# US 7,608,682 B2 · Substrates of N-end rule ubiquitylation and methods for measuring the ubiquitylation of these substrates

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- Number: US 7,608,682 B2
- Document ID: US7608682B2
- Status: Granted
- Kind: B2
- Application number: 10/693,999
- Filed: 2003-10-28
- Published: 2004-07-15
- Granted: 2009-10-27
- Earliest claimed: 2002-10-30
- Assignees: Meso Scale Technologies, LLC.
- Inventors: Ilia Davydov; John H. Kenten; Hans Biebuyck; Pankaj Oberoi
- Web page: https://patentgrep.com/patent/US7608682B2

## CPC

- G01N 33/573: for enzymes or isoenzymes
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 14/00: Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K 14/47: from mammals
- C07K 14/4702: {Regulators; Modulating activity}
- C07K 14/4703: {Inhibitors; Suppressors}
- C07K 14/4711: {Alzheimer's disease; Amyloid plaque core protein}
- C07K 14/4716: {Muscle proteins, e.g. myosin, actin}
- C07K 14/4738: {Cell cycle regulated proteins, e.g. cyclin, CDC, INK-CCR (cell cycle dependent kinases C12N9/12)}
- C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)}
- C12Q 1/25: involving enzymes not classifiable in groups C12Q1/26 {- C12Q1/66}
- C12Q 1/37: involving peptidase or proteinase
- G01N 2500/04: Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

The present invention relates to methods, compositions, compounds and kits for detecting, measuring and modulating protein ubiquitylation via the N-end rule pathway and for identifying novel substrates, enzymes and modulators of N-end rule ubiquitylation. The present invention also relates to specific substrates of N-end rule ubiquitylation as well as activated fragments of these substrates, proteases that expose N-degrons in these substrates, ubiquitin ligases that ubiquitylate these substrates and inhibitors of the ubiquitylation of these substrates.

## Claims

### Claim 1

An isolated conjugate comprising
(a) at least
(i) one ubiquitin or
(ii) ubiquitin fused with another molecule, which is employed for purification or visualization; and
(b) a protein selected from the group consisting of aprataxin, synaptogamin-like protein (SLP), high mobility group protein 17 (HMG17), PIN2-interacting protein 1 (PinX1), CBF1 interacting corepressor (CIR), high mobility group nucleosomal binding protein 3 (HMGN3), hematopoietic stem/progenitor cell 144 (HSPC144), Cullin 3, cell division cycle 6 (CDC6), and fragments and variants thereof;
wherein (i) said variants comprise polypeptides of at least 50 amino acids having at least 90% sequence identity to sequences within their corresponding proteins and (ii) said fragments comprise polypeptides of at least 50 amino acids having at least 90% identity to a sequence fragment of said protein; and said conjugate is formed via N-end rule ubiquitylation of a polypeptide comprising a destabilizing N-terminal residue and an internal Lys residue.

### Claim 2 (depends on 1)

The conjugate of claim 1, wherein said conjugate is immobilized on a support and/or linked to a label.

### Claim 3

A method for producing a conjugate comprising
(a) at least one
(i) ubiquitin or
(ii) ubiquitin derivatized with another molecule, which is employed for purification or visualization; and
(b) a protein selected from the group consisting of aprataxin, SLP, HMG17, PinX1, CIR, HMGN3, HSPC144, Cullin 3, CDC6, tau, and fragments and variants thereof; comprising:
A) forming a mixture comprising a vector containing a clone coding for said protein, or said fragment or variant thereof, an in vitro transcription/translation system, an N-rule ubiquitylation system and, optionally, a proteasome inhibitor; and
B) incubating said mixture to allow production of said conjugate;
wherein (i) said variants comprise polypeptides of at least 50 amino acids having at least 90% sequence identity to sequences within their corresponding proteins and (ii) said fragments comprise polypeptides of at least 50 amino acids having at least 90% identity to a sequence fragment of said protein; and said conjugate is formed via N-end rule ubiquitylation of a polypeptide comprising a destabilizing N-terminal residue and an internal Lys residue.

### Claim 4 (depends on 3)

The method of claim 3, further comprising:
C) isolating said conjugate.

### Claim 5 (depends on 4)

The method of claim 4, wherein said isolating is done by binding to an antibody specific to a poly-ubiquitin chain.

### Claim 6 (depends on 4)

The method of claim 4, wherein said isolating is done by binding to an antibody specific for said protein.

### Claim 7

A composition comprising a conjugate comprised of
(a) at least one
(i) ubiquitin or
(ii) ubiquitin derivatized with another molecule, which is employed for purification or visualization; and
(b) a protein selected from the group consisting of aprataxin, SLP, HMG17, PinX1, CIR, HMGN3, HSPC144, Cullin 3, CDC6, and fragments and variants thereof;
wherein (i) said variants comprise polypeptides of at least 50 amino acids having at least 90% sequence identity to sequences within their corresponding proteins and (ii) said fragments comprise polypeptides of at least 50 amino acids having at least 90% identity to a sequence fragment of said protein; said conjugate is formed via N-end rule ubiquitylation of a polypeptide comprising a destabilizing N-terminal residue plus an internal Lys residue, and said conjugate is immobilized on a support and/or linked to a label.

### Claim 8

An isolated conjugate comprising
(a) at least one
(i) ubiquitin or
(ii) ubiquitin derivatized with another molecule, which is employed for purification or visualization; and
(b) a recombinant protein selected from the group consisting of tau, and fragments and variants thereof;
wherein (i) said variants comprise polypeptides of at least 50 amino acids having at least 90% sequence identity to sequences within their corresponding proteins and (ii) said fragments comprise polypeptides of at least 50 amino acids having at least 90% identity to a sequence fragment of said protein; and said conjugate is formed via N-end rule ubiquitylation of a polypeptide comprising a destabilizing N-terminal residue plus an internal Lys residue.

### Claim 9 (depends on 8)

The conjugate of claim 8, wherein said conjugate is immobilized on a support and/or linked to a label.

### Claim 10

A composition comprising a conjugate comprised of
(a) at least one
(i) ubiquitin or
(ii) ubiquitin derivatized with another molecule, which is employed for purification or visualization; and
(b) a protein selected from the group consisting of tau, and fragments and variants thereof;
wherein (i) said variants comprise polypeptides of at least 50 amino acids having at least 90% sequence identity to sequences within tau and (ii) said fragments comprise polypeptides of at least 50 amino acids having at least 90% identity to a sequence fragment of said protein; and said conjugate is formed via N-end rule ubiquitylation of a polypeptide comprising a destabilizing N-terminal residue plus an internal Lys residue, and said conjugate is immobilized on a support and/or linked to a label.

### Claim 11 (depends on 1)

The conjugate of claim 1, wherein said fragment is an activated fragment of a protein, said fragment having an exposed N-degron.

### Claim 12 (depends on 11)

The conjugate of claim 11, wherein said activated fragment is immobilized on a support and/or linked to a label.

### Claim 13 (depends on 8)

The conjugate of claim 8, wherein said fragment is an activated fragment of a protein, said fragment having an exposed N-degron.

### Claim 14 (depends on 13)

The conjugate of claim 13, wherein said activated fragment is immobilized on a support and/or linked to a label.

### Claim 15 (depends on 4)

The method of claim 4, wherein said isolating is done by binding to said molecule.

### Claim 16

An isolated conjugate comprising
(a) at least one
(i) ubiquitin or
(ii) ubiquitin derivatized with another molecule, which is employed for purification or visualization; and
(b) a protein;
said conjugate made by a process comprising:
A) forming a mixture comprising
(i) a vector containing a clone coding for said protein, wherein said protein is selected from the group consisting of aprataxin, SLP, HMG17, PinX1, CIR, HMGN3, HSPC144, Cullin 3, CDC6, tau, and fragments and variants thereof; and (i) said variants comprise polypeptides of at least 50 amino acids having at least 90% sequence identity to sequences within their corresponding proteins and (ii) said fragments comprise polypeptides of at least 50 amino acids having at least 90% identity to a sequence fragment of said protein; and said fragment or variant thereof comprises a destabilizing N-terminal residue and an internal Lys residue;
(ii) an in vitro transcription/translation system;
(iii) an N-rule ubiquitylation system; and
(iv) optionally, a proteasome inhibitor; and
B) incubating said mixture to produce said conjugate.

### Claim 17 (depends on 16)

The conjugate of claim 16, wherein said conjugate is immobilized on a support and/or linked to a label.