Kristina Lindsten

Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells.

The ubiquitin/proteasome-dependent proteolytic pathway is an attractive target for therapeutics because of its critical involvement in cell cycle progression and antigen presentation. However, dissection of the pathway and development of modulators are hampered by the complexity of the system and the lack of easily detectable authentic substrates. We have developed a convenient reporter system by producing N-end rule and ubiquitin fusion degradation (UFD)-targeted green fluorescent proteins that allow quantification of ubiquitin/proteasome-dependent proteolysis in living cells. Accumulation of these reporters serves as an early predictor of G2/M arrest and apoptosis in cells treated with proteasome inhibitors. Comparison of reporter accumulation and cleavage of fluorogenic substrates demonstrates that the rate-limiting chymotrypsin-like activity of the proteasome can be substantially curtailed without significant effect on ubiquitin-dependent proteolysis. These reporters provide a new powerful tool for elucidation of the ubiquitin/proteasome pathway and for high throughput screening of compounds that selectively modify proteolysis in vivo.

Inhibition of proteasome deubiquitinating activity as a new cancer therapy

Ubiquitin-tagged substrates are degraded by the 26S proteasome, which is a multisubunit complex comprising a proteolytic 20S core particle capped by 19S regulatory particles. The approval of bortezomib for the treatment of multiple myeloma validated the 20S core particle as an anticancer drug target. Here we describe the small molecule b-AP15 as a previously unidentified class of proteasome inhibitor that abrogates the deubiquitinating activity of the 19S regulatory particle. b-AP15 inhibited the activity of two 19S regulatory-particle–associated deubiquitinases, ubiquitin C-terminal hydrolase 5 (UCHL5) and ubiquitin-specific peptidase 14 (USP14), resulting in accumulation of polyubiquitin. b-AP15 induced tumor cell apoptosis that was insensitive to TP53 status and overexpression of the apoptosis inhibitor BCL2. We show that treatment with b-AP15 inhibited tumor progression in four different in vivo solid tumor models and inhibited organ infiltration in an acute myeloid leukemia model. Our results show that the deubiquitinating activity of the 19S regulatory particle is a new anticancer drug target.

Mutant ubiquitin found in neurodegenerative disorders is a ubiquitin fusion degradation substrate that blocks proteasomal degradation

Loss of neurons in neurodegenerative diseases is usually preceded by the accumulation of protein deposits that contain components of the ubiquitin/proteasome system. Affected neurons in Alzheimers disease often accumulate UBB+1, a mutant ubiquitin carrying a 19–amino acid C-terminal extension generated by a transcriptional dinucleotide deletion. Here we show that UBB+1 is a potent inhibitor of ubiquitin-dependent proteolysis in neuronal cells, and that this inhibitory activity correlates with induction of cell cycle arrest. Surprisingly, UBB+1 is recognized as a ubiquitin fusion degradation (UFD) proteasome substrate and ubiquitinated at Lys29 and Lys48. Full blockade of proteolysis requires both ubiquitination sites. Moreover, the inhibitory effect was enhanced by the introduction of multiple UFD signals. Our findings suggest that the inhibitory activity of UBB+1 may be an important determinant of neurotoxicity and contribute to an environment that favors the accumulation of misfolded proteins.

The ER-resident ubiquitin-specific protease 19 participates in the UPR and rescues ERAD substrates

Ubiquitination regulates membrane events such as endocytosis, membrane trafficking and endoplasmic‐reticulum‐associated degradation (ERAD). Although the involvement of membrane‐associated ubiquitin‐conjugating enzymes and ligases in these processes is well documented, their regulation by ubiquitin deconjugases is less well understood. By screening a database of human deubiquitinating enzymes (DUBs), we have identified a putative transmembrane domain in ubiquitin‐specific protease (USP)19. We show that USP19 is a tail‐anchored ubiquitin‐specific protease localized to the ER and is a target of the unfolded protein response. USP19 rescues the ERAD substrates cystic fibrosis transmembrane conductance regulator (CFTR)ΔF508 and T‐cell receptor‐α (TCRα) from proteasomal degradation. A catalytically inactive USP19 was still able to partly rescue TCRα but not CFTRΔF508, suggesting that USP19 might also exert a non‐catalytic function on specific ERAD substrates. Thus, USP19 is the first example of a membrane‐anchored DUB involved in the turnover of ERAD substrates.

The ubiquitin specific protease 4 (USP4) is a new player in the Wnt signalling pathway.

The canonical Wnt signalling pathway is essential for cell fate determination during embryonic development and for the maintenance of adult tissue homeostasis. Deregulation of Wnt signalling leads to developmental defects and is associated with various types of cancer. Here we have used an RNA interference (RNAi) library specifically targeting human deubiquitinating enzymes (DUBs) to screen for new regulators of the canonical Wnt signalling pathway. We found that suppression of the ubiquitin specific protease 4 (USP4) activates β‐catenin dependent transcription. We also show that USP4 is a DUB with dual hydrolysing activity for K48‐ and K63‐conjugated polyubiquitin chains and interacts with two known Wnt signalling components: the Nemo like kinase (Nlk) and the transcription factor (T‐cell factor 4 [TCF4]). Overexpression of a catalytically active Nlk promotes nuclear accumulation of USP4 whereas a subpopulation of TCF4 is a substrate of USP4‐dependent deubiquitination. Thus, modulation of USP4 expression may provide a new means to interfere with canonical Wnt signalling in a variety of physiological and pathological conditions.

Stressing the ubiquitin-proteasome system

Unfolded and misfolded proteins are inherently toxic to cells and have to be quickly and efficiently eliminated before they intoxicate the intracellular environment. This is of particular importance during proteotoxic stress when, as a consequence of intrinsic or extrinsic factors, the levels of misfolded proteins are transiently or persistently elevated. To meet this demand, metazoan cells have developed specific protein quality control mechanisms that allow the identification and proper handling of non-native proteins. An important defence mechanism is the specific destruction of these proteins by the ubiquitin-proteasome system (UPS). A number of studies have shown that various proteotoxic stress conditions can cause functional impairment of the UPS resulting in cellular dysfunction and apoptosis. In this review, we will summarize our current understanding of proteotoxic stress-induced dysfunction of the UPS and some of its implications for human pathologies.

Ubiquitin-specific protease 19 (USP19) regulates hypoxia-inducible factor 1α (HIF-1α) during hypoxia.

Background: The highly regulated hypoxia-inducible factor 1α (HIF-1α) is a key player in the cellular response to hypoxia. Results: The ubiquitin-specific protease 19 (USP19) rescues HIF-1α from degradation in a non-catalytic manner. Conclusion: USP19 is required for cells to mount an appropriate response to hypoxia. Significance: Learning about HIF-1α regulation is essential for understanding the physiological and pathophysiological conditions of the hypoxic response. A proper cellular adaptation to low oxygen levels is essential for processes such as development, growth, metabolism, and angiogenesis. The response to decrease in oxygen supply, referred to as hypoxia, is also involved in numerous human diseases including cancer, inflammatory conditions, and vascular disease. The hypoxia-inducible factor 1-α (HIF-1α), a key player in the hypoxic response, is kept under stringent regulation. At normoxia, the levels are kept low as a consequence of the efficient degradation by the ubiquitin-proteasome system, and in response to hypoxia, the degradation is blocked and the accumulating HIF-1α promotes a transcriptional response essential for proper adaptation and survival. Here we show that the ubiquitin-specific protease-19 (USP19) interacts with components of the hypoxia pathway including HIF-1α and rescues it from degradation independent of its catalytic activity. In the absence of USP19, cells fail to mount an appropriate response to hypoxia, indicating an important role for this enzyme in normal or pathological conditions.

The ubiquitin-specific protease USP25 interacts with three sarcomeric proteins

Abstract.The biological functions of the more than one hundred genes coding for deubiquitinating enzymes in the human genome remain mostly unknown. The USP25 gene, located at 21q11.2, encodes three protein isoforms produced by alternative splicing. While two of the isoforms are expressed nearly ubiquituously, the expression of the longer USP25 isoform (USP25m) is restricted to muscular tissues and is upregulated during myogenesis. USP25m interacts with three sarcomeric proteins: actin alpha-1 (ACTA1), filamin C (FLNC), and myosin binding protein C1 (MyBPC1), which are critically involved in muscle differentiation and maintenance, and have been implicated in the pathogenesis of severe myopathies. Biochemical analyses demonstrated that MyBPC1 is a short-lived proteasomal substrate, and its degradation is prevented by over-expression of USP25m but not by other USP25 isoforms. In contrast, ACTA1 and FLNC appear to be stable proteins, indicating that their interaction with USP25m is not related to their turnover rate.

Epstein-Barr Virus Encodes Three Bona Fide Ubiquitin-Specific Proteases

ABSTRACT Manipulation of the ubiquitin proteasome system (UPS) is emerging as a common theme in viral pathogenesis. Some viruses have been shown to encode functional homologs of UPS enzymes, suggesting that a systematic identification of these products may provide new insights into virus-host cell interactions. Ubiquitin-specific proteases, collectively known as deubiquitinating enzymes (DUBs), regulate the activity of the UPS by hydrolyzing ubiquitin peptide or isopeptide bonds. The prediction of viral DUBs based on sequence similarity with known enzymes is hampered by the diversity of viral genomes. In this study sequence alignments, pattern searches, and hidden Markov models were developed for the conserved C- and H-boxes of the known DUB families and used to search the open reading frames (ORFs) of Epstein-Barr virus (EBV), a large gammaherpesvirus that has been implicated in the pathogenesis of a broad spectrum of human malignancies of lymphoid and epithelial cell origin. The searches identified a limited number of EBV ORFs that contain putative DUB catalytic domains. DUB activity was confirmed by functional assays and mutation analysis for three high scoring candidates, supporting the usefulness of this bioinformatics approach in predicting distant homologues of cellular enzymes.

Cell-Based Fluorescence Assay for Human Immunodeficiency Virus Type 1 Protease Activity

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) protease is essential for production of infectious virus and is therefore a major target for the development of drugs against AIDS. Cellular proteins are also cleaved by the protease, which explains its cytotoxic activity and the consequent failure to establish convenient cell-based protease assays. We have exploited this toxicity to develop a new protease assay that relies on transient expression of an artificial protease precursor harboring the green fluorescent protein (GFP-PR). The precursor is activated in vivo by autocatalytic cleavage, resulting in rapid elimination of protease-expressing cells. Treatment with therapeutic doses of HIV-1 protease inhibitors results in a dose-dependent accumulation of the fluorescent precursor that can be easily detected and quantified by flow cytometric and fluorimetric assays. The precursor provides a convenient and noninfectious model for high-throughput screenings of substances that can interfere with the activity of the protease in living cells.