Mikael Altun - Academia.edu (original) (raw)

Papers by Mikael Altun

Cancer research, 2005

Multiple myeloma is a B-cell malignancy for which no curative therapies exist to date, despite en... more 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 su...

PLOS ONE, 2015

Ovarian tumor domain containing proteases cleave ubiquitin (Ub) and ubiquitin-like polypeptides f... more Ovarian tumor domain containing proteases cleave ubiquitin (Ub) and ubiquitin-like polypeptides from proteins. Here we report the crystal structure of human otubain 2 (OTUB2) in complex with a ubiquitin-based covalent inhibitor, Ub-Br2. The ubiquitin binding mode is oriented differently to how viral otubains (vOTUs) bind ubiquitin/ISG15, and more similar to yeast and mammalian OTUs. In contrast to OTUB1 which has exclusive specificity towards Lys48 poly-ubiquitin chains, OTUB2 cleaves different poly-Ub linked chains. N-terminal tail swapping experiments between OTUB1 and OTUB2 revealed how the N-terminal structural motifs in OTUB1 contribute to modulating enzyme activity and Ub-chain selectivity, a trait not observed in OTUB2, supporting the notion that OTUB2 may affect a different spectrum of substrates in Ub-dependent pathways.

Muscle atrophy in many conditions share a common mechanism in the upregulation of the muscle-spec... more Muscle atrophy in many conditions share a common mechanism in the upregulation of the muscle-specific ubiquitin E3-ligases atrophy gene-1/muscle atrophy F-box (Atrogin-1/MAFbx) and muscle ring-finger protein 1 (MuRF1). E3-ligases are part of the ubiquitin proteasome pathway utilized for protein degradation during muscle atrophy. In this study, we provide new data to show that this is not the case in age-related

Virology Journal, 2011

A large quantitative study was carried out to compare the proteome of respiratory syncytial virus... more A large quantitative study was carried out to compare the proteome of respiratory syncytial virus (RSV) infected versus uninfected cells in order to determine novel pathways regulated during viral infection. RSV infected and mock-infected HEp2 cells were lysed and proteins separated by preparative isoelectric focussing using offgel fractionation. Following tryptic digestion, purified peptides were characterized using label-free quantitative expression profiling by nano-ultra performance liquid chromatography coupled to electrospray ionisation mass spectrometry with collision energy ramping for all-ion fragmentation (UPLC-MS E ). A total of 1352 unique cellular proteins were identified and their abundance compared between infected and non-infected cells. Ingenuity pathway analysis revealed regulation of several central cellular metabolic and signalling pathways during infection. Selected proteins that were found regulated in RSV infected cells were screened by quantitative real-time PCR for their regulation on the transcriptional level. Synthesis of interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) and 5'-3'-exoribonuclease 2 (XRN2) mRNAs were found to be highly induced upon RSV infection in a time dependent manner. Accordingly, IFIT3 protein levels accumulated during the time course of infection. In contrast, little variation was observed in XRN2 protein levels, but different forms were present in infected versus noninfected cells. This suggests a role of these proteins in viral infection, and analysis of their function will shed further light on mechanisms of RNA virus replication and the host cell defence machinery.

Physiology & Behavior, 2007

Several disturbances occurring during aging of humans and rodents alike stem from changes in sens... more Several disturbances occurring during aging of humans and rodents alike stem from changes in sensory and motor functions. Using a battery of behavioral tests we have studied alterations in performance with advancing age in female and male rats of some frequently used strains. In parallel, we collected survival and body weight data. The median survival age was similar for female and male Sprague-Dawley rats, inbred female Lewis and outbred male Wistar rats (29-30 months). In contrast, male Fisher 344 had a significantly shorter median life span. During aging there is a gradual decline in locomotor activity and explorative behavior while disturbances of coordination and balance first became evident at more advanced age. In old age, also weight carrying capacity, limb movement and temperature threshold were impaired. While whole body weight continues to increase over the better part of a rats' life span, the behavioral changes in old age associated with a decrease in both total body weight and muscle mass. Dietary restriction increases median life span expectancy; retards the pace of behavioral aging and impedes sarcopenia. Housing in enriched environment did not improve the scoring in the behavioral tests but tended to increase median life span. Finally, there was an agreement between behavioral data collected from longitudinal age-cohorts and those obtained from multiple age-cohorts.

Physiology & Behavior, 2007

Motor disturbances and wasting of skeletal muscles (sarcopenia) causes significant impairment of ... more 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.

Organic & Biomolecular Chemistry, 2012

Novel ubiquitin-based active site probes including a fluorescent tag have been developed and eval... more Novel ubiquitin-based active site probes including a fluorescent tag have been developed and evaluated. A new, functionalizable electrophilic trap is utilized allowing for late stage diversification of the probe. Attachment of fluorescent dyes allowed direct detection of endogenous deubiquitinating enzyme (DUB) activities in cell extracts by in-gel fluorescence imaging. † Electronic supplementary information (ESI) available: Synthesis of fluorescein azide used for creation of probe 4; MS/MS characterization of N-terminal M(ox) probe variants; HPLC purification of alkyne probe 2. See

Muscle & Nerve, 2007

Loss of skeletal muscle mass (sarcopenia) is a major contributor to disability in old age. We use... more 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. Thirtyfive 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.

Journal of Neuroscience Methods, 2001

In this study we have used fluorescent microspheres to retrogradely label primary sensory neurons... more In this study we have used fluorescent microspheres to retrogradely label primary sensory neurons in dorsal root ganglia (DRGs). Following injection into peripheral nerves, the animals were allowed to survive up to 480 days. Simple profile count indicates that there is a substantial retention of the labeling still after at least 480 days, i.e. about two-thirds of a rat's life span. Moreover, the appearance of the labeling remains quite distinct. Using established markers for axon damage of DRG neurons, we could detect a slight and transient effect of the peripheral nerve injection on the gene expression pattern. It is concluded that fluorescent microspheres represents an attractive means of tagging neurons in experiments covering long time periods.

The Journals of Gerontology Series A: Biological Sciences and Medical Sciences, 2006

Muscle atrophy in many conditions share a common mechanism in the upregulation of the muscle-spec... more Muscle atrophy in many conditions share a common mechanism in the upregulation of the muscle-specific ubiquitin E3-ligases atrophy gene-1/muscle atrophy F-box (Atrogin-1/MAFbx) and muscle ring-finger protein 1 (MuRF1). E3-ligases are part of the ubiquitin proteasome pathway utilized for protein degradation during muscle atrophy. In this study, we provide new data to show that this is not the case in age-related loss of muscle mass (sarcopenia). On the contrary, Atrogin-1/MAFbx and MuRF1 are downregulated in skeletal muscle of 30-month-old rats, and our results suggest that AKT (protein kinase B)-mediated inactivation of forkhead box O 4 (FOXO4) underlies this suppression. The data also suggest that activation of AKT is mediated through the insulin-like growth factor-1 (IGF-1) receptor, signaling via ShcA-Grb2-GAB. Using dietary restriction, we find that it impedes sarcopenia as well as the effects of aging on AKT phosphorylation, FOXO4 phosphorylation, and Atrogin-1/MAFbx and MuRF1 transcript regulation. We conclude that sarcopenia is mechanistically different from acute atrophies induced by disuse, disease, and denervation.

Journal of Biological Chemistry, 2012

The highly regulated hypoxia-inducible factor 1␣ (HIF-1␣) is a key player in the cellular respons... more The highly regulated hypoxia-inducible factor 1␣ (HIF-1␣) is a key player in the cellular response to hypoxia. Results: The ubiquitin-specific protease 19 (USP19) rescues HIF-1␣ from degradation in a non-catalytic manner. Conclusion: USP19 is required for cells to mount an appropriate response to hypoxia. Significance: Learning about HIF-1␣ regulation is essential for understanding the physiological and pathophysiological conditions of the hypoxic response.

EMBO reports, 2009

Ubiquitination regulates membrane events such as endocytosis, membrane trafficking and endoplasmi... more Ubiquitination regulates membrane events such as endocytosis, membrane trafficking and endoplasmic-reticulum-associated degradation (ERAD). Although the involvement of membraneassociated 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)DF508 and T-cell receptor-a (TCRa) from proteasomal degradation. A catalytically inactive USP19 was still able to partly rescue TCRa but not CFTRDF508, 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.

Chemistry & Biology, 2001

Background: The 26S proteasome is responsible for most cytosolic proteolysis, and is an important... more Background: The 26S proteasome is responsible for most cytosolic proteolysis, and is an important protease in major histocompatibility complex class I-mediated antigen presentation. Constitutively expressed proteasomes from mammalian sources possess three distinct catalytically active species, L1, L2 and L5, which are replaced in the Q-interferon-inducible immunoproteasome by a different set of catalytic subunits, L1i, L2i and L5i, respectively. Based on preferred cleavage of short fluorogenic peptide substrates, activities of the proteasome have been assigned to individual subunits and classified as`chymotryptic-like' (L5), tryptic-like' (L2) and`peptidyl-glutamyl peptide hydrolyzing' (L1). Studies with protein substrates indicate a far more complicated, less strict cleavage preference. We reasoned that inhibitors of extended size would give insight into the extent of overlapping substrate specificity of the individual activities and subunits.

Chemistry & Biology, 2013

Posttranslational modification with ubiquitin (Ub) controls many cellular processes, and aberrant... more 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.

Chemistry & Biology, 2011

Converting lead compounds into drug candidates is a crucial step in drug development, requiring e... more 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.

Chemistry & Biology, 2011

2-oxoglutarate (2-OG)-dependent oxygenases have diverse roles in human biology. The inhibition of... more 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.

Cancer research, 2005

Multiple myeloma is a B-cell malignancy for which no curative therapies exist to date, despite en... more 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 su...

PLOS ONE, 2015

Ovarian tumor domain containing proteases cleave ubiquitin (Ub) and ubiquitin-like polypeptides f... more Ovarian tumor domain containing proteases cleave ubiquitin (Ub) and ubiquitin-like polypeptides from proteins. Here we report the crystal structure of human otubain 2 (OTUB2) in complex with a ubiquitin-based covalent inhibitor, Ub-Br2. The ubiquitin binding mode is oriented differently to how viral otubains (vOTUs) bind ubiquitin/ISG15, and more similar to yeast and mammalian OTUs. In contrast to OTUB1 which has exclusive specificity towards Lys48 poly-ubiquitin chains, OTUB2 cleaves different poly-Ub linked chains. N-terminal tail swapping experiments between OTUB1 and OTUB2 revealed how the N-terminal structural motifs in OTUB1 contribute to modulating enzyme activity and Ub-chain selectivity, a trait not observed in OTUB2, supporting the notion that OTUB2 may affect a different spectrum of substrates in Ub-dependent pathways.

Muscle atrophy in many conditions share a common mechanism in the upregulation of the muscle-spec... more Muscle atrophy in many conditions share a common mechanism in the upregulation of the muscle-specific ubiquitin E3-ligases atrophy gene-1/muscle atrophy F-box (Atrogin-1/MAFbx) and muscle ring-finger protein 1 (MuRF1). E3-ligases are part of the ubiquitin proteasome pathway utilized for protein degradation during muscle atrophy. In this study, we provide new data to show that this is not the case in age-related

Virology Journal, 2011

A large quantitative study was carried out to compare the proteome of respiratory syncytial virus... more A large quantitative study was carried out to compare the proteome of respiratory syncytial virus (RSV) infected versus uninfected cells in order to determine novel pathways regulated during viral infection. RSV infected and mock-infected HEp2 cells were lysed and proteins separated by preparative isoelectric focussing using offgel fractionation. Following tryptic digestion, purified peptides were characterized using label-free quantitative expression profiling by nano-ultra performance liquid chromatography coupled to electrospray ionisation mass spectrometry with collision energy ramping for all-ion fragmentation (UPLC-MS E ). A total of 1352 unique cellular proteins were identified and their abundance compared between infected and non-infected cells. Ingenuity pathway analysis revealed regulation of several central cellular metabolic and signalling pathways during infection. Selected proteins that were found regulated in RSV infected cells were screened by quantitative real-time PCR for their regulation on the transcriptional level. Synthesis of interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) and 5'-3'-exoribonuclease 2 (XRN2) mRNAs were found to be highly induced upon RSV infection in a time dependent manner. Accordingly, IFIT3 protein levels accumulated during the time course of infection. In contrast, little variation was observed in XRN2 protein levels, but different forms were present in infected versus noninfected cells. This suggests a role of these proteins in viral infection, and analysis of their function will shed further light on mechanisms of RNA virus replication and the host cell defence machinery.

Physiology & Behavior, 2007

Several disturbances occurring during aging of humans and rodents alike stem from changes in sens... more Several disturbances occurring during aging of humans and rodents alike stem from changes in sensory and motor functions. Using a battery of behavioral tests we have studied alterations in performance with advancing age in female and male rats of some frequently used strains. In parallel, we collected survival and body weight data. The median survival age was similar for female and male Sprague-Dawley rats, inbred female Lewis and outbred male Wistar rats (29-30 months). In contrast, male Fisher 344 had a significantly shorter median life span. During aging there is a gradual decline in locomotor activity and explorative behavior while disturbances of coordination and balance first became evident at more advanced age. In old age, also weight carrying capacity, limb movement and temperature threshold were impaired. While whole body weight continues to increase over the better part of a rats' life span, the behavioral changes in old age associated with a decrease in both total body weight and muscle mass. Dietary restriction increases median life span expectancy; retards the pace of behavioral aging and impedes sarcopenia. Housing in enriched environment did not improve the scoring in the behavioral tests but tended to increase median life span. Finally, there was an agreement between behavioral data collected from longitudinal age-cohorts and those obtained from multiple age-cohorts.

Physiology & Behavior, 2007

Motor disturbances and wasting of skeletal muscles (sarcopenia) causes significant impairment of ... more 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.

Organic & Biomolecular Chemistry, 2012

Novel ubiquitin-based active site probes including a fluorescent tag have been developed and eval... more Novel ubiquitin-based active site probes including a fluorescent tag have been developed and evaluated. A new, functionalizable electrophilic trap is utilized allowing for late stage diversification of the probe. Attachment of fluorescent dyes allowed direct detection of endogenous deubiquitinating enzyme (DUB) activities in cell extracts by in-gel fluorescence imaging. † Electronic supplementary information (ESI) available: Synthesis of fluorescein azide used for creation of probe 4; MS/MS characterization of N-terminal M(ox) probe variants; HPLC purification of alkyne probe 2. See

Muscle & Nerve, 2007

Loss of skeletal muscle mass (sarcopenia) is a major contributor to disability in old age. We use... more 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. Thirtyfive 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.

Journal of Neuroscience Methods, 2001

In this study we have used fluorescent microspheres to retrogradely label primary sensory neurons... more In this study we have used fluorescent microspheres to retrogradely label primary sensory neurons in dorsal root ganglia (DRGs). Following injection into peripheral nerves, the animals were allowed to survive up to 480 days. Simple profile count indicates that there is a substantial retention of the labeling still after at least 480 days, i.e. about two-thirds of a rat's life span. Moreover, the appearance of the labeling remains quite distinct. Using established markers for axon damage of DRG neurons, we could detect a slight and transient effect of the peripheral nerve injection on the gene expression pattern. It is concluded that fluorescent microspheres represents an attractive means of tagging neurons in experiments covering long time periods.

The Journals of Gerontology Series A: Biological Sciences and Medical Sciences, 2006

Muscle atrophy in many conditions share a common mechanism in the upregulation of the muscle-spec... more Muscle atrophy in many conditions share a common mechanism in the upregulation of the muscle-specific ubiquitin E3-ligases atrophy gene-1/muscle atrophy F-box (Atrogin-1/MAFbx) and muscle ring-finger protein 1 (MuRF1). E3-ligases are part of the ubiquitin proteasome pathway utilized for protein degradation during muscle atrophy. In this study, we provide new data to show that this is not the case in age-related loss of muscle mass (sarcopenia). On the contrary, Atrogin-1/MAFbx and MuRF1 are downregulated in skeletal muscle of 30-month-old rats, and our results suggest that AKT (protein kinase B)-mediated inactivation of forkhead box O 4 (FOXO4) underlies this suppression. The data also suggest that activation of AKT is mediated through the insulin-like growth factor-1 (IGF-1) receptor, signaling via ShcA-Grb2-GAB. Using dietary restriction, we find that it impedes sarcopenia as well as the effects of aging on AKT phosphorylation, FOXO4 phosphorylation, and Atrogin-1/MAFbx and MuRF1 transcript regulation. We conclude that sarcopenia is mechanistically different from acute atrophies induced by disuse, disease, and denervation.

Journal of Biological Chemistry, 2012

The highly regulated hypoxia-inducible factor 1␣ (HIF-1␣) is a key player in the cellular respons... more The highly regulated hypoxia-inducible factor 1␣ (HIF-1␣) is a key player in the cellular response to hypoxia. Results: The ubiquitin-specific protease 19 (USP19) rescues HIF-1␣ from degradation in a non-catalytic manner. Conclusion: USP19 is required for cells to mount an appropriate response to hypoxia. Significance: Learning about HIF-1␣ regulation is essential for understanding the physiological and pathophysiological conditions of the hypoxic response.

EMBO reports, 2009

Ubiquitination regulates membrane events such as endocytosis, membrane trafficking and endoplasmi... more Ubiquitination regulates membrane events such as endocytosis, membrane trafficking and endoplasmic-reticulum-associated degradation (ERAD). Although the involvement of membraneassociated 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)DF508 and T-cell receptor-a (TCRa) from proteasomal degradation. A catalytically inactive USP19 was still able to partly rescue TCRa but not CFTRDF508, 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.

Chemistry & Biology, 2001

Background: The 26S proteasome is responsible for most cytosolic proteolysis, and is an important... more Background: The 26S proteasome is responsible for most cytosolic proteolysis, and is an important protease in major histocompatibility complex class I-mediated antigen presentation. Constitutively expressed proteasomes from mammalian sources possess three distinct catalytically active species, L1, L2 and L5, which are replaced in the Q-interferon-inducible immunoproteasome by a different set of catalytic subunits, L1i, L2i and L5i, respectively. Based on preferred cleavage of short fluorogenic peptide substrates, activities of the proteasome have been assigned to individual subunits and classified as`chymotryptic-like' (L5), tryptic-like' (L2) and`peptidyl-glutamyl peptide hydrolyzing' (L1). Studies with protein substrates indicate a far more complicated, less strict cleavage preference. We reasoned that inhibitors of extended size would give insight into the extent of overlapping substrate specificity of the individual activities and subunits.

Chemistry & Biology, 2013

Posttranslational modification with ubiquitin (Ub) controls many cellular processes, and aberrant... more 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.

Chemistry & Biology, 2011

Converting lead compounds into drug candidates is a crucial step in drug development, requiring e... more 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.

Chemistry & Biology, 2011

2-oxoglutarate (2-OG)-dependent oxygenases have diverse roles in human biology. The inhibition of... more 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.