Mikael Altun

A Small Molecule Inhibitor of Ubiquitin-Specific Protease-7 Induces Apoptosis in Multiple Myeloma Cells and Overcomes Bortezomib Resistance

Bortezomib therapy has proven successful for the treatment of relapsed/refractory, relapsed, and newly diagnosed multiple myeloma (MM); however, dose-limiting toxicities and the development of resistance limit its long-term utility. Here, we show that P5091 is an inhibitor of deubiquitylating enzyme USP7, which induces apoptosis in MM cells resistant to conventional and bortezomib therapies. Biochemical and genetic studies show that blockade of HDM2 and p21 abrogates P5091-induced cytotoxicity. In animal tumor model studies, P5091 is well tolerated, inhibits tumor growth, and prolongs survival. Combining P5091 with lenalidomide, HDAC inhibitor SAHA, or dexamethasone triggers synergistic anti-MM activity. Our preclinical study therefore supports clinical evaluation of USP7 inhibitor, alone or in combination, as a potential MM therapy.

Activity-Based Chemical Proteomics Accelerates Inhibitor Development for Deubiquitylating Enzymes

Converting lead compounds into drug candidates is a crucial step in drug development, requiring early assessment of potency, selectivity, and off-target effects. We have utilized activity-based chemical proteomics to determine the potency and selectivity of deubiquitylating enzyme (DUB) inhibitors in cell culture models. Importantly, we characterized the small molecule PR-619 as a broad-range DUB inhibitor, and P22077 as a USP7 inhibitor with potential for further development as a chemotherapeutic agent in cancer therapy. A striking accumulation of polyubiquitylated proteins was observed after both selective and general inhibition of cellular DUB activity without direct impairment of proteasomal proteolysis. The repertoire of ubiquitylated substrates was analyzed by tandem mass spectrometry, identifying distinct subsets for general or specific inhibition of DUBs. This enabled identification of previously unknown functional links between USP7 and enzymes involved in DNA repair.

Effects of PS-341 on the Activity and Composition of Proteasomes in Multiple Myeloma Cells

Multiple myeloma is a B-cell malignancy for which no curative therapies exist to date, despite enormous research efforts. The remarkable activity of the proteasome inhibitor bortezomib (PS-341, Velcade) observed in clinical trials of patients with relapsed refractory myeloma has led to investigations of the role of the ubiquitin-proteasome pathway in the pathogenesis of myeloma. Here we report a biochemical analysis of proteasome activity and composition in myeloma cells exposed to PS-341 in the presence or absence of cytokines present in the bone marrow milieu. We observed that the myeloma cell lines MM1.S, RPMI8226, and U266 contain active immunoproteasomes, the amount of which is enhanced by IFN-gamma and tumor necrosis factor-alpha. Using a radiolabeled active site-directed probe specific for proteasome catalytic subunits, we show that PS-341 targets the beta5 and beta1 subunits in a concentration-dependent manner. Furthermore, PS-341 also targeted the corresponding catalytic subunits of the immunoproteasome, beta5i and beta1i, respectively. These data suggest that PS-341 targets both normal and immunoproteasome species to a similar extent in myeloma cells.

Factors contributing to neuromuscular impairment and sarcopenia during aging.

Motor disturbances and wasting of skeletal muscles (sarcopenia) causes significant impairment of daily life activities and is a major underlying cause for hospitalization in senescence. Herein we review data and present new findings on aging-specific changes in motoneurons, skeletal muscle and the interplay between motoneurons and target muscle fibers. Although many of the changes occurring during aging may be specific to motoneurons and myofibers, respectively, evidence indicates that myofiber regeneration in sarcopenic muscle is halted at the point where reinnervation is critical for the final differentiation into mature myofibers. Combined, evidence suggests that sarcopenia to a significant extent depend on a decreased capacity among motoneurons to innervate regenerating fibers. There are also conspicuous changes in the expression of several cytokines known to play important roles in establishing and maintaining neuromuscular connectivity during development and adulthood. We also present data showing the usefulness of rodent models in studies of successful and unsuccessful patterns of aging. Finally, we show that not only dietary restriction (DR) but also activity and social environment may modulate the pattern of aging.

Muscle Wasting in Aged, Sarcopenic Rats Is Associated with Enhanced Activity of the Ubiquitin Proteasome Pathway

Among the hallmarks of aged organisms are an accumulation of misfolded proteins and a reduction in skeletal muscle mass (“sarcopenia”). We have examined the effects of aging and dietary restriction (which retards many age-related changes) on components of the ubiquitin proteasome system (UPS) in muscle. The hindlimb muscles of aged (30 months old) rats showed a marked loss of muscle mass and contained 2–3-fold higher levels of 26S proteasomes than those of adult (4 months old) controls. 26S proteasomes purified from muscles of aged and adult rats showed a similar capacity to degrade peptides, proteins, and an ubiquitylated substrate, but differed in levels of proteasome-associated proteins (e.g. the ubiquitin ligase E6AP and deubiquitylating enzyme USP14). Also, the activities of many other deubiquitylating enzymes were greatly enhanced in the aged muscles. Nevertheless, their content of polyubiquitylated proteins was higher than in adult animals. The aged muscles contained higher levels of the ubiquitin ligase CHIP, involved in eliminating misfolded proteins, and MuRF1, which ubiquitylates myofibrillar proteins. These muscles differed from ones rapidly atrophying due to disease, fasting, or disuse in that Atrogin-1/MAFbx expression was low and not inducible by glucocorticoids. Thus, the muscles of aged rats showed many adaptations indicating enhanced proteolysis by the UPS, which may enhance their capacity to eliminate misfolded proteins and seems to contribute to the sarcopenia. Accordingly, dietary restriction decreased or prevented the aging-associated increases in proteasomes and other UPS components and reduced muscle wasting.

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.

Deubiquitinating Enzyme Specificity for Ubiquitin Chain Topology Profiled by Di-Ubiquitin Activity Probes

Summary Posttranslational modification with ubiquitin (Ub) controls many cellular processes, and aberrant ubiquitination can contribute to cancer, immunopathology, and neurodegeneration. The versatility arises from the ability of Ub to form polymer chains with eight distinct linkages via lysine side chains and the N terminus. In this study, we engineered Di-Ub probes mimicking all eight different poly-Ub linkages and profiled the deubiquitinating enzyme (DUB) selectivity for recognizing Di-Ub moieties in cellular extracts. Mass spectrometric profiling revealed that most DUBs examined have broad selectivity, whereas a subset displays a clear preference for recognizing noncanonical over K48/K63 Ub linkages. Our results expand knowledge of Ub processing enzyme functions in cellular contexts that currently depends largely on using recombinant enzymes and substrates.

Iron load and redox stress in skeletal muscle of aged rats.

Loss of skeletal muscle mass (sarcopenia) is a major contributor to disability in old age. We used two‐dimensional gel electrophoresis and mass spectrometry to screen for changes in proteins, and cDNA profiling to assess transcriptional regulations in the gastrocnemius muscle of adult (4 months) and aged (30 months) male Sprague‐Dawley rats. Thirty‐five proteins were differentially expressed in aged muscle. Proteins and mRNA transcripts involved in redox homeostasis and iron load were increased, representing novel components that were previously not associated with sarcopenia. Tissue iron levels were elevated in senescence, paralleling an increase in transferrin. Proteins involved in redox homeostasis showed a complex pattern of changes with increased SOD1 and decreased SOD2. These results suggest that an elevated iron load is a significant component of sarcopenia with the potential to be exploited clinically, and that mitochondria of aged striated muscle may be more vulnerable to radicals produced in cell respiration. Muscle Nerve, 2007

A Photoreactive Small-Molecule Probe for 2-Oxoglutarate Oxygenases

2-oxoglutarate (2-OG)-dependent oxygenases have diverse roles in human biology. The inhibition of several 2-OG oxygenases is being targeted for therapeutic intervention, including for cancer, anemia, and ischemic diseases. We report a small-molecule probe for 2-OG oxygenases that employs a hydroxyquinoline template coupled to a photoactivable crosslinking group and an affinity-purification tag. Following studies with recombinant proteins, the probe was shown to crosslink to 2-OG oxygenases in human crude cell extracts, including to proteins at endogenous levels. This approach is useful for inhibitor profiling, as demonstrated by crosslinking to the histone demethylase FBXL11 (KDM2A) in HEK293T nuclear extracts. The results also suggest that small-molecule probes may be suitable for substrate identification studies.

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.