DeUBiquitinating enzymes that regulate cell growth

Details DeUBiquitinating enzymes that regulate cell growth DeUBiquitinating enzymes that regulate cell growth

1998-02-05

2001-09-11

Elliott, George C. (Department: 1635)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of regulating cell metabolism or physiology

C435S006120, C435S069100, C435S183000, C435S226000, C435S320100, C435S325000, C435S355000, C435S366000, C536S023100, C536S023200, C536S023500

Reexamination Certificate

active

06287858

ABSTRACT:

BACKGROUND OF THE INVENTION
Recently, a large superfamily of genes encoding deubiquitinating enzymes, called ubps, has been identified. Ubps are ubiquitin-specific thiol proteases. Deubiquitinating enzymes have multiple roles within the cell, including stabilization of some ubiquitin (Ub) conjugated substrates, degradation of other Ub-conjugated substrates and recycling of the cell's free monomeric Ub pool. Some deubiquitinating enzymes remove Ub from cellular target proteins and thereby prevent proteasome mediated degradation (UBP2). Other deubiquitinating enzymes remove Ub from Ub-peptide degradation products produced by the proteasomes and thereby accelerate proteasome mediated degradation (Doa-4). Little is known about the specific cellular functions of the ubp family members. The presence of multiple family members suggests considerable functional diversity. Disruption of ubp genes, in general, has not resulted in phenotypic variation, suggesting considerable functional redundancy among members of the superfamily.
Protein ubiquitination also serves regulatory functions that do not involve protein degradation (Hochstrasser al.,
Cell
84:813-815 (1995)). For example, Hicke and Riezman (Hicke and Riezman,
Cell
84:277-287 (1996)) have recently demonstrated ligand inducible ubiquitination of the Ste2 receptor in yeast. Ubiquitination of the Ste2 receptor results in receptor endocytosis and receptor targeting to vacuoles, not proteosomes. Chen, et at. (Chen al.,
Cell
84:853-862 (1996)) have demonstrated that activation of the IKBa Kinase requires a rapid, inducible ubiquitination event. This ubiquitination event is a prerequisite for the specific phosphorylation of IKBa and does not result in subsequent proteolysis of the complex. Whether or not the ubiquitination of Ste2 or IKBa Kinase is reversible through the action of a specific deubiquitinating enzyme is not known.
SUMMARY OF THE INVENTION
The present invention relates to ubiquitin-specific thiol proteases or deubiquitinating enzymes, referred to as DUB (DeUBiquitinating) enzymes, of eukaryotic origin which are members of a superfamily of deubiquitinating enzymes and which comprise a new subfamily of deubiquitinating enzymes which are similar in size and amino acid sequence to one another. DUB enzymes of the present invention are of eukaryotic origin, such as vertebrate origin, including mammalian (e.g., murine, human) origin, as well as yeast origin. All DUB enzymes of the present invention are inducible by at least one cytokine. Further, DUB enzymes of the present invention are of three types: (a) interleukin-3 (IL-3), interleukin-5 (IL-5) and/or granulocyte macrophage colony stimulating factor (GM-CSF)-inducible DUB enzymes; (b) interleukin-2 (IL-2)-inducible DUB enzymes, both (a) and (b) being specifically expressed in hematopoietic cells; and (c) those DUB enzymes induced by at least one cytokine, also referred to as other cytokineinducible DUB enzymes. It was originally thought that the IL-3, IL-5 and GM-CSF-inducible DUB enzymes, described above, were inducible only with IL-3. However, as described herein, this type of DUB enzyme is also inducible with IL-3, IL-5 and/or GM-CSF. The IL-3, IL-5 and GM-CSF receptors share a &bgr; common (&bgr;c) subunit, which appears to be responsible for induction of this type of DUB enzyme.
As described herein, DUB enzymes of the present invention are encoded by nucleic acid sequences which cross hybridize. Therefore, additional DUB enzymes can be identified through the use of DNAs or RNAs described herein and known hybridization methods. DUB enzymes encoded by DNAs which hybridize, under low stringency, with nucleic acids described herein (e.g., cDNAs, genomic DNAs such as DUB-1, DUB-2, DUB-3, DUB-4, DUB-5, human DUB D38378 and fragments thereof) and which can be confirmed by further analysis, are within the scope of the present invention. It should be noted at this time that DUB-2 of the present application was referred to as (is the same as) DUB-3 of the provisional application U.S. Ser. No. 60/002066 and DUB-3 of the present application was referred to as (is the same as) DUB-2 of the provisional application U.S. Serial No. 60/002066.
DUB enzymes of the present invention include the two conserved domains (one containing an active cysteine residue (CYS domain) and one containing conserved histidine residues (HIS domain)) present in superfamily members. In addition, they include variable regions (e.g., a hypervariable region, a basic region) within the carboxy terminal regions. The DUB enzymes described herein are smaller than previously-described ubiquitin-specific thiol proteases of the ubp superfamily. DUB enzymes of the present invention generally comprise 400-700 amino acid residues, and typically comprise 475-625 amino acid residues and even more typically average 500 to 550 amino acid residues. DUB enzymes of the present invention show substantial identity to one another not only in the conserved regions (i.e., the CYS domain and HIS domain), but throughout their entire amino acid sequences. The DUB enzymes of the present invention generally exhibit at least 30% homology to one another throughout the primary amino acid sequence and, typically, at least 50% identity, and more typically at least 65% identity, and most typically at least 80% identity. The present invention also relates to DUB enzyme variants (also referred to as DUB enzyme mutant polypeptides) and fusion proteins containing a DUB enzyme or DUB enzyme variant.
The present invention further encompasses nucleic acids, including DNA encoding DUB enzymes and DUB enzyme variants and RNA transcribed from or encoded by DNA encoding DUB enzymes or DUB enzyme variants; nucleic acid constructs comprising DNA or RNA encoding a DUB enzyme of the present invention; promoters and enhancers present in DUB genes; and host cells containing the nucleic acid constructs. It further relates to methods of producing heterologous proteins through the use of such promoters and/or enhancers.
The present invention also relates to antibodies (monoclonal and polyclonal) which bind a DUB enzyme or DUB enzyme variant; methods of producing DUB enzymes and DUB enzyme variants and uses thereof, including methods of altering (reducing or inhibiting, enhancing) cell proliferation and diagnostic methods, such as for diagnosing leukemias; methods of identifying inhibitors or enhancers of DUB enzymes; and inhibitors and enhancers of DUB enzymes.
In one embodiment, a DUB enzyme is a ubiquitin-specific thiol protease which is specifically expressed in hematopoietic cells induced with IL-3, IL-5 and/or GM-CSF. IL-3, IL-5 and/or GM-CSF-inducible DUB enzymes specifically expressed in hematopoietic cells include DUB-1 (SEQ ID NO. 2) and other DUB enzymes which are IL-3, IL-5 and/or GM-CSF inducible and have ubiquitin-specific thiol protease activity. Additional IL-3, IL-5 and/or GM-CSF inducible DUB enzymes are encoded by nucleic acids (DNA or RNA) which hybridize under conditions of low stringency with DUB-1-encoding DNA or RNA or a fragment thereof, which can be further confirmed by additional analysis known to those of skill in the art, and/or have amino acid sequences sufficiently similar, as described herein, to that of the DUB-1 enzyme amino acid sequence presented herein that they are IL-3, IL-5 and/or GM-CSF inducible ubiquitin-specific thiol proteases.
In a second embodiment, a DUB enzyme is a ubiquitin-specific thiol protease which is specifically expressed in hematopoietic cells induced with IL-2. IL-2 inducible DUB enzymes specifically expressed in hematopoietic cells include DUB-2 (SEQ. ID NO. 38) and other DUB enzymes which are IL-2 inducible and have ubiquitin-specific thiol protease activity. Additional IL-2 inducible DUB enzymes are encoded by nucleic acids (DNA or RNA) which hybridize under conditions of low stringency to DUB-2-encoding DNA or RNA or a fragment thereof and/or have amino acid sequences sufficiently similar, as described herein, to that of the DUB-2 enzyme amino acid sequence presented herein that they are IL-2

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