense of include, that is to say permitting the presence of one or more features or components. As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise.
As used herein the terms "subject" or "patient" are well-recognized in the art, and, are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human,
"Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder, for example cancer, as well as those in which the disorder, for example cancer, is to be prevented. Hence, the mammal to be treated herein may have been diagnosed as having the disorder, for example cancer, or may be predisposed or susceptible to the disorder, for example cancer.
"Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals or pet animals, such as dogs, horses, cats, cows, monkeys etc. Preferably, the mammal is human.
The term "therapeutically effective amount" refers to an amount of a drag effective to treat a disease or disorder in a mammal. In the case of cancer, the therapeutically effective amount of the drag may reduce the number of cancer cells; reduce the tumour size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumour metastasis; inhibit, to some extent, tumour growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. To the extent the drag may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. The phrase "therapeutically effective amount" is used herein to mean an amount sufficient to prevent, or preferably reduce by at least about 30 percent, preferably by at least 50 percent, preferably by at least 70 percent, preferably by at least 80 percent, preferably by at least 90%, a clinically significant change in the growth or progression or mitotic activity of a target cellular mass, group of cancer cells or tumour, or other feature of pathology.
The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals mammals that is typically characterized by unregulated cell growth. According to the present invention, cancer refers preferably to.
Surprisingly, Applicants have identified and characterized a new family of membrane proteins (pfam UPF0041) whose members are necessary and sufficient for the transport of pyruvate into mitochondria. Applicants have renamed this family the Mitochondrial Pyruvate Carrier (MPC) family. Applicants previously identified MPC 1 (formerly Brp44L) in a proteomic analysis of the IMM of mouse liver (4). MPCl and its paralog MPC2 (formerly Brp44) have unknown function. Both are IMM proteins with three predicted transmembrane a-hclices on the basis of secondary structure predictions and hydropathy profiling (Fig. 5). They share sequence similarity with yeast Mpcl (Ygl080w), Mpc2 (Yhrl62w) and Mpc3 (Ygr243w) (Fig. 5 C and D).
In particular, the present invention provides an isolated and purified mitochondrial Pyruvate carrier (MPC ) family comprising the heterocomplex association of two members of a family of membrane proteins consisting of an isolated and purified MPCl member (Brp44L) together with an isolated and purified MPC2 member (Brp44), wherein said isolated and purified MPC family is capable of transporting Pyruvate into mitochondria.
According to an embodiment of the invention, the mitochondrial pyruvate carrier consists in the mandator)' association of a heterodimer of MPC1/MPC2. Preferably the isolated and purified mitochondrial Pyruvate carrier (MPC) is a heterodimer of human MPC1/MPC2.
In a particular embodiment of the invention, the isolated and purified mitochondrial Pyruvate carrier (MPC) family further comprises the association of a third member of a family of membrane proteins consisting of an isolated and purified MPC3 member.
The modulation of the transport of Pyruvate into mitochondria has direct consequences in the treatment or prevention of cancer or diabetes as well as in treating or preventing ischemia reperfusion injury.
Cancers to be treated according to the present invention include osteosarcoma, Breast cancer, Small lung cell cancer, Gliomas and glioblastoma, Colon cancer as well as leukemia The term "diabetes" or "insulin resistance" refers to a state in which a normal amount of insulin produces a subnormal biologic response relative to the biological response in a subject that does not have insulin resistance. An "insulin resistance disorder" refers to any disease or condition that is caused by or contributed to by insulin resistance.
The term "protein" or "polypeptide" refers to a polymer of amino acids and its equivalent and does not refer to a specific length of the product; thus, peptides, oligopeptides and proteins are included within the definition of a polypeptide. This term also does not refer to, or exclude modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations, and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, natural amino acids, etc.), polypeptides with substituted linkages as well as other modifications known in the art, both naturally and non-natural ly occurring.
In the context of the present invention, a homologous sequence is taken to include an amino acid sequence which is at least 60, 70, 80 or 90% identical, preferably at least 95 or 98% identical at the amino acid level over at least 20, 50, 100, 200, 300 or 400 amino acids with the amino acid sequences of Brp44L and Brp44. In particular, homology should typically be considered with respect to those regions of the sequence known to be essential for the function of the protein rather than non-essential neighbouring sequences.
Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity. The terms "substantial homology" or "substantial identity", when referring to polypeptides, indicate that the polypeptide or protein in question exhibits at least about 70% identity with an entire naturally-occurring protein or a portion thereof, usually at least about 80*% identity, and preferably at least about 90 or 95% identity.
Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences. Percentage (%) homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues (for example less than 50 contiguous amino acids).
The terms "isolated", "substantially pure", and "substantially homogeneous" are used interchangeably to describe a protein or polypeptide that has been separated from components that accompany it in its natural state. A monomerie protein is substantially purified when at least about 60 to 75% of a sample exhibits a single polypeptide sequence. A substantially purified protein will typically comprise about 60 to 90% W/W of a protein sample, more usually about 95%, and preferably will be over about 99% pure. Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel. For certain puiposes, higher resolution may be provided by using HPLC or other means well known in the art which are utilized for application.
A MPC protein is substantially free of naturally associated components when it is separated from the native contaminants that accompany it in its natural state. Thus, a polypeptide that is chemically synthesised or synthesised in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components. A protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.
A polypeptide produced as an expression product of an isolated and manipulated genetic sequence is an "isolated polypeptide," as used herein, even if expressed in a homologous cell type.
Synthetically made forms or molecules expressed by heterologous cells are inherently isolated molecules.
In particular, the isolated and purified mitochondrial Pyruvate carrier (MPC) family of the invention is capable of transporting Pyruvate into mitochondria of mammalian cells, preferably human cells. However, the isolated and purified mitochondrial Pyruvate carrier (MPC) family of the invention is also capable of transporting Pyruvate into mitochondria of yeast. In the latter, three members of a family of membrane proteins are required and consist of Mpcl (Ygl OSOw), Mpc2 (Yhrl62w) and
Mpc3 (Ygr243w).
It is another object of the invention to provide a modulator of the Pyruvate import activity in a patient, wherein that said modulator of the Pyruvate import activity is capable of either activating or inhibiting the activity of the isolated and purified mitochondrial Pyruvate carrier (MPC) family. The modulator of the pyruvate import can be either a shRNA directed against MPC 1 or MPC2 or a small molecule.
As used herein, a modulator compound of the invention can include, but is not limited to: nucleic acids, peptides, proteins such as antibodies, sugars, polysaccharides, glycoproteins, lipids, and small organic molecules. A "modulator compound" is a compound that can be tested in the screening assay of the present invention.
The term "small molecule or small organic molecule" typically refers to molecules of a size comparable to those organic molecules generally used in pharmaceuticals. Small organic molecules generally exclude biological polymers (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da., more preferably up to 2000 Da., and most preferably up to about 1000 Da.
In some embodiments, the modulator compound can be at least one of a metal, a peptide, a protein, a lipid, a polysaccharide, a nucleic acid, a library of small organic molecules, and a drug.
In some embodiments the modulator compound of the invention can include a fluorescent molecule, a radionuclide, a protein tag or combination thereof.
In one embodiment, modulator of the Pyruvate import activity of the invention can be an inhibitor in the form of a nucleic acid. As used herein, the nucleic acids modulator of the invention encompasses siRNA molecules, shRNA molecules, miRNA molecules, and antisense nucleic acid molecules. In some embodiments, a shRNA nucleic acid molecule refers to an RNA agent having a stem-loop structure, comprising a first and second region of complementary sequence, the degree of complementary sequence, the degree of complementarity and orientation of the regions being sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region.
In particular, a preferred modulator of the Pyruvate import activity of the invention acting as an inhibitor is a sliRNA of SEQ ID NO: 6 directed against MPCl .
Preferably said shRNA is capable of reducing the growth of osteosarcoma cells.
In particular, a lentiviras expressing a shRNA against MPCl is capable of reducing the growth of osteosarcoma cells.
As used herein, orthologs, homologs, functional fragments, or mutant forms of shRNA of SEQ ID
NO: 6 directed against MPCl refer to nucliec acids having at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 100%, identity to, or homologous with the SEQ ID NO: 6 or nucleic acid sequence that hybridizes to the coding region of the nucleic acid sequence in SEQ II) NO: 6 or complementary sequences thereof under "stringent hybridization conditions" as is defined herein and commonly used in the art of molecular biology, for example, in: Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridisation with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993).
The modulator of the Pyruvate import activity of the invention when acting as an inhibitor may be used in a method of treating or preventing cancer or diabetes (see example 4.3 ).
Cancers to be treated according to the present invention include osteosarcoma, Breast cancer, Small lung cell cancer, Gliomas and glioblastoma, Colon cancer as well as leukemia.
In addition, the modulator of the Pyruvate import activity of the invention, when acting as an inhibitor, may be used in a method of treating or preventing ischemia rcperfusion injury (see example 4.4). shRNAs directed against MPC1 or MPC2 can alter expression of the MFC and thereby decrease the activity of the carrier. Expression of these shRNA is mediated by lenti viruses (see example 4.3).
Another object of the invention is to provide an in vitro method (or screening method) of identifying modulator candidates of the Pyruvate import activity comprising the steps of:
- culturing a medium of bacteria that express a human MPCI /MPC2 heterodimer in their cell membrane (namely the purified human mitochondrial pyruvate carrier as defined above);
- adding labeled pyruvate together with said modulator candidates to said medium of bacteria;
- isolating the bacteria from said culture medium and measuring the amount of label associated with the bacteria;
wherein modulator candidates that decrease the import of pyruvate are inhibitors of the MPC activity whereas modulator candidates that increase the import of pyruvate are activators of the MPC activity.
According to one embodiment of the invention the added labeled pyruvate of the invention can include a fluorescent molecule, a radionuclide, a protein tag or combination thereof. Preferably the added labeled pyruvate is radioactive.
The modulator candidates acting as inhibitors of the MPC activity are used in a method of treating or preventing cancer or diabetes as well as in a method of treating or preventing ischemia reperfusion injury. Preferred cancers to be treated according to the present invention include osteosarcoma, Breast cancer, Small lung cell cancer, Gliomas and glioblastoma, Colon cancer as well as leukemia.
The surprising advantage of the in vitro method (or screening method) of identifying modulator candidates resides in the use of Lactococcus lactis as an expression system and in the expression of said two human proteins in this artificial system. Importantly, these two human proteins are able to reconstitute a functional pyruvate earner in the bacteria. Thus the screening method according to the present invention requires the expression of both MPCl and MPC2 in the membrane of bacteria. Thus the use of the entire heterodimer according to the invention is required for the screening method to be functional and effective.
In addition, the screening method according to the present invention requires that the heterodimer be expressed in a membrane, such as the cell membrane of a bacterium. The assay cannot function with purified soluble proteins as the mitochondrial pyruvate transporter heterodimer has to reside in a membrane to be functional and as pyruvate transport can only be measured across a membrane.
This method is also characterized by the use of bacteria that do not have mitochondria and do not express the mitochondrial pyruvate carrier endogeneously. In particular the screening method according to the invention is based on the expression of the human mitochondrial pyruvate carrier in bacteria that naturally do not contain the latter (i.e. introduction into a cell that does not contain it). This allows the measurement of pyruvate transport by the exogeneously introduced mitochondrial pyruvate transporter with insignificant background noise from endogeneous pyruvate transport.
Preferably the bacteria or prokaryotic cells able to express both MPCl and MPC2 members at their surface and preferably expressing a human MPC1/MPC2 heterodimer in their cell membrane is lactococcus lactis.
In brief, bacteria (i.e. Lactoccus lactis) expressing both human MPCl and MPC2 will be used for the in vitro method of identifying modulator candidates of the Pyruvate import activity (screen). Radioactive pyruvate will be added to the culture medium. Thanks to the expression of the two proteins in their membrane, the bacteria are able to import radioactive pyruvate. To assess the transport of pyruvate, the bacteria are isolated from the culture medium by filtration and the radioactivity is measured. The higher the radioactivity, the higher the import of pyruvate into the bacteria. The goal is to identify chemical compounds that can modulate pyruvate import. Modulator candidates (i.e. chemical compounds) will be added in the culture medium together with radioactive pyruvate. Modulators compounds that either decrease the import of pyruvate (inhibitors) or that increase the import of pyruvate (activators) are selected. In some embodiments, the screening assay of the present invention includes the screening of one or more modulator candidate compounds in the form of a library of compounds, The libraiy of compounds comprises a combinatorial chemical library. Combinatorial chemical libraries can include a plurality of small organic molecules. Typically, the combinatorial chemical library can contain at least 1000 candidate compounds. In various embodiments, the candidate compound is a small organic molecule.
A further object of the invention is a kit for identifying modulator candidates of the Pyruvate import activity comprising bacteria able to express both MPC1 and MPC2 members at their surface and optionally radioactive pyruvate.
Preferably the bacteria able to express both MPC1 and MPC2 members at their surface is lactococcus lactis.
The identified modulator candidates acting as inhibitors of the MFC activity are useful in the treatment or prevention of cancers or diabetes as well as in the treatment or prevention of ischemia reperfusion injury. Preferred cancers to be treated according to the present invention include osteosarcoma, Breast cancer, Small lung cell cancer, Gliomas and glioblastoma, Colon cancer as well as leukemia.
Generally, the kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds the compound's composition or the pro-drug composition or pharmaceutically acceptable salts thereof that are effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert indicates that the composition is used for treating the condition of choice, such as cancer.
The present invention also includes pharmaceutical compositions and formulations that include the modulator compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications without departing from the spirit or essential characteristics thereof. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.
Various references are cited throughout this Specification, each of which is incorporated herein by reference in its entirety.
The foregoing description will be more fully understood with reference to the following Examples. Such Examples, are, however, exemplary of methods of practicing the present invention and are not intended to limit the scope of the invention.
EXAMPLES
Example 1 :
Applicants have identified a family of membrane proteins (pfam UPF0041) whose members are necessary and sufficient for the transport of pyruvate into mitochondria. Applicants have renamed this family the Mitochondrial Pyruvate Carrier (MPC) family.
Applicants previously identified MPC1 (formerly Brp44L) in a proteomic analysis of the IMM of mouse liver (J). MPC1 and its paralog MPC2 ( formerly Brp44) have unknown function. Both are IMM proteins with three predicted transmembrane a -helices on the basis of secondary structure predictions and hydropathy profiling (Fig. 5). They share sequence similarity with yeast Mpcl (YglOSOw), Mpc2 (Yhrl62w) and Mpc3 (Ygr243w) (Fig. 5 C and D). Applicants used D). Applicants used Saccharomyces cerevisiae as a model organism to investigate the function of this family of proteins.
In yeast, Mpcl localized in the I. MM (Fig. 6 S2). Growth of the knockout mutants mpclA, mpc2\, mpc3A and mpc2Ampc3A was normal on rich medium, either containing fermentable (YPD) or non-fermentable carbon sources (YPG) (Fig. 1A). In contrast, mpcl A and mpc2,\mpc3A cells grew more slowly in amino acid-free medium (SD) than did the cells of the isogenic wild-type (WT) strain (Fig. 1 A and 7 A). Deletion of MFC 2 alone led to a minor growth defect whereas deletion of
MPC3 had no visible effect on growth.
Expression of WT MPC1 restored growth of mpcl A cells (Fig. 1 B and C). Suppressor screens for genes that restored growth of mpcl A cells in SD identified only MFC I (SOM text), suggesting that its function cannot be complemented by other genes. Expression of either MPC2 or MPC3 restored growth of mpc2A mpc3A cells, but not that of cells lacking MPC1 (Fig. 1 B and C). Thus the growth of yeast in SD required a combination of Mpcl with Mpc2 or Mpc3. Applicants found that Mpcl coimmunoprecipitated with Mpc2, suggesting that they form a heteroeomplex (Fig. 7 B).
The growth defect in SD was relieved by addition of valine or leucine, with an additive effect of both amino acids (Fig. 1A). In contrast, addition of all amino acids except leucine and valine did not restore growth of the mutant strains (SC -V · F; Fig. 1A). This finding prompted Applicants to investigate the role of MPC proteins in the metabolism of valine and leucine. Applicants assayed their decarboxylation by monitoring the release of 14C02 by cells grown in SD supplemented with either 1-14C valine or 1-] 4C leucine. Release of !4C02 by mpcl A cells was less than 2% of that in WT cells (Fig. ID). A similar defect occurs in an IpdlA strain (Fig. IE), lacking a lipoamide dehydrogenase essential for the function of the mitochondrial dehydrogenase complexes, PDH, a - KDH and BCKDH. To test whether mpcl A cells also had dysfunctional PDH and a -KDH, Applicants assessed their activities in mitochondria. The activity of these two complexes was impaired in mitochondria from mpcl A cells grown in SD (Fig. IF), suggesting that the function of Mpcl extends to several lipoyl-dcpendent complexes.
I .ipoie acid is covalently attached to the E2 subunits of PDH (Latl) and a -KDH (Kgd2), the E3- binding protein of PDH (Pdxl) and the H subunit of the glycine cleavage system (Gcv3). This modification can be readily assessed by protein immunoblotting with antibodies to lipoic acid (10). In rich medium, WT, mpcl A, mpc2A, mpc3A and mpc2A mpcSA cells had similar amounts of lipoylated complexes (Fig. 2 A). However, when cells were grown in SD, lipoylated proteins were lipoylated proteins were virtually absent from mpcl A, rnpc2\ and mpc2Ampc3A but not mpc3A cell lysates (Fig, 2A). Addition of valine or leucine, or both, to SD or expression of MPC1 restored lipoylation in mpclA (Fig. 2B and Fig. 7 C). The defect in lipoylation was correlated with decreased abundance of lipoic acid in mpcl A cells grown in SD (Fig. 7 D). In contrast, lipoic acid was abundant in mpclA cells grown in rich medium (Fig. 7 D). Thus, Mpcl is essential for the production of lipoic acid and the function of lipoyl-dependent complexes only when cells are grown in SD and this defect is relieved by the addition of valine and leucine.
Applicants tested at which level the pathway leading to lipoic acid synthesis was defective in mpcl A cells grown in SD (Fig. 8). Applicants excluded defects in the mitochondrial acetyl-CoA carboxylase HFA1, which converts acetyl-CoA into malonyl-CoA, or in the lipoic acid synthase Lip5, which converts octanoic acid into lipoic acid, as cells lacking these enzymes cannot produce lipoic acid even when grown in rich medium. In addition, no defect in the synthesis of octanoic acid from malonyl-CoA was detected in mitochondria isolated from mpcl A ceils grown in SD medium (Fig. 7 E). Thus, the function of Mpcl was likely to be upstream of acetyl-CoA (Fig. 8). As it is impossible to measure amounts of acetyl-CoA in mitochondria accurately, we tested the impact of reduced amount of mitochondrial acetyl-CoA on protein lipoylation by inhibiting PDH, the main source of acetyl-CoA from pyruvate. PDFI deficient cells (pdxlA) were defective in protein lipoylation in SD, which was restored by addition of valine and leucine, similarly to mpcl A cells (Fig. 2B). However, PDH activity was almost normal in mpcl A cells grown in rich medium (i.e. when the E2 subunit was lipoylated (Fig. 2C). Thus the MFC proteins appeared to act upstream of PDH and may function in the transport of pyruvate into mitochondria. Applicants measured uptake of !4C pyruvate in mitochondria isolated from WT, mpcl A, mpc2A, mpcSA and mpc2A mpc3A cells grown in lactate medium (Fig. 3 A). The specificity of uptake was assessed by the use of UK5099, an non specific inhibitor of the mitochondrial pyruvate carrier (4). Uptake of pyruvate in WT mitochondria was sensitive to the proton ionophore CCCP (Fig. 3B). Mitochondria from mpcl A and mpc2A mpc3A cells showed decreased pyruvate uptake (Fig. 3A and B) despite a normal mitochondrial membrane potential (Fig 9). Surprisingly, deletion of MPC3 alone impaired pyruvate uptake in mitochondria whereas mitochondria from the mpc2A mutant transported pyruvate normally. As this result did not correlate with the phenotypes of mpc2A and mpc3A single mutants grown in SD, Applicants investigated the expression of Mpc2 and Mpc3 in SD and lactate media. In SD, yeast expressed mainly Mpc2 whereas in lactate medium, they mainly expressed Mpc3 (Fig. whereas in lactate medium, they mainly expressed Mpc3 (Fig, 3C). This expression pattern could be explained, at least in part, by the presence of binding sites for Gcn4, a transcription factor activated by amino acid starvation, upstream of MPC2. This raises that under certain growth conditions, these two proteins have specific, non-redundant, functions.
Applicants next assessed whether murine MPC1 (mMPCl ) and MPC2 (mMPC2) could restore growth of yeast cells lacking a functional pyruvate transporter (Fig. 4A and B). mMPCl alone restored growth of mpcl Δ cells but mMPC2 failed to restore growth of the double deletion strain of its orthologous genes MFC 2 and MFC 3. However, growth of the triple deletion strain m
Figure imgf000020_0001
pc I Λ mpc2 \ cells was restored by coexpression of both mMPCl and mMPC2 (Fig. 4A). Thus, mMPCl and mMPC2 together functionally complement the absence of pyruvate transport. Applicants next expressed mMPCl and mMPC2, alone and in combination, in the bacterium Lactococcus lactis (Fig. 4C), which has been successfully used to express and characterize mitochondrial transporters (5). No pyruvate uptake was observed in bacteria expressing either protein alone compared to the empty vector control. However, a four-fold increase in pyruvate uptake was detected when mMPCl and mMPC2 were coexpressed (Fig. 4D and E). This uptake was sensitive to the mitochondrial pyruvate carrier inhibitor UK5099 and to 2- deoxyglucose, which collapses the proton electrochemical gradient (Fig. 4E). Moreover, artificially increasing the membrane potential by lowering the pH in the import buffer from 7.2 to 6.2 significantly increased pyruvate uptake (two-tailed T-test, p<().()5. Fig. 4E). Thus, coexpression of mMPCl and mMPC2 in bacteria is sufficient to allow import of pyruvate with similar properties to the mitochondrial pyruvate carrier (1). Applicants therefore conclude that the mitochondrial pyruvate carrier is composed of Mpcl and either Mpc2 or Mpc3 in yeast and of MPC 1 and MPC2 in mammals.
Example 2:
Materials and Methods
Yeast strains and growth conditions
Yeast strains used in this study are derived from BY4741 requiring only uracil (ura) or uracil and histidine (his ura) and are listed in table SI . Cells were grown according to standard procedure. SD medium was supplemented with 20 mg/1 uracil and 100 mg/1 valine or leucine when indicated (SD when indicated (SD +V and SD + L respectively). For cells containing a plasmid, uracil was oinitted. SC -V -L contains all amino acids (at 50 mg/l) except valine and leucine. MPC1 (ygl080w), MPC2 (yhrl62w) and MPC3 (ygr243w) genes were cloned by PCR from BY4741 yeast genomic DNA into p2U vector (2μ, GPD promoter). Mouse MPC1 (NM_018819.4) and mouse MPC2 (NM 027430.2) coding sequences were optimized for yeast codon usage and synthesized by GenScript Inc (USA) and cloned downstream up the GPD promoter into pRS423 and pRS426 vectors respectively. Wild type his ura cells were cotransfonned with two vectors to generate cells expressing no protein, either protein alone or both proteins together.
Growth assays
For spot assays, overnight grown cells in appropriate medium were diluted to an OD600 of 0.5.
Serial 5-fold dilutions were spotted on plate, grown for 2 days at 30°C and photographed. Alternatively, cells were diluted to an OD600 of 0.1 in liquid medium and growth, curves were recorded in a 96-well plate reader by monitoring the OD600 at 30-minute intervals.
BCAA decarboxylation. Briefly, 500 μΐ of exponentiaily growing cells were incubated for 15 min with 20 μΜ 1-14C valine or leucine in SD medium at 30°C during which 14C02 was trapped by a filter paper pre- soaked in NaOH. The reaction was terminated with acetic acid and the incubation was continued for 1 hour to trap all the 14C02. The filters were then transferred to a scintillation vial and radioactivity measured by liquid scintillation counting. The reaction proved to be linear over this time frame. The decarboxylation is expressed as nmol C02 released per OD unit per hour.
Mitochondria isolation
Yeast mitochondria were isolated from cells grown in lactate medium, unless otherwise stated. Mammalian mitochondria were isolated from 293T cells using a Dounce homogenizer. Nuclei and cell debris were removed (2000g) and mitochondria were recovered by centrifugation at 10'OOOg.
Submitochondrial local ization
Ι ΟμΙ of mitochondria (5mg/ml) isolated from yeast or 293T cells were added to 90μ1 of isolation buffer or 90μ1 of 2()m.Vl Hepes (swelling) or 90μ1 of isolation buffer containing 1% Triton XI 00. 5\ig proteinase K was added when required and incubated on ice for 30 minutes. ImM PMSF was 1 mM PMSF was added to inhibit the proteinase K and all samples were precipitated with 12% TCA and resolved on SDS-PAGE followed by protein immunoblotting. For sodium carbonate extraction Ι ΟμΙ mitochondria were mixed with 90μ] 20 mM Hepes and incubated for 20 minutes on ice. Sodium carbonate was added at a final concentration of 1 QOmM, vortexed and further incubated 2 minutes on ice before ultracentrif ligation at 100'OOOg for 15 minutes. The pellet (P) and supernatant (S) were precipitated with 12% TCA and resolved on SDS-PAGE followed by protein immunoblotting.
Immunoprecipitation
For immunoprecipitation, mitochondria were solubilized in IP buffer (150 mM NaCl, 10 mM I ris pH 7.4, 10% glycerol, 1 % digitonin) on ice for 30 minutes and centrifuged at 100Ό00 g for 1 hour. Protein G beads (GE healthcare) were incubated with 2 μΐ anii HA (Covance Inc.) for 2 hours at
4°C and added to the mitochondrial lysate. Following incubation, beads were washed and the immunoprecipitate was analyzed by Western blot analysis after elution in Ix SDS loading buffer.
Dehydrogenase activity
PDH and a-KDH activities were assessed using 3-Acetylpyridine adenine dinucleotide (APAD) in place of NAD+ to avoid reoxidation. Reactions were initiated by the addition of 1 mM substrate (pyruvate or u-ketoglutarate respectively). Activities are expressed as nmol reduced APAD per minute per mg of mitochondrial protein (mU/mg).
Pyruvate import in isolated mitochondria
Pyruvate import was assessed in isolated mitochondria by the inhibitor-stop method as described previously (I) with minor modifications. Briefly, mitochondria were suspended at 10 mg/ml in import buffer (120 mM KG, 5m M NaCl, 2 mM potassium phosphate pf 17.4) and transferred to the same buffer at pi I 6.8 containing 50 uM 2-14C pyruvate to initiate import. UK5099 was used to terminate the reaction or was present before mitochondria as control. Mitochondria were spun down, washed with import buffer containing 50 mM cold pyruvate and radioactivity was quantified in the pellet by liquid scintillation counting. Radioactivity in control sample was subtracted from ail time points to determine the inhibitor-sensitive import. Rate of pyruvate import were calculated from the first two minutes of the reaction. Pyruvate import in bacteria
Mouse MFC I (mMPCl) or MPC2 (mMPC2) were codon optimized (GenScript),
subcloned in pNZ8148 vector and transformed in L. lactis. For coexpression (mMPCl +
mMPC2), the chloramphenicol resistance cassette of pNZ8148 mMPCl was replaced by the erythromycin resistance cassette of the vector pli .253 thus generating pNZ8148e mMPCl vector. This vector was cotransformed together with pNZ8148 mMPC2 (See Table 1).
Table 1. Strains used in this study
Figure imgf000023_0001
Bacteria were grown overnight in Ml 7 containing 1% glucose, 1 Opg/ml chloramphenicol and lO^tg/ml erythromycin when needed, diluted to an 01)600 of 0.1 , further grown for 3 hours before induction of expression with lng/1 Nisin A. Import was measured after 20 hours of induction by incubating bacteria in PBS with 37.5μΜ [2-,4C] pyruvate for 10, 20, 30 or 60 minutes at room temperature followed by filtration over nitrocellulose ester filters. The filters were washed twice with cold PBS, transferred to a scintillation vial followed by liquid scintillation counting. Protein immunoblotting was performed on the same cells to assess the expression level of the proteins.
Example 3 :
Multicopy suppressor screen (MCS)
Two MCS were performed to find genes able to rescue the growth defect of mpclA strain. mpclA cells were transformed with a homemade gDNA library in YEP 13 vector and plated on medium lacking leucine, in order to select for both transformation events and rescue of growth without leucine. 23 independent clones were isolated, which all contained a plasmid with MPC1 gene.
Next, Applicants transformed mpclA cells with a pool of 1588 plasm ids covering 97% of the S. cere vis iae genome (Open Biosystems). Cells were plated on SD plate containing isoleucine, a condition where the growth defect of mpclA was stronger and allowed better identification of rescued clones. In this screen, 38 independent clones were isolated and all contained the WT MFC 7 ORF.
Applicants obtained a more than 50-fold coverage of the genome in the two screens and the only gene found to rescue the growth defect of mpclA strain was MPC1 itself, suggesting that loss of this gene cannot be compensated by any other.
Example 4:
1. Screen to identify small molecule inhibitors of the mitochondrial pyruvate carrier 1. Use of Lactococcus lactis as a primary screen Use of lactococcus lactis (L. lactis) as a screening tool for the identification of small molecule inhibitors of the mitochondrial pyruvate carrier. L. lactis has been previously used to characterize mitochondrial carriers (5).
1.1 Coexpression of MFC 1 and MPC2 in L. lactis
Human MPC1 (hMPCl) and MPC2 (hMPC2) are codon optimized for expression in L. lactis. KMPC2 is subcloncd downstream of the nisin promoter in pNZ8148 vector that contains a chloramphenicol resistance cassette. hMPCl is subcloncd downstream of the nisin promoter in a modified pNZ8148 vector, in which the chloramphenicol cassette is replaced by the erythromycin resistance cassette of the vector pi 1, 253. These constructs are cotransformed together in L. lactis by electroporation. Bacteria are grown overnight in Ml 7 Broth containing 1 % glucose, 10 ug/ml chloramphenicol and 10tug/ml erythromycin, diluted to an OD600 of 0.1, further grown for 3 hours before induction of expression with lng/1 Nisin A.
1.2 Principle of pyruvate import into L. lactis
Import is measured after 20 hours of induction by incubating bacteria for 60 minutes at room temperature in PBS containing 37.5 μΜ [2-!4C | pyruvate (Mix). Bacteria are recovered by filtration over nitrocellulose ester filters. The filters are washed twice with cold PBS and radioactivity detected by liquid scintillation counting. For inhibitor sensitivity, is added to the mix before the bacteria.
1 .3 Screening of small molecules in 96 or 384 well format
In each well of a multiScreennTS 96- or 384-well plate (Millipore), a compound of a chemical library is added at 20 μΜ in 50 or 25 μΐ Mix before addition of 50 or 25 μΐ bacteria (10 OD/ml).
The mitochondrial pyruvate inhibitor, UK5099 (Sigma), is used as a control. After incubation for 1 hour, the plate is transferred to a MultiScreenHTS vacuum manifold and the bacteria isolated by filtration. 50 or 25 μΐ scintillation fluid is added to each well before counting using a microplate reader. IC50s are determined for each compound that shows an inhibitory activity in the first screen. Compounds with an IC50 < 1 mM (Hits) are selected for further assays.
2. Validation of hits using cultured cells and isolated mitochondria from cultured cells 2.1 Use of cultured cells
Osteosarcoma cells (143 B cells) are cultured in 96 well plates. Compounds are tested at 10 μΜ and the pi 1 and oxygen consumption measured during a 12-hour period using a Seahorse XF-96 apparatus. Compounds that significantly decrease the pH and oxygen consumption are tested for their specific activity on the mitochondrial pyruvate carrier using isolated mitochondria.
2.2 Use of isolated mitochondria
Mitochondria from 143B cells are isolated using a standard procedure and used to test the activity of the compounds on pyruvate import and oxygen consumption.
2.2.1 Pyruvate import
500 iig mitochondria are incubated with 37.5 μΜ [2-14C] pyruvate in 120 mM KC1, 5 mM NaCl and 2 mM KH2P04, pH 6.8 (KC1 buffer) for 2 min. Mitochondria are eentrifuged at 16Ό00 g for 1 min. The pellet is washed in 500 ml KC1 buffer containing 50 mM cold pyruvate. Mitochondria are eentrifuged as above and the pellet resuspended in 200 ml ¾0 before addition of 3 ml of scintillation fluid and counting using a scintillation counter.
2.2.2. Oxygen consumption
Oxygen consumption is measured on isolated mitochondria in the presence or absence of compounds, using a Seahorse XF-96 apparatus. 5 mM Pyruvate or succinate is used as a respiratory substrate.
Compounds that specifically inhibit pyruvate-driven respiration and pyruvate import are considered as inhibitors of the mitochondrial pyruvate carrier.
3. Applications for inhibitors of the mitochondrial pyruvate carrier in breast cancer
In some types of cancer, lactate produced in the hypoxic region of the tumor has been suggested to feed the respiratory chain of cells in more oxygenated regions of the tumor and this effect was shown to be essential for tumor cell viability. For example, pyruvate oxidation was reported to be was reported to be essential for tumor proliferation in breast cancers (6), Interestingly, MPC1 upregulation was shown to be a marker of tumorigenesis and metastasis in breast cancer, where MPC1 mRNA and protein levels were higher in cancer cells than in control cells (7). Therefore, the observation that pyruvate fuels mitochondrial respiration in breast cancer and that MPC1 is overexpressed in tumorigenic and metastatic breast cancer cells strongly suggest that mitochondrial pyruvate transport is a key determinant in breast cancer progression. Specific inhibition of MFC proteins could be used as a therapeutic strategy for this type of cancer.
Inhibition of MPC1 slows down cancer cells proliferation
Applicants transduced 143B osteosarcoma cells with lentiviruses expressing a doxycycline- inducible shRNA against MPC1 (shMPCl) or carrying an empty vector as a control. Cells were then seeded at a density of 20Ό00 cells per well in a 6- well plate and grown for 6 days in the absence (0) or with the indicated concentration of doxycycline. The doxycycline was changed every 48 hours. The number of cells in the wells was then counted. As shown in Fig 10A, there was a decrease in the number of cells per well in the presence of doxycycline. Applicants did not observe cell death in these wells and thus concluded that the proliferation of these cells decreased. Of note, the proliferation was not altered by doxycycline alone and can thus be attributed to the induction of the MPC1 shRNA. The level of MPC1 protein after 6 days of shRNA induction with the different doses of doxycycline is shown in Fig. l OB. A strong reduction in the amount of MPC1 (arrow) was observed for all the doses tested compared to no doxycycline or to the empty vector in the presence of the highest doxycycline dose. Thus, downregulation of MPC1 correlated with a decrease in the proliferation of 143 B osteocarcoma cells.
4. Inhibition of the mitochondrial pyruvate carrier to prevent cell damage during ischemia- reperfpsion injury
Upon reperfusion of ischemic tissues (brain, myocardic tissue, kidney) cells produce high amounts of reactive oxygen species (ROS) that may cause more damage than the ischemic accident itself. ROS are mainly produced by the mitochondrial respiratory chain. Inhibiting ROS is one of the approaches that has been proposed to prevent ischemia-reperfusion injury. However, ROS However, ROS scavengers are not highly efficient. Their ability to scavenge ROS is limited, in part because these molecules oxidize various substrates (proteins, lipids, DNA) with an extreme rapidity. Ideally, one would like to prevent the formation of ROS. Inhibition of mitochondrial pyruvate import is expected to diminish ROS production. By preventing pyruvate to enter into mitochondria, the cells will produce energy mainly through glycolysis and their mitochondrial respiratory chain activity will be significantly decreased. This will decrease the formation of ROS and therefore protect cells from ischemia-reperfusion injur}'. Mitochondrial pyruvate inhibitors are expected to be of key interest in myocardial infarction and in stroke.
References
1. A. P. Halestrap, The mitochondrial pyruvate carrier. Kinetics and specificity for substrates and inhibitors. Biochem J 148, 85 (Apr, 1975).
2. Papa, S., Francavilla, A., Paradies, G., and Meduri, B. (1971). The transport of pyruvate in rat liver mitochondria. FEBS Lett 12, 285-288.
3. S. Da Cruz et al , Proteomic analysis of the mouse liver mitochondrial inner membrane. J Biol Chem 278, 41566 (Oct 17, 2003).
4. A. P. Halestrap, R. M. Denton, The specificity and metabolic implications of the inhibition of pyruvate transport in isolated mitochondria and intact tissue preparations by alpha- Cyano-4-hydroxycinnamate and related compounds. Biochem J " 148, 97 (Apr, 1975).
5. E. R. Kunji et al, Lactococcus lactis as host for overproduction of functional membrane proteins. Biochimica et biophysica acta 1610, 97 (Feb 17, 2003).
6 Diers, A.R., Broniowska, K.A., Chang, C.F., and Hogg, N. (2012). Pyruvate fuels mitochondrial respiration and proliferation of breast cancer cells: effect of monocarboxylate transporter inhibition. Biochem J.
7. Xu, X., Qiao, M., Zhang, Y., Jiang, Y., Wei, P., Yao, J., Gu, B., Wang, Y„ Lu, I, Wang, Z„ et al. (2010). Quantitative proteomics study of breast cancer cell lines isolated from a single patient: Discovery of TIMM17A as a marker for breast cancer. Proteomics.

Claims

1. An isolated and purified mitochondrial Pyruvate carrier (MPC) family comprising the heterocomplex association of two members of a family of membrane proteins consisting of an isolated and purified MPC1 member (Brp44L) together with an isolated and purified MPC2 member (Brp44). wherein said isolated and purified MPC family is capable of transporting
Pyruvate into mitochondria.
2. The isolated and purified mitochondrial Pyruvate carrier (MPC) family of claim 1, further comprising the association of a third member of a family of membrane proteins consisting of an isolated and purified MPC3 member.
3. The isolated and purified mitochondrial Pyruvate carrier (MPC) family of claims 1-2, wherein said isolated and purified MPC family is capable of transporting Pyruvate into
mitochondria of mammalian cells.
4. The isolated and purified mitochondrial Pyruvate carrier (MPC) family of claim 2, wherein said isolated and purified MPC family is capable of transporting Pyruvate into mitochondria of yeast, characterized in that said three members of a family of membrane proteins consist of Mpcl (Ygl080w), Mpc2 (Yhrl 62w) and Mpc3 (Ygr243w).
5. A modulator of the Pyruvate import activity in a patient, characterized in that said modulator of the Pyruvate import activity is capable of either activating or inhibiting the activity of the isolated and purified mitochondrial Pyruvate carrier (MPC) family of any of claims 1-3.
6. The modulator of the Pyruvate import activity of claim 5, wherein said modulator act as an inhibitor and whereas said inhibitor is a shRNA of SEQ ID NO: 5 directed against MPC1.
7. The modulator of the Pyruvate import activity of claim 6, wherein said shRNA is capable of reducing the growth of osteosarcoma cells.
8. The modulator of the Pyruvate import activity of claim 5, for use in a method of treating or preventing cancer or diabetes.
9. The modulator of the Pyruvate import activity of claim 5, for use in a method of treating or preventing ischemia reperfusion injury.
10. The modulator of the Pyruvate import activity for use according to any one of claims 8 or 9, characterized in that said modulator of the Pyruvate import activity is an inhibitor of the activity of the isolated and purified mitochondrial Pyruvate carrier (MFC) family of any one of claims 1-4.
1 1. An in vitro method of identifying modulator candidates of the Pyruvate import activity comprising the steps of:
- cul taring a medium of bacteria that express a human MPC1/MPC2 heterodimer in their cell membrane;
- adding labeled pyruvate together with said modulator candidates to said medium of bacteria;
- isolating the bacteria from said culture medium and measuring the amount of label associated with the bacteria;
wherein modulator candidates that decrease the import of pyruvate are inhibitors of the MFC activity whereas modulator candidates that increase the import of pyruvate are activators of the MFC activity.
12. The method of claim 1 1 , characterized in that the bacteria able to express both MPC1 and MPC2 members at their surface is lactococcus lactis.
13. The in vitro method according to any one of claims 1 1 to 12, wherein the identified modulator candidates of the Pyruvate import activity is acting as an inhibitor effective against cancers, diabetes or ischemia reperfusion injury.
14. The in vitro method according to any one of claims 1 1 to 13, wherein the added labelled pyruvate is radioactive.
15. A kit for identifying modulator candidates of the Pyruvate import activity comprising bacteria able to express both MPCl and MPC2 members at their surface and optionally radioactive pyruvate.
16. The kit of claim 15, characterized in that the bacteria able to express both MPCl and MPC2 members at their surface is lactococcus lactis.
PCT/EP2013/060815 2012-05-24 2013-05-24 Identification and functional expression of the mitochondrial pyruvate carrier, mpc1 WO2013175015A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP13727081.5A EP2855532A1 (en) 2012-05-24 2013-05-24 Identification and functional expression of the mitochondrial pyruvate carrier, mpc1

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP12169216.4 2012-05-24
EP12169216 2012-05-24

Publications (1)

Publication Number Publication Date
WO2013175015A1 true WO2013175015A1 (en) 2013-11-28

Family

ID=48576966

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2013/060815 WO2013175015A1 (en) 2012-05-24 2013-05-24 Identification and functional expression of the mitochondrial pyruvate carrier, mpc1

Country Status (2)

Country Link
EP (1) EP2855532A1 (en)
WO (1) WO2013175015A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2853602A1 (en) * 2013-09-25 2015-04-01 Samsung Electronics Co., Ltd Yeast cell with increased pyruvate pool in cytosol and method of producing pyruvate-based metabolite using the same
WO2015049365A3 (en) * 2013-10-03 2015-10-01 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods and pharmaceutical compositions for modulating autophagy in a subject in need thereof

Non-Patent Citations (13)

* Cited by examiner, † Cited by third party
Title
A. P. HALESTRAP: "The mitochondrial pyruvate carrier. Kinetics and specificity for substrates and inhibitors", BIOCHEM J, vol. 148, April 1975 (1975-04-01), pages 85
A. P. HALESTRAP; R. M. DENTON: "The specificity and metabolic implications of the inhibition of pyruvate transport in isolated mitochondria and intact tissue preparations by alpha-Cyano-4-hydroxycinnamate and related compounds", BIOCHEM J, vol. 148, April 1975 (1975-04-01), pages 97
D. K. BRICKER ET AL: "A Mitochondrial Pyruvate Carrier Required for Pyruvate Uptake in Yeast, Drosophila, and Humans", SCIENCE, vol. 337, no. 6090, 24 May 2012 (2012-05-24), pages 96 - 100, XP055076457, ISSN: 0036-8075, DOI: 10.1126/science.1218099 *
DANIEL K. BRICKER: "MPC1 plays an essential role in pyruvate metabolism in yeast, Drosophila and human disease.", 72, 27 January 2012 (2012-01-27), pages 145, XP002711805, Retrieved from the Internet <URL:http://www.drosophila-conf.org/2012/book/Abstracts_Web.pdf> [retrieved on 20130826] *
DATABASE UniProt [Online] 1 May 1999 (1999-05-01), "RecName: Full=Mitochondrial pyruvate carrier 2; AltName: Full=Brain protein 44;", XP002711807, retrieved from EBI accession no. UNIPROT:O95563 Database accession no. O95563 *
DATABASE UniProt [Online] 1 November 1999 (1999-11-01), "RecName: Full=Mitochondrial pyruvate carrier 1; AltName: Full=Brain protein 44-like protein;", XP002711806, retrieved from EBI accession no. UNIPROT:Q9Y5U8 Database accession no. Q9Y5U8 *
DIERS, A.R.; BRONIOWSKA, K.A.; CHANG, C.F.; HOGG, N.: "Pyruvate fuels mitochondrial respiration and proliferation of breast cancer cells: effect of monocarboxylate transporter inhibition", BIOCHEM J., 2012
E. R. KUNJI ET AL.: "Lactococcus lactis as host for overproduction of functional membrane proteins", BIOCHIMICA ET BIOPHYSICA ACTA, vol. 1610, 17 February 2003 (2003-02-17), pages 97
PAPA, S.; FRANCAVILLA, A.; PARADIES, G.; MEDURI, B.: "The transport of pyruvate in rat liver mitochondria", FEBS LETT, vol. 12, 1971, pages 285 - 288
S. DA CRUZ ET AL.: "Proteomic analysis of the mouse liver mitochondrial inner membrane", J BIOL CHEM, vol. 278, 17 October 2003 (2003-10-17), pages 41566
S. HERZIG ET AL: "Identification and Functional Expression of the Mitochondrial Pyruvate Carrier", SCIENCE, vol. 337, no. 6090, 24 May 2012 (2012-05-24), pages 93 - 96, XP055076460, ISSN: 0036-8075, DOI: 10.1126/science.1218530 *
TIJSSEN: "Overview of principles of hybridization and the strategy of nucleic acid assays", TECHNIQUES IN BIOCHEMISTRY MOLECULAR BIOLOGY-HYBRIDISATION WITH NUCLEIC PROBES, 1993
XU, X.; QIAO, M.; ZHANG, Y.; JIANG, Y.; WEI, P.; YAO, J.; GU, B.; WANG, Y.; LU, J.; WANG, Z. ET AL.: "Quantitative proteomics study of breast cancer cell lines isolated from a single patient: Discovery of TIMM17A as a marker for breast cancer", PROTEOMICS, 2010

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2853602A1 (en) * 2013-09-25 2015-04-01 Samsung Electronics Co., Ltd Yeast cell with increased pyruvate pool in cytosol and method of producing pyruvate-based metabolite using the same
WO2015049365A3 (en) * 2013-10-03 2015-10-01 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods and pharmaceutical compositions for modulating autophagy in a subject in need thereof
US11040052B2 (en) 2013-10-03 2021-06-22 Inserm (Institut National De La Sante Et De La Recherche Medicale) Methods and pharmaceutical compositions for modulating autophagy in a subject in need thereof

Also Published As

Publication number Publication date
EP2855532A1 (en) 2015-04-08

Similar Documents

Publication Publication Date Title
Mo et al. Targeting Ubc9 for cancer therapy
Kooij et al. Small-molecule activity-based probe for monitoring ubiquitin C-terminal hydrolase L1 (UCHL1) activity in live cells and zebrafish embryos
AU2008333810B2 (en) Compositions and methods to modulate cell membrane resealing
Kurisu et al. MDG1/ERdj4, an ER‐resident DnaJ family member, suppresses cell death induced by ER stress
CN110064056A (en) Cell permeable peptide, the conjugate comprising the peptide and the composition comprising the conjugate
US20140288162A1 (en) Methods, compositions and screens for therapeutics for the treatment of synovial sarcoma
EP2868326A1 (en) Peptide inhibitors of TEAD/YAP-TAZ interaction
US20100105625A1 (en) Product and Methods for Diagnosis and Therapy for Cardiac and Skeletal Muscle Disorders
Wang et al. Methyltransferase-like 21c methylates and stabilizes the heat shock protein Hspa8 in type I myofibers in mice
Cullen et al. AarF domain containing kinase 3 (ADCK3) mutant cells display signs of oxidative stress, defects in mitochondrial homeostasis and lysosomal accumulation
Milan et al. Pancreatic cancer cells require the transcription factor MYRF to maintain ER homeostasis
CA3003262C (en) Spexin-based galanin receptor type 2 agonists and use thereof
EP2855532A1 (en) Identification and functional expression of the mitochondrial pyruvate carrier, mpc1
CA2471307A1 (en) Protein complexes and methods for their use
US20140165222A1 (en) High affinity sumo traps
WO2017192761A1 (en) Propionyl-coa carboxylase compositions and uses thereof
Salvi et al. Programmed cell death protein 5 (PDCD5) is phosphorylated by CK2 in vitro and in 293T cells
US9766241B2 (en) PGC-1beta-protein-function regulator, mitochondria-function regulator, anti-obesity agent, and screening method therefor
EP2841449B1 (en) Archease as rna ligase complex member
CA2679435C (en) Use of inhibitors of the degradation of p27, in particular argyrin and derivatives thereof, for the treatment of proliferative diseases
KR20080080863A (en) A novel use of kiaa0317 as an apoptosis regulator
EP2619330B1 (en) Method for identifying compounds to increase the expression or activity of aspartyl aminopeptidase
CN117756909A (en) Improved anti-aging compounds and their use in cancer treatment
US9079968B2 (en) Establishment of motif comprising acidic amino acid, capable of stabilizing protein in cells, and applicable to protein therapy, control of differentiation/undifferentiation of cell and antibody therapy
CN112359034B (en) Polypeptide inhibitor and application thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13727081

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2013727081

Country of ref document: EP