Autophagy inhibitors
11229680 · 2022-01-25
Assignee
Inventors
- Mingjie Zhang (Hong Kong, CN)
- Jianchao Li (Sai Kung, CN)
- Ruichi Zhu (Hong Kong, CN)
- Chao Wang (Anhui, CN)
Cpc classification
A61P35/00
HUMAN NECESSITIES
International classification
A61K38/16
HUMAN NECESSITIES
Abstract
Provided herein are recombinant peptides useful for inhibiting the function of autophagy-related 8 (Atg8) proteins. The recombinant peptides can be used in the preparation of imaging agents for monitoring autophagy in a cell or subject and treating autophagy related diseases, such as cancer.
Claims
1. A recombinant peptide comprising the amino acid sequence of SEQ ID NO: 1, wherein position 7, 12, 13 or 16 of SEQ ID NO: 1 is substituted with Arg or Gln, and wherein the peptide binds GABA Type A Receptor-Associated Protein (GABARAP).
2. The recombinant peptide of claim 1, wherein position 7 or 12 of SEQ ID NO:1 is substituted with Arg, or position 13 or 16 of SEQ ID NO:1 is substituted with Gln.
3. The recombinant peptide of claim 1, wherein position 7 or 12 of SEQ ID NO:1 is substituted with Arg.
4. The recombinant peptide of claim 1, wherein position 13 or 16 of SEQ ID NO:1 is substituted with Gln.
5. The recombinant peptide of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:2.
6. The recombinant peptide of claim 5, wherein the peptide comprises the amino acid sequence of SEQ ID NO:14.
7. The recombinant peptide of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:15.
8. The recombinant peptide of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:16.
9. The recombinant peptide of claim 1, further comprising an affinity tag or detectable label.
10. The recombinant peptide of claim 9, wherein the detectable label is selected from the group consisting of chromogenic enzymes, radioactive isotopes, chromophores, luminescent compounds, fluorescent compounds, magnetic resonance imaging compounds, superparamagnetic particles, and ultra-small superparamagnetic particles.
11. A polynucleotide encoding the recombinant peptide of claim 1.
12. A method of inhibiting autophagy in a cell comprising the step of contacting the cell with the recombinant peptide of claim 1 thereby inhibiting autophagy in the cell.
13. The method of claim 12, wherein the peptide comprises the amino acid sequence of SEQ ID NO:2.
14. The method of claim 12, wherein the peptide comprises the amino acid sequence of SEQ ID NO:14.
15. The method of claim 12, wherein the peptide comprises the amino acid sequence of SEQ ID NO:15.
16. The method of claim 12, wherein the peptide comprises the amino acid sequence of SEQ ID NO:16.
17. The method of claim 12, wherein the cell is within a person's body.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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(45) expressing mCherry::AnkB WT (SEQ ID NO: 3) (left panel) showing a large number of SQST-1::GFP aggregates accumulate in hypodermal cells in embryos (right panel).
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DETAILED DESCRIPTION
(82) 270/480 kD ankyrin-G (AnkG), a neuronal specific isoform of AnkG enriched in axon initial segment and nodes of Ranvier, binds to GABARAP with a K.sub.d ˜10-20 nM. The region of AnkG identified for binding to GABARAP contains a LIR motif. The superior affinity of the binding between AnkG and GABARAP prompted an investigation as to whether it might be possible to develop super strong Atg8 binding peptides as autophagy inhibitors based on the LIR motif found in AnkG. This research identified recombinant peptides containing a 23-27 amino acid sequence with surprisingly high affinity to GABARAP and LC3 proteins. By modifying the structure of the recombinant peptides, recombinant peptides with improved selectivity against GABARAP or LC3 can be prepared.
Definitions
(83) The definitions of terms used herein are meant to incorporate the present state-of-the-art definitions recognized for each term in the field of biotechnology. Where appropriate, exemplification is provided. The definitions apply to the terms as they are used throughout this specification, unless otherwise limited in specific instances, either individually or as part of a larger group.
(84) The term “attach” or “attached” as used herein, refers to connecting or uniting by a bond or non-bonding interaction in order to keep two or more compounds together, which encompasses either direct or indirect attachment such that for example where a first polypeptide is directly bound to a second polypeptide or other molecule, and the embodiments wherein one or more intermediate compounds (e.g., a linker), such as a polypeptide, is disposed between the first polypeptide and the second polypeptide or other molecule.
(85) The term “protein”, “polypeptide”, or “peptide” as used herein indicates an organic polymer composed of two or more amino acid monomers and/or analogs thereof. The term “polypeptide” or “peptide” includes amino acid polymers of any length including full length proteins and peptides, as well as analogs and fragments thereof. A polypeptide of three or more amino acids is also called an oligopeptide. As used herein, the term “amino acid”, “amino acidic monomer”, or “amino acid residue” refers to any of the twenty naturally occurring amino acids including synthetic amino acids with unnatural side chains and including both D and L optical isomers. The term “amino acid analog” refers to an amino acid in which one or more individual atoms have been replaced, either with a different atom, isotope, or with a different functional group but is otherwise identical to its natural amino acid analog.
(86) The term “amino acid” refers to naturally occurring and non-natural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrrolysine and selenocysteine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (such as, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
(87) As used herein, the term “unnatural amino acid” or the like refers to any amino acid, modified amino acid, and/or amino acid analogue that is not one of the 20 common naturally occurring amino acids, selenocysteine or pyrrolysine.
(88) Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
(89) As used herein, the term “variant” refers to a polynucleotide or nucleic acid differing from a reference nucleic acid or polypeptide, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the reference nucleic acid or polypeptide.
(90) A variant can, for example, comprise the amino acid sequence of the parent polypeptide sequence with at least one conservative amino acid substitution. Alternatively or additionally, the variant can comprise the amino acid sequence of the parent polypeptide sequence with at least one non-conservative amino acid substitution. In this case, it is preferable for the non-conservative amino acid substitution to not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution may enhance the biological activity of the variant, such that the biological activity of the variant is increased as compared to the parent polypeptide.
(91) Amino acid substitutions of the described polypeptides can be conservative amino acid substitutions. Conservative amino acid substitutions are known in the art, and include amino acid substitutions in which one amino acid having certain physical and/or chemical properties is exchanged for another amino acid that has the same or similar chemical or physical properties. For instance, the conservative amino acid substitution can be an acidic/negatively charged polar amino acid substituted for another acidic/negatively charged polar amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), a basic/positively charged polar amino acid substituted for another basic/positively charged polar amino acid (e.g. Lys, His, Arg, etc.), an uncharged amino acid with a polar side chain substituted for another uncharged amino acid with a polar side chain (e.g., Asn, Gln, Ser, Thr, Tyr, etc.), an amino acid with a beta-branched side-chain substituted for another amino acid with a beta-branched side-chain (e.g., Ile, Thr, and Val), an amino acid with an aromatic side-chain substituted for another amino acid with an aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.
(92) The terms “percentage homology” and “percentage sequence identity”, when used in reference to a polypeptide or polynucleotide sequence, are used interchangeably herein to refer to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Homology is evaluated using any of the variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW [Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85(8):2444-2448; Altschul et al., 1990, J. Mol. Biol. 215(3):403-410; Thompson et al., 1994, Nucleic Acids Res. 22(2):4673-4680; Higgins et al. 1996, Methods Enzymol. 266:383-402; Altschul et al., 1990, J. Mol. Biol. 215(3):403-410; Altschul et al., 1993, Nature Genetics 3:266-272]. In certain embodiments, protein and nucleic acid sequence homologies are evaluated using the Basic Local Alignment Search Tool (“BLAST”) which is well known in the art (see, e.g., Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2267-2268; Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al., 1993, Nature Genetics 3:266-272; Altschul et al., 1997, Nuc. Acids Res. 25:3389-3402).
(93) As used herein, the terms “treat”, “treating”, “treatment”, and the like refer to reducing or ameliorating a disorder/disease and/or symptoms associated therewith. It will be appreciated, although not precluded, treating a disorder or condition does not require that the disorder, condition, or symptoms associated therewith be completely eliminated. In certain embodiments, treatment includes prevention of a disorder or condition, and/or symptoms associated therewith. The term “prevention” or “prevent” as used herein refers to any action that inhibits or at least delays the development of a disorder, condition, or symptoms associated therewith. Prevention can include primary, secondary and tertiary prevention levels, wherein: a) primary prevention avoids the development of a disease; b) secondary prevention activities are aimed at early disease treatment, thereby increasing opportunities for interventions to prevent progression of the disease and emergence of symptoms; and c) tertiary prevention reduces the negative impact of an already established disease by restoring function and reducing disease-related complications.
(94) As used herein, the term “subject” refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, canines, felines, bovines, and rodents.
(95) The phrase “consisting essentially of is herein meant to define the scope of the recombinant peptides to the specified material amino acids, and to only include additional amino acids or changes that do not materially affect the claimed invention's basic and novel characteristics, namely, e.g., the binding affinity of the recombinant peptides to Atg8 protein family members.
(96) As used herein, the term “recombinant protein”, “recombinant peptide”, or the like refers to any protein of interest that can be manufactured using recombinant DNA technology.
(97) Recombinant host cells may be any of the host cells used for recombinant protein production, including, but not limited to, bacteria, yeast, insect and mammalian cell lines. Recombinant host cells may be made by transforming host cells with expression vectors containing DNA coding for the recombinant peptide of interest.
(98) The recombinant peptides comprising the Formula I and other sequences provided herein specifically exclude naturally occurring proteins that include the sequences described herein (e.g., naturally occurring full length AnkB, AnkG, and FAM134B). In certain embodiments, the recombinant peptides described herein exclude peptides sequences that comprise the recombinant peptide of Formula I and other sequences provided herein and are equal to or longer than 35, 30, 35, 40, 45, or 50 amino acids of any naturally occurring AnkB, AnkG, and FAM134B protein. In certain embodiments, the recombinant peptides described herein exclude peptides sequences that comprise the recombinant peptide of Formula I having more than 5, 10, 15, or 20 flanking amino acids at the n-terminus and/or the c-terminus of the recombinant peptide of Formula I that are naturally flanking amino acids at the equivalent extended LIR peptide sequence in naturally occurring AnkB, AnkG, and FAM134B proteins.
(99) The AnkG LIR motif is located at the N-terminal part of the giant insertion shared by the two giant AnkG isoforms (270/480 kD AnkG) (
(100) In order to elucidate the molecular basis governing the super strong and selective binding between AnkG LIR and GABARAPs, we determined the crystal structures of AnkG LIR in complex with GABARAPL1 and with LC3B, respectively. The crystals of the AnkG LIR/GABARAPL1 complex were diffracted to 1.85 Å resolution and the structure was solved by the molecular replacement methods. In the complex, 23 (Pro1985-His2007) out of the total 26 residues of AnkG LIR can be clearly traced in the electron density map. The GABARAPL1-bound AnkG LIR is composed of two parts: the N-terminal extended structure formed by the LIR core containing the signature “ΦXXΨ”-motif and a C-terminal extension containing a three-turn α-helix (termed as “C-helix”, covering Glu1996 to Ala2003), which is missing in most of the reported LIRs (
(101) The structure of AnkG LIR/LC3B complex was determined at the 2.6 Å resolution. The overall structure and the binding mode of the AnkG LIR/LC3B complex are generally similar to that of AnkG LIR/GABARAPL1 (RMSD of 0.86 Å;
(102) The super strong binding of AnkG LIR to GABARAP prompted us to search for possible existence of other naturally occurring, strong Atg8 binding proteins. A BLAST search using AnkG LIR as the template against the human proteome returned a similar extended LIR sequence from the 440 kD giant AnkB as the top candidate, and we refer it as AnkB LIR (SEQ ID NO:3)(
(103) To understand the mechanisms governing the strong interactions between AnkB LIR (SEQ ID NO:3) and Atg8s, we solved the crystal structures of the AnkB LIR/GABARAP and AnkB LIR/LC3B complexes at 2.75 Å and 2.2 Å resolutions, respectively (
(104) There are several subtle but significant differences when comparing the bindings of AnkB LIR (SEQ ID NO:3) and AnkG LIR (SEQ ID NO:1) to the Atg8 members, which can explain why AnkB LIR binds to Atg8s uniformly stronger than AnkG LIR does. The two middle residues in the “ΦXXΨ” LIR-motif of AnkB LIR are also hydrophobic (Val1593 and Ile1594, corresponding to Thr1990 and Glu1991 in AnkG LIR;
(105) A defining feature for the strong Atg8 binding sequences from AnkB and AnkG LIR revealed from the above structural studies is the presence of a ˜10-residue amphipathic α-helix (the C-helix) immediately following the canonical LIR-motif (
(106) We noted that a recently identified LC3 binding sequence from FYCO1 (SEQ ID NO: 20) has a sequence feature partially fitting the super strong Atg8 binding motif by having a one-turn helix extension following the canonical LIR-motif and with a Glu followed by a Leu at the beginning of the helix (Cheng et al., 2016; Olsvik et al., 2015) (
(107) The consensus sequence motif for the extended LIRs with super strong Atg8 binding affinities: “(D/E).sub.2-3X.sub.0-2ΦXXΨXXXEΨρρΨρρρΨ” can also be represented by a recombinant peptide comprising a sequence represented by Formula I:
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X.sub.10-X.sub.11-X.sub.12-X.sub.13-R.sub.1 I
or a pharmaceutically acceptable salt or zwitterion thereof, wherein X.sub.1, X.sub.2, and X.sub.3, are independently aspartate, glutamate, or absent; X.sub.4 and X.sub.5 are independently any amino acid or absent; X.sub.6 is an amino acid having a side chain comprising an aromatic or heteroaromatic moiety; X.sub.7 and X.sub.8 are independently any amino acid; X.sub.9 is an amino acid having a side chain comprising an acyclic aliphatic or an aromatic moiety; X.sub.10, X.sub.11, and X.sub.12 are independently any amino acid; X.sub.13 is glutamate or arginine; and R.sub.1 is an amphipathic alpha helix comprising between 7 and 15 amino acids, wherein at least two of X.sub.1, X.sub.2, and X.sub.3, are selected from aspartate and glutamate.
(108) The recombinant peptides described herein can generally have high binding affinity to both GABARAP and LC3 protein families and as such can be used as inhibitors of the function of both protein families. However, the selectivity of the recombinant peptides described herein between GABARAP and LC3 can be modified by the appropriate structural modification of the recombinant peptides described herein to afford recombinant peptides that are capable of binding to more selectively to one of GABARAP or LC3 over the other. Such selective recombinant peptides can be useful as biochemical probes for studying the function GABARAP or LC3. For example, as demonstrated in
(109) In certain embodiments, when X.sub.13 is arginine, the recombinant peptide must comprise a peptide having at least an 84% sequence homology with SEQ ID NO: 12 or SEQ ID NO: 14. In certain embodiments, X.sub.13 is arginine and the recombinant peptide comprises a peptide having at least 88%, at least 92%, at least 96% or 100% sequence homology with SEQ ID NO: 12. In certain embodiments, X.sub.13 is arginine and the recombinant peptide comprises a peptide having at least 88%, at least 92%, at least 96% or 100% sequence homology with SEQ ID NO: 14.
(110) In certain embodiments, X.sub.13 is glutamate.
(111) Amino acids having a side chain comprising an aliphatic moiety include glycine, alanine, leucine, isoleucine, valine, and proline.
(112) Amino acids having a side chain comprising an acyclic aliphatic moiety include glycine, alanine, leucine, isoleucine, and valine.
(113) Amino acids having a side chain comprising an aromatic moiety include phenylalanine and tyrosine.
(114) Amino acids having a side chain comprising a heteroaromatic moiety include tryptophan and histidine.
(115) Amino acids having a side chain comprising a polar moiety can include both uncharged amino acids, such as serine, threonine, cysteine, asparagine, glutamine, and tyrosine; and charged amino acids, such as arginine, glutamate, aspartate, lysine, and histidine.
(116) Amino acids having a side chain comprising a carboxylic moiety can include aspartate and glutamate.
(117) Amino acids having a side chain comprising an amide moiety can include asparagine and glutamine.
(118) In certain embodiments, X.sub.1, X.sub.2, and X.sub.3 are independently selected from aspartate and glutamate and X.sub.4 and X.sub.5 are absent. In certain embodiments, X.sub.1, X.sub.2, and X.sub.3 are independently selected from aspartate and glutamate; X.sub.4 and X.sub.5 are absent; and X.sub.13 is glutamate.
(119) In certain embodiments, X.sub.1, X.sub.2, and X.sub.3 are glutamate, aspartate, and aspartate, respectively and X.sub.4 and X.sub.5 are absent. In certain embodiments, X.sub.1, X.sub.2, and X.sub.3 are glutamate, aspartate, and aspartate, respectively; X.sub.4 and X.sub.5 are absent; and X.sub.13 is glutamate.
(120) In certain embodiments, X.sub.1, X.sub.2, and X.sub.3 are each glutamate and X.sub.4 and X.sub.5 are absent. In certain embodiments, X.sub.1, X.sub.2, and X.sub.3 are each glutamate; X.sub.4 and X.sub.5 are absent; and X.sub.13 is glutamate.
(121) In certain embodiments, X.sub.1 is absent; X.sub.2, and X.sub.3 are from aspartate; and X.sub.4 and X.sub.5 are absent. In certain embodiments, X.sub.1 is absent; X.sub.2, and X.sub.3 are from aspartate; X.sub.4 and X.sub.5 are absent; and X.sub.13 is glutamate.
(122) In certain embodiments, X.sub.1 is absent; X.sub.2, and X.sub.3 are aspartate; and X.sub.4 and X.sub.5 alanine and valine, respectively. In certain embodiments, X.sub.1 is absent; X.sub.2, and X.sub.3 are aspartate; X.sub.4 and X.sub.5 alanine and valine, respectively; and X.sub.13 is glutamate.
(123) In certain embodiments, X.sub.6 is tryptophan or phenyl alanine. In certain embodiments, X.sub.6 is tryptophan or phenyl alanine; and X.sub.13 is glutamate.
(124) In certain embodiments, X.sub.7 and X.sub.8 are independently threonine, valine, isoleucine, leucine, arginine, or glutamate. In certain embodiments, X.sub.7 and X.sub.8 are independently threonine, valine, isoleucine, leucine, arginine, or glutamate; and X.sub.13 is glutamate.
(125) In certain embodiments, X.sub.9 is phenylalanine, valine, leucine, or isoleucine. In certain embodiments, X.sub.9 is phenylalanine, valine, leucine, or isoleucine; and X.sub.13 is glutamate.
(126) In certain embodiments, X.sub.10, X.sub.11, and X.sub.12 are independently glutamate, aspartate, glutamine, serine, or threonine. In certain embodiments, X.sub.10, X.sub.11, and X.sub.12 are independently glutamate, aspartate, glutamine, serine, or threonine; and X.sub.13 is glutamate
(127) In certain embodiments, X.sub.1, X.sub.2, and X.sub.3 are each glutamate and X.sub.4 and X.sub.5 are absent; X.sub.4 and X.sub.5 alanine and valine, respectively; X.sub.6 is tryptophan; X.sub.7 and X.sub.8 are independently valine or isoleucine; X.sub.9 is valine; and X.sub.10, X.sub.11, and X.sub.12 are independently glutamate, aspartate, or serine. In certain embodiments, X.sub.1, X.sub.2, and X.sub.3 are each glutamate and X.sub.4 and X.sub.5 are absent; X.sub.4 and X.sub.5 alanine and valine, respectively; X.sub.6 is tryptophan; X.sub.7 and X.sub.8 are independently valine or isoleucine; X.sub.9 is valine; X.sub.10, X.sub.11, and X.sub.12 are independently glutamate, aspartate, or serine; and X.sub.13 is glutamate.
(128) In certain embodiments, X.sub.1, X.sub.2, and X.sub.3 are each glutamate and X.sub.4 and X.sub.5 are absent; or X.sub.1, X.sub.2, and X.sub.3 are aspartate, glutamate, and glutamate, respectively and X.sub.4 and X.sub.5 are absent; X.sub.6 is tryptophan; X.sub.7 and X.sub.8 are independently valine, isoleucine, or leucine; X.sub.9 is valine or leucine; and X.sub.10, X.sub.11, and X.sub.12 are independently glutamate, aspartate, glutamine, threonine, or serine. In certain embodiments, X.sub.1, X.sub.2, and X.sub.3 are each glutamate and X.sub.4 and X.sub.5 are absent; or X.sub.1, X.sub.2, and X.sub.3 are aspartate, glutamate, and glutamate, respectively and X.sub.4 and X.sub.5 are absent; X.sub.6 is tryptophan; X.sub.7 and X.sub.8 are independently valine, isoleucine, or leucine; X.sub.9 is valine or leucine; X.sub.10, X.sub.11, and X.sub.12 are independently glutamate, aspartate, glutamine, threonine, or serine; and X.sub.13 is glutamate.
(129) In certain embodiments, X.sub.1, X.sub.2, and X.sub.3 are glutamate, aspartate, and aspartate, respectively and X.sub.4 and X.sub.5 are absent; or X.sub.1, X.sub.2, and X.sub.3 are glutamate, aspartate, and glutamate, respectively and X.sub.4 and X.sub.5 are absent; X.sub.6 is tryptophan; X.sub.7 and X.sub.8 are independently valine, isoleucine, threonine, or glutamate; X.sub.9 is phenylalanine; and X.sub.10, X.sub.11, and X.sub.12 are independently lysine, glutamate, asparagine, aspartate, threonine, or serine. In certain embodiments, X.sub.1, X.sub.2, and X.sub.3 are glutamate, aspartate, and aspartate, respectively and X.sub.4 and X.sub.5 are absent; or X.sub.1, X.sub.2, and X.sub.3 are glutamate, aspartate, and glutamate, respectively and X.sub.4 and X.sub.5 are absent; X.sub.6 is tryptophan; X.sub.7 and X.sub.8 are independently valine, isoleucine, threonine, or glutamate; X.sub.9 is phenylalanine; X.sub.10, X.sub.11, and X.sub.12 are independently lysine, glutamate, asparagine, aspartate, threonine, or serine; and X.sub.13 is glutamate.
(130) In certain embodiments, X.sub.1, X.sub.2, and X.sub.3 are glutamate, aspartate, and aspartate, respectively and X.sub.4 and X.sub.5 are absent; X.sub.6 is tryptophan; X.sub.7 and X.sub.8 are independently isoleucine, threonine, or glutamate; X.sub.9 is phenylalanine; and X.sub.10, X.sub.11, and X.sub.12 are independently glutamate or serine. In certain embodiments, X.sub.1, X.sub.2, and X.sub.3 are glutamate, aspartate, and aspartate, respectively and X.sub.4 and X.sub.5 are absent; X.sub.6 is tryptophan; X.sub.7 and X.sub.8 are independently isoleucine, threonine, or glutamate; X.sub.9 is phenylalanine; X.sub.10, X.sub.11, and X.sub.12 are independently glutamate or serine; and X.sub.13 is glutamate.
(131) In certain embodiments, X.sub.1, X.sub.2, and X.sub.3 are glutamate, aspartate, and aspartate, respectively and X.sub.4 and X.sub.5 are absent; X.sub.6 is tryptophan; X.sub.7 and X.sub.8 are threonine and arginine respectively; X.sub.9 is phenylalanine; and X.sub.10, X.sub.11, and X.sub.12 are independently glutamate or serine. In certain embodiments, X.sub.1, X.sub.2, and X.sub.3 are glutamate, aspartate, and aspartate, respectively and X.sub.4 and X.sub.5 are absent; X.sub.6 is tryptophan; X.sub.7 and X.sub.8 are threonine and arginine respectively; X.sub.9 is phenylalanine; X.sub.10, X.sub.11, and X.sub.12 are independently glutamate or serine; and X.sub.13 is glutamate
(132) Alpha helices are well known in the art, as are amino acids that have a propensity to form alpha helices. For example, the amino acids methionine, alanine, leucine, glutamate and/or lysine are known to have a high propensity to form alpha helix structures when included in peptides. Other amino acids that are known to have a propensity to form an alpha helix include phenyl alanine, glycine, tryptophan, isoleucine, and valine.
(133) R.sub.1 can be an amphipathic alpha helix. In an amphipathic alpha helix, one face of the helix comprises mainly hydrophilic amino acids and the other face of the helix comprises mainly hydrophobic amino acids. The amino acid sequence of amphipathic alpha helix can generally alternate between hydrophilic and hydrophobic residues every about 3 to 4 residues, since the alpha helix makes a turn every 3.6 amino acids.
(134) In certain embodiments, R.sub.1 is a 3, 4, 5, 6, 7, 8, 9, or 10 turn amphipathic alpha helix. In certain embodiments, the amphipathic alpha helix comprises between 7 and 36; 7 and 34; 7 and 32; 7 and 30; 7 and 28; 7 and 26; 7 and 24; 7 and 22; 7 and 20; 7 and 18; 7 and 15; 7 and 13; 7 and 11; or 7 and amino acids. In certain embodiments, the amphipathic alpha helix comprises 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.
(135) In certain embodiments, R.sub.1 comprises an amphipathic alpha helix comprising a sequence represented by Formula II:
X.sub.14-X.sub.15-X.sub.16-X.sub.17-X.sub.18-X.sub.19-X.sub.20-X.sub.21 II
(136) wherein X.sub.14 and X.sub.17 are independently an amino acid having a side chain comprising an acyclic aliphatic moiety; X.sub.16 is an amino acid having a side chain comprising a polar moiety or is absent; X.sub.15 and X.sub.18-X.sub.20 are independently an amino acid having a side chain comprising a polar moiety; and X.sub.21 is an amino acid having a side chain comprising an acyclic aliphatic moiety or is absent.
(137) In certain embodiments, X.sub.14 and X.sub.17 are independently alanine, leucine, isoleucine, or valine. In certain embodiments, X.sub.14 is isoleucine or leucine. In certain embodiments, X.sub.17 is alanine or isoleucine.
(138) In certain embodiments, X.sub.15 and X.sub.18-X.sub.20 are independently aspartate, arginine, glutamate, glutamine, lysine, serine, or cysteine.
(139) In certain embodiments X.sub.21 is alanine, leucine, or glycine.
(140) In certain embodiments, X.sub.14 and X.sub.17 are independently alanine, leucine, isoleucine, or valine; X.sub.15 and X.sub.18-X.sub.20 are independently aspartate, arginine, glutamate, glutamine, lysine, serine, or cysteine; and X.sub.21 is alanine, leucine, or glycine.
(141) In certain embodiments, R.sub.1 comprises a peptide having at least 75%, at least 87%, or 100% sequence homology with SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19.
(142) In certain embodiments the recombinant peptide comprises an extended LIR motif found in AnkB or AnkG proteins from any organism including, but not limited to non-human primate, human (e.g., AnkB: SEQ ID NO:3 and AnkG: SEQ ID NO:4), mouse (e.g., AnkB: SEQ ID NO:8), rat (e.g., AnkG: SEQ ID NO:1), canine, chicken (e.g., AnkB: SEQ ID NO: 9 and AnkG: SEQ NO:5), rabbit, rat (e.g., AnkG: SEQ ID NO:1), zebra fish (e.g., AnkB SEQ ID NO:10 and AnkG: SEQ ID NO:7), frog (e.g., AnkG SEQ ID NO:6), etc.
(143) In other embodiments, the recombinant peptide is any non-naturally occurring protein comprising a polypeptide sequence as described herein. Such peptides can be derived from proteins unrelated to AnkB and AnkG. For example, FAM134B (SEQ ID NO:21) exhibits strong binding affinities to ATG8 family members.
(144) In certain embodiments, the recombinant peptide comprising a sequence represented by Formula I comprises a peptide having at least 88%, at least 92%, at least 97% or 100% sequence homology with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 20, or SEQ ID NO: 21.
(145) In certain embodiments, the recombinant peptide comprising a sequence represented by Formula I comprises a peptide having at least 88%, at least 92%, at least 97% or 100% sequence homology with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 20, or SEQ ID NO: 21.
(146) In certain embodiments, the recombinant peptide comprising a sequence represented by Formula I comprises a peptide having at least 88%, at least 92%, at least 97% or 100% sequence homology with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.
(147) In certain embodiments, the recombinant peptide of Formula I consists essentially of any of the peptide sequences described herein.
(148) The recombinant peptides described herein exhibit strong binding to GABARAP, GABARAPL1, GABARAPL2, LC3A, LC3B, and LC3C. The recombinant peptides described herein can bind to GABARAP and LC3 family proteins with K.sub.d in the nanomolar and sub-nanomolar range. In certain embodiments, the recombinant peptides described herein can bind to GABARAP family members with a K.sub.d between 0.1 nM to 200 nM; 0.1 nM to 150 nM; 0.1 nM to 100 nM; 0.1 nM to 80 nM; 0.1 nM to 60 nM; 0.1 nM to 40 nM; 0.1 nM to 30 nM; 0.1 nM to 25 nM; 0.1 nM to 20 nM; 0.1 nM to 15 nM; 0.1 nM to 10 nM; or 0.1 nM to 1 nM. In certain embodiments, the recombinant peptides described herein can bind to LC3 family members with a K.sub.d between 1 nM to 100 nM; 1 nM to 75 nM; 1 nM to 50 nM; 1 nM to 25 nM; 1 nM to 20 nM; or 1 nM to 15 nM. Optionally, these values may be determined by the assays described herein and depicted in the Examples below.
(149) In certain embodiments, the recombinant peptides described herein exhibit a K.sub.d for one or more GABARAP proteins up to 2, 3, 5, 10, 100, 500, or 1,000 times stronger than at least one LC3 protein.
(150) The inhibitory effect of the recombinant peptides described herein may occur in a cell-free system, in cell or tissue culture and/or in a cell and/or tissue in a patient. Thus, in certain embodiments, provided is a method of inhibiting autophagy in a cell comprising the step of contacting the cell with a recombinant peptide described herein thereby inhibiting or at least partially inhibiting autophagy in the cell.
(151) The recombinant peptides described herein can be for imaging autophagic processes by the appropriate modification of the recombinant peptide with the appropriate detectable tag. The recombinant peptides can also be modified with affinity tags, which enable the recombinant peptides to be used for pull down experiments to isolate binding targets (e.g., Atg8 proteins) involved in autophagic processes. Thus, in certain embodiments, the recombinant peptides described herein further comprise an affinity tag and/or detectable label. The affinity tag or detectable label can be covalently attached directly to the n-terminal or c-terminal of the recombinant peptide via a peptide bond or covalently attached via a linker. In alternative embodiments, affinity tag or detectable label can be covalently attached to an appropriately functionalized side chain, such as a lysine, cysteine, glutamate, aspartate, tyrosine, serine, or threonine side chain. In certain embodiments, the detectable label is covalently attached to the n-terminal of the recombinant peptide indirectly via a (Gly-Ser).sub.n (SEQ ID NO: 27), (Gly-Gly-Gly-Ser).sub.n (SEQ ID NO: 22), (Gly-Gly-Ser-Gly).sub.n (SEQ ID NO: 23), (Gly-Gly-Gly-Gly-Ser).sub.n (SEQ ID NO: 24), (Gly-Gly-Ser)n (SEQ ID NO: 28), (Gly-Ser).sub.n (SEQ ID NO: 27) or Gly.sub.n linker (SEQ ID NO: 29), wherein n is 1-10. In certain embodiments, the detectable label is attached indirectly to the n-terminal of the recombinant peptide via a (Gly-Ser).sub.n (SEQ ID NO: 30), (Gly-Gly-Gly-Ser).sub.n (SEQ ID NO: 31), (Gly-Gly-Ser-Gly).sub.n (SEQ ID NO: 32), (Gly-Gly-Gly-Gly-Ser).sub.n (SEQ ID NO: 33), (Gly-Gly-Ser)n (SEQ ID NO: 34), (Gly-Ser).sub.n (SEQ ID NO: 30) or Gly.sub.n linker (SEQ ID NO: 35), wherein n is 1-4. In certain embodiments, the detectable label is attached indirectly to the n-terminal of the recombinant peptide via a SGLRSGS (SEQ ID NO: 25) or YSDLDGS (SEQ ID NO: 26). In certain embodiments, the recombinant peptide further comprises a mCherry detectable label covalently attached at the n-terminal via a SGLRSGS (SEQ ID NO: 25) linker. In certain embodiments, the recombinant peptide further comprises a green fluorescent protein detectable label covalently attached at the n-terminal via an YSDLDGS (SEQ ID NO: 26) linker.
(152) The term “affinity tag” as used herein denotes a polypeptide segment that can be attached to a one or more polypeptides to provide for, e.g., purification or detection of the one or more polypeptides. In principal, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag. Affinity tags include, but are not limited to: a poly-histidine, protein, glutathione S transferase, Glu-Glu affinity tag, substance P, streptavidin binding peptide, or other antigenic epitope, such as a hemagglutinin (HA) polypeptide. See, in general, Ford et al., Protein Expression and Purification 2: 95-107, 1991.
(153) Detectable labels can include chromogenic enzymes, radioactive isotopes, chromophores, luminescent compounds, fluorescent compounds, magnetic resonance imaging compounds, superparamagnetic particles, and ultra-small superparamagnetic particles. In certain embodiments, the detectable label is fluorescent protein, such as green fluorescent protein or mCherry.
(154) Suitable detectable labels include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Suitable detectable labels include, but are not limited to, magnetic beads (e.g. Dynabeads™), fluorescent dyes (e.g., fluorescein, Texas Red, rhodamine, a green fluorescent protein, a red fluorescent protein, a yellow fluorescent protein, and the like), radiolabels (e.g., .sup.3H, .sup.125I, .sup.35S, .sup.14C, or .sup.32P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase, luciferase, and others commonly used in an enzyme-linked immunosorbent assay (ELISA)), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads.
(155) Also provided is a polynucleotide encoding a recombinant peptide as described herein. In certain embodiments, the polynucleotide encodes the recombinant peptide::fluorescent protein conjugate. The polynucleotide can be included in a plasmid expression vector. Expression of the plasmid expression vector encoding the recombinant peptide can be driven in a tissue-specific or development-stage-specific manner or induced by chemical-regulation or physical-regulation using the appropriate promoter. Any promoter known in the art can be used to drive the expression of the plasmid encoding the recombinant peptides described herein. Such promoters include, but are not limited to, nfya-1, col-19, myo-3, vha-6, hyp7, y37A1B.5 CMV, CAG, SV40, and the like.
(156) The present disclosure also provides a pharmaceutical composition comprising any one of the recombinant peptides described herein and at least one pharmaceutically acceptable excipient.
(157) The recombinant peptides described herein and their pharmaceutically acceptable salts are can be administered to a subject either alone or in combination with pharmaceutically acceptable carriers or diluents in a pharmaceutical composition according to standard pharmaceutical practice. The compounds can be administered orally or parenterally, preferably parenterally. Parenteral administration includes intravenous, intramuscular, intraperitoneal, subcutaneous and topical, the preferred method being intravenous administration.
(158) Accordingly, the present disclosure provides pharmaceutically acceptable compositions, which comprise a therapeutically-effective amount of one or more of the recombinant peptides described herein, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. The pharmaceutical compositions of the present disclosure may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; and (2) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue. The preferred method of administration of compounds of the present invention is parental administration (intravenous).
(159) As set out herein, certain embodiments of the recombinant peptides described herein may contain a basic functional group, such as amino, and are, thus, capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable acids. The term “pharmaceutically-acceptable salts” in this respect, refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present disclosure. These salts can be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed during subsequent purification. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like.
(160) The pharmaceutically acceptable salts of the recombinant peptides of the present disclosure include the conventional nontoxic salts or quaternary ammonium salts of the compounds, e.g., from nontoxic organic or inorganic acids. For example, such conventional nontoxic salts include those derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like.
(161) In other cases, the recombinant peptides described herein may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable bases. The term “pharmaceutically-acceptable salts” in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of compounds of the present invention. These salts can likewise be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.
(162) Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives, solubilizing agents, buffers and antioxidants can also be present in the compositions.
(163) Methods of preparing these formulations or recombinant peptides include the step of bringing into association a recombinant peptide described herein with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers (liquid formulation), liquid carriers followed by lyophylization (powder formulation for reconstitution with sterile water or the like), or finely divided solid carriers, or both, and then, if necessary, shaping or packaging the product.
(164) Pharmaceutical compositions of the present disclosure suitable for parenteral administration comprise one or more recombinant peptides described herein in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, chelating agents, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. In the examples, the active ingredients are brought together with the pharmaceutically acceptable carriers in solution and then lyophilized to yield a dry powder. The dry powder is packaged in unit dosage form and then reconstituted for parental administration by adding a sterile solution, such as water or normal saline, to the powder.
(165) Examples of suitable aqueous and non-aqueous carriers which may be employed in the pharmaceutical compositions of the disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
(166) These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms upon the recombinant peptides of the present disclosure may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
(167) The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
(168) The phrases “systemic administration,” “administered systemically,” “peripheral administration” and “administered peripherally” as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
(169) The recombinant peptides described herein are potent inhibitors of Atg8 and thus can be used to treat a disease or condition in which inhibition of autophagy has a therapeutic effect. Thus, in certain embodiments, provided herein is a method of inhibiting autophagy in a subject for whom inhibition of autophagy is beneficial, comprising administering to the subject a therapeutically effective amount of the recombinant peptide described herein thereby inhibiting autophagy activity in the subject. Such disease diseases or conditions in which inhibition of autophagy is beneficial include, but are not limited to cancer (e.g. breast, ovarian and prostate cancers), metabolic diseases (e.g. atherosclerosis, and diabetes), immunity disorders (e.g. celiac disease, multiple sclerosis) and neurodegenerative diseases (e.g. alzheimer's Diseases, Parkinson diseases).
(170) Inhibition of autophagy has been proposed to be a new anticancer therapy by promoting radiosensitization and chemosensitization. Thus, the recombinant peptides described herein can be used to sensitize tumors to cancer drugs and/or radiation therapy. Accordingly, the recombinant peptides described herein can be co-administered with one or more cancer drugs to treat subjects suffering from cancer.
(171) A recombinant peptide described and a cancer drug may be administered concurrently (e.g., simultaneously, essentially simultaneously or within the same treatment protocol) or sequentially, depending upon the nature of the cancer, the condition of the patient, and the actual choice of cancer drug to be administered in conjunction (i.e., within a single treatment protocol) with a recombinant peptide described herein.
(172) If a recombinant peptide described herein and the cancer drug are not administered simultaneously or essentially simultaneously, then the optimum order of administration of the recombinant peptide described herein and the cancer drug, may be different for different types of cancer. Thus, in certain situations the recombinant peptide described herein may be administered first followed by the administration of the cancer drug; and in other situations the cancer drug may be administered first followed by the administration of a recombinant peptide described herein. This alternate administration may be repeated during a single treatment protocol. The determination of the order of administration, and the number of repetitions of administration of each therapeutic agent during a treatment protocol, is well within the knowledge of the skilled physician after evaluation of the disease being treated and the condition of the patient.
EXAMPLES
(173) Ank-Derived Peptides can Potently Inhibit Autophagy in Heterologous Cells
(174) We used the well-established cultured COS7 cells for assessing autophagy inhibitions by the AnkB/G LIR peptides over-expressed in the cells. We quantified the numbers of endogenous LC3- or GABARAP-positive puncta, which represent LC3 or GABARAP-containing autophagic structures, as readouts to quantify autophagy inhibitions (Klionsky et al., 2016; Mizushima et al., 2010). After nutrients deprivation, obvious accumulations of LC3- or GABARAP-positive puncta could be observed in cells over-expressing mCherry only, indicating induction of autophagy by starvation (FIG. 6C6&E6). Over-expression of the mCherry-AnkB WT reduced the LC3- and GABARAP-puncta to the background level (i.e. the level of LC3- and GABARAP-puncta when cells were not starved, data not shown). In contrast, expression of the mCherry-AnkB WR peptide had no impact on the LC3- or GABARAP-puncta numbers (i.e. with the same puncta numbers as expressing the mCherry vector control;
(175) Entirely consistent with our biochemical data, the AnkG WT peptide potently inhibited GABARAP puncta formation, but only modestly reduced LC3-positive puncta when over-expressed in COS7 cells. Satisfyingly, the AnkG ER peptide had essentially no impact on the LC3 puncta number reduction, but potently inhibited the GABARAP puncta formation (
(176) We further quantified the level of p62, one of the best studied selective autophagy substrates, to monitor the autophagic flux in COS7 cells expressing various AnkB/G LIR peptides under the starvation condition (Klionsky et al., 2016; Mizushima et al., 2010). Upon autophagy induction, p62 forms aggregates via its LIR motif-mediated interactions to Atg8s and subsequent incorporation into autophagosomes (Bjorkoy et al., 2005; Pankiv et al., 2007). Only a relatively low and steady level of p62 aggregates is formed if autophagy flux is normal (i.e. p62-positive autophagosomes are steadily cleared by fusing with lysosomes; (Bjorkoy et al., 2005)), and this is indeed the case when cells were expressed with the mCherry vector control (
(177) Expression of the Ank-Derived Peptides Impairs Autophagy in C. elegans
(178) C. elegans provides an ideal platform to study autophagy and especially to investigate roles of different members of Atg8 in autophagy, because worms contain both GABARAP and LC3 subfamily Atg8s and yet simple enough with one gene for each subfamily (worm lgg-1 and lgg-2 are mammalian orthologues of GABARAP and LC3, respectively). It has also been shown earlier on that lgg-1 and lgg-2 act non-redundantly in autophagy (Wu et al., 2015). Prior to performing autophagy assays in worms, we measured the bindings of various AnkB/G LIR peptides to purified LGG-1 and LGG-2 using the same method as we have described for
(179) We next determined whether the AnkB WT peptide blocks autophagy in C. elegans. Autophagy is required for degradation of a variety of protein substrates, including the C. elegans p62 homolog SQST-1 during development (Tian et al., 2010). In the wild type animals, SQST-1::GFP is weakly expressed and diffusely localized in the cytoplasm, while numerous SQST-1 aggregates accumulate in autophagy mutants (Tian et al., 2010). The AnkB WT peptide fused with the Cherry reporter (Cherry::AnkB WT) or Cherry::AnkB WR was expressed under the control of the nfya-1 promoter, which is expressed ubiquitously from the embryonic to adult stages. The expression plasmids were injected into animals carrying integrated SQST-1::GFP reporter (bpIs151) and transgenic lines were obtained and analyzed. We found that animals expressing Cherry::AnkB WT accumulated a large number of SQST-1::GFP aggregates in multiple tissues from the embryonic to adult stages (
(180) Autophagy plays critical roles in various physiological processes occurring during development or in adult C. elegans (Zhang et al., 2015). Fewer autophagy mutants develop into larvae. Animals expressing Pnfya-1::Cherry::AnkB WT showed a significant reduction in hatching rate. 60.8% embryos expressing the AnkB WT peptide hatched, compared to 96.9% of embryos expressing AnkB WR (
(181) Similarly, we also tested the blocking effect of AnkG peptides in C. elegans. Under control of hyp7 promoter, the epidermis expressing Cherry::AnkG WT or ER peptides rather than Cherry::AnkG WR peptide caused accumulation of SQST-1::GFP aggregates from the embryonic to adult stages (
(182) C. Tissue- and Temporal-Specific Depletion of Autophagy by Expression of the Ank-Derived Peptides
(183) Although a large collection of autophagy mutants were isolated from genetic screens (Tian et al., 2010), assays to inhibit autophagy activity in a spatiotemporal manner are not available. We investigated whether the AnkB peptide can block autophagy in a tissue- and temporal-specific manner. The AnkB peptide was expressed under the control of tissue-specific promoters, including the promoter of myo-3 for body wall muscle cells, y37A1B.5 for hypodermal cells and vha-6 for intestinal cells. The expression constructs were injected into animals carrying the corresponding tissue-specific SQST-1::GFP expression integrated lines. We found that a large number of SQST-1::GFP aggregates accumulated in animals expressing AnkB WT peptide in different tissues from the embryonic to adult stages, while animals expressing WR peptide showed no accumulation (
(184) We also examined whether the AnkB peptide can impair autophagy in a temporal control manner. The expression of AnkB WT was driven by the promoter of col-19, which is expressed in hypodermal cells from the young adult stage onwards. We found that the animals expressing this peptide exhibited adult-specific accumulation of SQST-1::GFP aggregates phenotype, while no aggregates were found from the embryonic to L4 larval stages (
(185) Autophagy is known to regulate the adult worm aging process. We found that worms expressing Pnfya-1::Cherry::AnkB WT dramatically shortened the life span compared to animals expressing Cherry::AnkB WR (
(186) We next tried to explore the potential applications of the Ank-derived peptides as autophagosome markers. The COS7 cells were co-transfected with GFP-RFP-LC3B and Myc tagged AnkB WT or AnkB WR peptides. In such design, the LC3B on the autophagosome will exhibit both green and red fluorescence signal, while the LC3B on the lysosome will only show red fluorescence as GFP is quenched in the acidic environment. Since high amount of AnkB WT peptides expressed in the cell can effectively inhibit autophagy as we demonstrated above, we only focused on the COS7 cells with low expression level of Myc-AnkB. In the cells with AnkB WT expression, a significant number of yellow (green signal merged with red signal) and red only puncta are observed, suggesting that the autophagic flux remains normal in the presence of such low amount of AnkB WT peptides (
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