Ubiquitination Activity Research Articles

Somatic gain-of-function HIF2A mutations in sporadic central nervous system hemangioblastomas

Central nervous system hemangioblastomas (CNS-HBs) occur sporadically or as a component of von Hippel-Lindau-VHL syndrome. CNS-HBs share some molecular similarities with pheochromocytomas/paragangliomas (PPGLs) and renal cell carcinomas (RCCs). Recently, hypoxia-inducible factors, particularly somatic HIF2A mutations, have been found to play an important role in the pathogenesis of PPGLs. Somatic mutations in HIF2A have been reported in PPGLs associated with polycythemia, which have been reported to also be present in patients with RCCs and HBs. However, whether CNS-HBs is associated with the presence of a HIF2A mutation is currently uknown. We analyzed somatic HIF2A and VHL mutations in a series of 28 sporadic CNS-HBs. We also investigated the expression of HIF target proteins and hypoxia-associated factor (HAF). Two sporadic CNS-HBs were found to have somatic HIF2A mutations. One tumor had 2 HIF2A missense mutations, one of which was previously described in a PPGL (c.1121 T>A, F374Y). The second patient had coexistence of somatic truncated mutations (c.1669 C>T, Q557*) in HIF2A together with a VHL mutation. Neither of the two patients had polycythemia at the time of diagnosis. We demonstrate that the novel truncated mutation in HIF2A (Q557*) affects HIF-2α prolyl hydroxylation with its reduced ubiquitination but intact transcriptional activity, resulting in an activating effect. Both CNS-HB samples showed positive expression of VEGFR2/CA9/Glut1 and HAF. Our data support the unique central role of the VHL/HIF-2α signaling pathway in the molecular pathogenesis of CNS-HBs and show for the first time the presence of HIF2A mutations in sporadic HB.

Andreas Marx

Andreas Marx

Proteomic Analysis of the Ubiquitin Landscape in the Drosophila Embryonic Nervous System and the Adult Photoreceptor Cells.

BackgroundUbiquitination is known to regulate physiological neuronal functions as well as to be involved in a number of neuronal diseases. Several ubiquitin proteomic approaches have been developed during the last decade but, as they have been mostly applied to non-neuronal cell culture, very little is yet known about neuronal ubiquitination pathways in vivo.Methodology/Principal FindingsUsing an in vivo biotinylation strategy we have isolated and identified the ubiquitinated proteome in neurons both for the developing embryonic brain and for the adult eye of Drosophila melanogaster. Bioinformatic comparison of both datasets indicates a significant difference on the ubiquitin substrates, which logically correlates with the processes that are most active at each of the developmental stages. Detection within the isolated material of two ubiquitin E3 ligases, Parkin and Ube3a, indicates their ubiquitinating activity on the studied tissues. Further identification of the proteins that do accumulate upon interference with the proteasomal degradative pathway provides an indication of the proteins that are targeted for clearance in neurons. Last, we report the proof-of-principle validation of two lysine residues required for nSyb ubiquitination.Conclusions/SignificanceThese data cast light on the differential and common ubiquitination pathways between the embryonic and adult neurons, and hence will contribute to the understanding of the mechanisms by which neuronal function is regulated. The in vivo biotinylation methodology described here complements other approaches for ubiquitome study and offers unique advantages, and is poised to provide further insight into disease mechanisms related to the ubiquitin proteasome system.

Structural basis for the indispensable role of a unique zinc finger motif in LNX2 ubiquitination.

LNX (Ligand of Numb Protein-X) proteins, LNX1 and LNX2, are RING- and PDZ-based E3-ubiquitin ligases known to interact with Numb. Silencing of LNX2 has been reported to down-regulate WNT and NOTCH, two key signaling pathways in tumorigenesis. Here we report the identification of the domain boundary of LNX2 to confer its ubiquitination activity, its crystal structure along with functional studies. We show that the RING domain in LNX2 is flanked by two Zinc-binding motifs (Zn-RING-Zn), in which the N-terminal Zinc-binding motif adopts novel conformation. Although this motif follows the typical Cys2His2-type zinc finger configuration, it is devoid of any secondary structure and forms an open circle conformation, which has not been reported yet. This unique N-terminal Zn-finger motif is indispensable for the activity and stability of LNX2, as verified using mutational studies. The Zn-RING-Zn domain of LNX2 is a dimer and assumes a rigid elongated structure that undergoes autoubiquitination and undergoes N-terminal polyubiquitination. The ubiquitin chains consist of all seven possible isopeptide linkages. These results were validated using full-length LNX2. Moreover we have demonstrated the ubiquitination of cell fate determinant protein, Numb by LNX2. Our study provides a structural basis for the functional machinery of LNX2 and thus provides the opportunity to investigate suitable drug targets against LNX2.

Photoreceptor Specificity in the Light-Induced and COP1-Mediated Rapid Degradation of the Repressor of Photomorphogenesis SPA2 in Arabidopsis.

The Arabidopsis COP1/SPA E3 ubiquitin ligase is a key negative regulator that represses light signaling in darkness by targeting transcription factors involved in the light response for degradation. The COP1/SPA complex consists of COP1 and members of the four-member SPA protein family (SPA1-SPA4). Genetic analysis indicated that COP1/SPA2 function is particularly strongly repressed by light when compared to complexes carrying the other three SPAs, thereby promoting a light response after exposure of plants to extremely low light. Here, we show that the SPA2 protein is degraded within 5–15 min after exposure of dark-grown seedlings to a pulse of light. Phytochrome photoreceptors are required for the rapid degradation of SPA2 in red, far-red and also in blue light, whereas cryptochromes are not involved in the rapid, blue light-induced reduction in SPA2 protein levels. These results uncover a photoreceptor-specific mechanism of light-induced inhibition of COP1/SPA2 function. Phytochrome A (phyA) is required for the severe blue light responsiveness of spa triple mutants expressing only SPA2, thus confirming the important role of phyA in downregulating SPA2 function in blue light. In blue light, SPA2 forms a complex with cryptochrome 1 (cry1), but not with cryptochrome 2 (cry2) in vivo, indicating that the lack of a rapid blue light response of the SPA2 protein is only in part caused by a failure to interact with cryptochromes. Since SPA1 interacts with both cry1 and cry2, these results provide first molecular evidence that the light-regulation of different SPA proteins diverged during evolution. SPA2 degradation in the light requires COP1 and the COP1-interacting coiled-coil domain of SPA2, supporting that SPA2 is ubiquitinated by COP1. We propose that light perceived by phytochromes causes a switch in the ubiquitination activity of COP1/SPA2 from ubiquitinating downstream substrates to ubiquitinating SPA2, which subsequently causes a repression of COP1/SPA2 function.

Pharmacogenomics of Bortezomib in Multiple Myeloma Patients Reveals that the Ubiquitin Ligase SCF-Skp2 Promotes Drug Resistance

Abstract The ability to couple cancer patients with genetically-targeted therapeutic strategies remains a major challenge due to the heterogeneous and evolvable nature of individual tumors. This is particularly true of the molecularly diverse and complex disease multiple myeloma (MM). Clinical efficacy of the proteasome inhibitor (PI) Bortezomib (VelcadeTM) established the Ub+proteasome system (UPS) as a key target in MM. While the therapeutic benefit of PIs in MM remains unchallenged, many patients do not respond to therapy and drug resistance develops nearly uniformly. Despite marked therapeutic advances for most patients with MM, outcomes remain poor for 15% to 20% of patients presenting with high-risk disease defined by host-related and tumor-related factors and expert guidelines recommend enrollment in a clinical trial for the majority of patients who will ultimately fail approved therapies.The Skp1-Cul1-F-box protein (SCF) ubiquitin ligase SCFSkp2 promotes ubiquitination and degradation of the cell-cycle inhibitor p27. Increased SKP2 expression and reduced p27 levels are frequent in human cancers and are associated with therapeutic resistance and poor prognosis. SCFSkp2 ubiquitination activity is increased by Commd1, a copper metabolism MURR1 domain-containing 1 protein that binds Cul1 and by Cks1B that increases binding of phosphorylated p27 to Skp2. Bortezomib is a proteasome inhibitor that has remarkably improved multiple myeloma (MM) patient outcome, but dose-limiting toxicities and development of resistance limit its long-term utility. Here, we observed higher CUL1 and COMMD1 mRNA levels in Bortezomib-resistant patient MM cells and correlated higher CUL1, COMMD1, SKP2 and CKS1B levels with decreased survival. Genetic knockdown of CUL1 or COMMD1 disrupted the SCFSkp2 complex, stabilized p27, synergistically increased the generation of annexin-positive cells by bortezomib and enhanced the anti-myeloma effect of PIs against resistant cells. Chemical library screening identified a novel compound, DT204, that reduced Skp2 association with Cul1 and Commd1, inhibited p27 degradation and induced synergistic anti-MM activity combined with Bortezomib in patient tumor cells and chemoresistant stem cell-like cells to overcame Bortezomib resistance. Bortezomib treatment with DT204 also reduced the growth of xenotransplanted myeloma cells in mouse models and prolongs host survival. Taken together, our results demonstrate that the SCFSkp2 -p27 axis represents a major determinant in the ability of Bortezomib to induce apoptosis in MM and provide proof-of-concept for the combination of SCFSkp2 inhibitors with Bortezomib as a strategy to overcome Bortezomib resistance. Disclosures No relevant conflicts of interest to declare.

E4-Ubiquitin ligase Ufd2 stabilizes Yap8 and modulates arsenic stress responses independent of the U-box motif.

ABSTRACTAdaptation of Saccharomyces cerevisiae cells to arsenic stress is mediated through the activation of arsenic detoxification machinery by the Yap8 transcription factor. Yap8 is targeted by the ubiquitin proteasome system for degradation under physiological conditions, yet it escapes proteolysis in arsenic-injured cells by a mechanism that remains to be elucidated. Here, we show that Ufd2, an E4-Ubiquitin (Ub) ligase, is upregulated by arsenic compounds both at mRNA and protein levels. Under these conditions, Ufd2 interacts with Yap8 mediating its stabilization, thereby controlling expression of ACR3 and capacity of cells to adapt to arsenic injury. We also show that Ufd2 U-box domain, which is associated to the ubiquitination activity of specific ubiquitin ligases, is dispensable for Yap8 stability and has no role in cell tolerance to arsenic stress. Thus, our data disclose a novel Ufd2 role beyond degradation. This finding is further supported by genetic analyses showing that proteins belonging to Ufd2 proteolytic pathways, namely Ubc4, Rad23 and Dsk2, mediate Yap8 degradation.

Abstract 968: Co-occurring genomic alterations define major subsets of KRAS-mutant lung adenocarcinoma (LUAC) with distinct biology and therapeutic vulnerabilities

Abstract Introduction The development of more effective treatment strategies for LUAC bearing activating mutations in KRAS is hampered by the biological heterogeneity of KRAS-mutant tumors. The molecular underpinnings that drive this process are poorly characterized. Here, we implemented an integrated approach to the discovery of biologically distinct subsets of KRAS-mutant LUAC and explored their molecular vulnerabilities. Methods Our datasets consisted of 68 KRAS-mutant tumors from TCGA, 88 additional chemo-naive KRAS-mutant LUACs (PROSPECT and Chitale datasets) and 36 platinum-refractory LUACs from the BATTLE-2 clinical trial. Non-negative matrix factorization (NMF) consensus clustering was applied to RNASeq data as previously described. Signature enrichment was assessed using Gene Set Enrichment Analysis (GSEA). Results NMF consensus clustering identified three robust subsets of KRAS-mutant LUAC that were reproducible across diverse clinical datasets of early-stage, chemotherapy-naive and metastatic, chemo-refractory tumors. Distinct KRAS-mutant alleles were not differentially represented in the three subgroups (P = 0.3). In contrast, the subgroups were dominated, respectively, by co-occurring genetic events in STK11/LKB1 (termed the KL subgroup) (P = 1.03e−05), TP53 (KP) (P = 3.8e-06) and low expression of TTF1 coupled with frequent CDKN2A/B inactivation (KC) (P = 0.004 and P = 0.002). Distinct patterns of intracellular signaling were detected in the three subsets. KL tumors showed evidence of LKB1-AMPK pathway inactivation and adaptation to energetic, proteotoxic and oxidative stress, the latter exemplified by near ubiquitous inactivation of KEAP1 and up-regulation of a NRF2-driven antioxidant signature. KP tumors carried a higher somatic mutation load and were characterized by prominent inflammation and up-regulation of several immune checkpoint effector molecules, including PD-L1. KC tumors frequently displayed a GI-like differentiation program, suppression of MTORC1 signaling and elevated wild-type p53 transcriptional output. Using a large panel of KRAS-mutant NSCLC cell lines we detected co-mutation-dependent patterns of drug sensitivity. Specifically, KL cell lines showed enhanced sensitivity to several structurally distinct HSP90 inhibitors. These results were confirmed in panels of isogenic cell lines. Mechanistically, treatment with ganetespib resulted in concurrent degradation of several molecules with established role in supporting the fitness of LKB1-deficient cells. Conclusions Our work identifies three major subsets of KRAS-mutant LUAC - dominated by co-occurring genetic events - with distinct biology and therapeutic vulnerabilities. Citation Format: Ferdinandos Skoulidis, Lauren Byers, Lixia Diao, Vassiliki Papadimitrakopoulou, Pan Tong, Julie Izzo, Carmen Behrens, Humam Kadara, Edwin R. Parra, Jaime Rodriguez-Canales, Jianjun Zhang, Uma Giri, Jayanthi Gudikote, Maria Angelica Cortez, Chao Yang, You Hong Fan, Michael Peyton, Luc Girard, Kevin R. Coombes, Carlo Toniatti, Timothy P. Heffernan, Murim Choi, Garrett M. Frampton, Vincent Miller, John N. Weinstein, Roy S. Herbst, Kwok-Kin Wong, Jianhua Zhang, Padmanee Sharma, Gordon M. Mills, Waun Ki Hong, John D. Minna, James P. Allison, Andrew Futreal, Jing Wang, Ignacio Wistuba, John V. Heymach. Co-occurring genomic alterations define major subsets of KRAS-mutant lung adenocarcinoma (LUAC) with distinct biology and therapeutic vulnerabilities. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 968. doi:10.1158/1538-7445.AM2015-968

Cell regulation by phosphotyrosine-targeted ubiquitin ligases.

Three classes of E3 ubiquitin ligases, members of the Cbl, Hakai, and SOCS-Cul5-RING ligase families, stimulate the ubiquitination of phosphotyrosine-containing proteins, including receptor and nonreceptor tyrosine kinases and their phosphorylated substrates. Because ubiquitination frequently routes proteins for degradation by the lysosome or proteasome, these E3 ligases are able to potently inhibit tyrosine kinase signaling. Their loss or mutational inactivation can contribute to cancer, autoimmunity, or endocrine disorders, such as diabetes. However, these ligases also have biological functions that are independent of their ubiquitination activity. Here we review relevant literature and then focus on more-recent developments in understanding the structures, substrates, and pathways through which the phosphotyrosine-specific ubiquitin ligases regulate diverse aspects of cell biology.

Analysis of RNA Interference Lines Identifies New Functions of Maternally-Expressed Genes Involved in Embryonic Patterning inDrosophila melanogaster

Embryonic patterning in Drosophila melanogaster is initially established through the activity of a number of maternally expressed genes that are expressed during oogenesis. mRNAs from some of these genes accumulate in the posterior pole plasm of the oocyte and early embryo and localize further into RNA islands, which are transient ring-like structures that form around the nuclei of future primordial germ cells (pole cells) at stage 3 of embryogenesis. As mRNAs from several genes with known functions in anterior–posterior patterning and/or germ cell specification accumulate in RNA islands, we hypothesized that some other mRNAs that localize in this manner might also function in these developmental processes. To test this, we investigated the developmental functions of 51 genes whose mRNAs accumulate in RNA islands by abrogating their activity in the female germline using RNA interference. This analysis revealed requirements for ttk, pbl, Hip14, eIF5, eIF4G, and CG9977 for progression through early oogenesis. We observed dorsal appendage defects in a proportion of eggs produced by females expressing double-stranded RNA targeting Mkrn1 or jvl, implicating these two genes in dorsal–ventral patterning. In addition, posterior patterning defects and a reduction in pole cell number were seen in the progeny of Mkrn1 females. Because the mammalian ortholog of Mkrn1 acts as an E3 ubiquitin ligase, these results suggest an additional link between protein ubiquitination and pole plasm activity.

Protein-pyridinol thioester precursor for biosynthesis of the organometallic acyl-iron ligand in [Fe]-hydrogenase cofactor.

The iron-guanylylpyridinol (FeGP) cofactor of [Fe]-hydrogenase contains a prominent iron centre with an acyl-Fe bond and is the only acyl-organometallic iron compound found in nature. Here, we identify the functions of HcgE and HcgF, involved in the biosynthesis of the FeGP cofactor using structure-to-function strategy. Analysis of the HcgE and HcgF crystal structures with and without bound substrates suggest that HcgE catalyses the adenylylation of the carboxy group of guanylylpyridinol (GP) to afford AMP-GP, and subsequently HcgF catalyses the transesterification of AMP-GP to afford a Cys (HcgF)-S-GP thioester. Both enzymatic reactions are confirmed by in vitro assays. The structural data also offer plausible catalytic mechanisms. This strategy of thioester activation corresponds to that used for ubiquitin activation, a key event in the regulation of multiple cellular processes. It further implicates a nucleophilic attack onto the acyl carbon presumably via an electron-rich Fe(0)- or Fe(I)-carbonyl complex in the Fe-acyl formation.

The dominant negative ARM domain uncovers multiple functions of PUB13 in Arabidopsis immunity, flowering, and senescence.

Regulating the intensity and duration of immune responses is crucial to combat infections without deleterious side effects. Arabidopsis FLS2, the receptor for bacterial flagellin, activates immune signalling by association with its partner BAK1. Upon flagellin (flg22) perception, the plant U-box E3 ubiquitin ligases PUB12 and PUB13 complex with FLS2 in a BAK1-dependent manner, and ubiquitinate FLS2 for protein degradation, thereby down-regulating flagellin signalling. Domain deletion analysis indicates that the ARM domain of PUB13 interacts with the FLS2-BAK1 complex and is phosphorylated by BAK1. Overexpression of the PUB13 ARM domain alone inhibits flg22-induced FLS2-PUB13 association and PUB12/13-mediated FLS2 ubiquitination and degradation in Arabidopsis, suggesting that ectopic expression of the ARM domain in planta generates a dominant negative effect via blocking the ubiquitination activity. Similar to the pub12pub13 double mutant, transgenic plants expressing the PUB13 ARM domain display enhanced immune responses compared with wild-type plants. Moreover, PUB13ARM transgenic plants and the pub12pub13 mutant are more sensitive to stress-induced leaf senescence accompanied by elevated expression of stress-induced senescence marker genes. The resemblance between PUB13ARM transgenic plants and the pub12pub13 mutant provides genetic evidence that ectopic expression of the PUB ARM domain serves as an alternative approach to dissect the overlapping functions of closely related PUB genes.

Hypertension Causing Mutations in Cullin3 Decrease RhoA Ubiquitination and Sequester Substrate Adaptors

Cullin-Ring ubiquitin Ligases (CRL) regulate protein turnover by promoting ubiquitination of substrates. Impaired Cullin3 (Cul3) activity in vascular smooth muscle causes increased RhoA/Rho kinase signaling leading to augmented contraction. PHAII patients with de novo Cul3 mutations which cause skipping of exon 9 (Cul3Δ9) have early onset hypertension, but the mechanistic basis remains unclear. We hypothesize that Cul3Δ9 reduces RhoA ubiquitination and degradation. There was no defect in neddylation of Cul3Δ9. However, binding of Cul3Δ9 to Rbx1, which is necessary for delivering ubiquitin to the substrate was impaired. Consistent with this, ubiquitination of RhoA by Cul3Δ9 was significantly impaired compared with Cul3WT. Thus Cul3Δ9 exhibits impaired ubiquitination of Cul3 substrates. Interestingly, substrate recognition proteins Bacurd1, RhoBTB1 and KLHL3 bound more efficiently to Cul3Δ9 than Cul3WT. Moreover, we observed a significant increase in Cul3Δ9:Cul3WT heterodimers compared to Cul3Δ9:Cul3Δ9 homodimers. This is important because dimerization of CRL complexes is required for efficient ubiquitination activity. Thus Cul3Δ9 may act dominantly by sequestering substrate adaptors from the CRL3 complex or by directly interfering with Cul3WT. We conclude that Cul3Δ9 exhibits impaired ubiquitin ligase activity. In smooth muscle, this would increase the pool of activatable RhoA in response to contractile agonists. When combined with our previous data, we suggest that Cul3-mediated regulation of RhoA protein turnover controls both vascular function and arterial blood pressure. Patients carrying Cul3Δ9 mutations may exhibit elevated smooth muscle RhoA/Rho kinase activity significantly contributing to their hypertension.

Ziram and sodium N,N-dimethyldithiocarbamate inhibit ubiquitin activation through intracellular metal transport and increased oxidative stress in HEK293 cells.

Ubiquitin activating enzyme E1 plays a pivotal role in ubiquitin based protein signaling through regulating the initiating step of the cascade. Previous studies demonstrated that E1 is inhibited by covalent modification of reactive cysteines contained within the ubiquitin-binding groove and by conditions that increase oxidative stress and deplete cellular antioxidants. In this study, we determined the relative contribution of covalent adduction and oxidative stress to E1 inhibition produced by ziram and sodium N,N-dimethyldithiocarbamate (DMDC) in HEK293 cells. Although no dithiocarbamate-derived E1 adducts were identified on E1 using shotgun LC/MS/MS for either ziram or DMDC, both dithiocarbamates significantly decreased E1 activity, with ziram demonstrating greater potency. Ziram increased intracellular levels of zinc and copper, DMDC increased intracellular levels of only copper, and both dithiocarbamates enhanced oxidative injury evidenced by elevated levels of protein carbonyls and expression of heme oxygenase-1. To assess the contribution of intracellular copper transport to E1 inhibition, coincubations were performed with the copper chelator triethylenetetramine hydrochloride (TET). TET significantly protected E1 activity for both of the dithiocarbamates and decreased the associated oxidative injury in HEK293 cells as well as prevented dithiocarbamate-mediated lipid peroxidation assayed using an ethyl aracidonate micelle system. Because TET did not completely ameliorate intracellular transport of copper or zinc for ziram, TET apparently maintained E1 activity through its ability to diminish dithiocarbamate-mediated oxidative stress. Experiments to determine the relative contribution of elevated intracellular zinc and copper were performed using a metal free incubation system and showed that increases in either metal were sufficient to inhibit E1. To evaluate the utility of the HEK293 in vitro system for screening environmental agents, a series of additional pesticides and metals was assayed, and eight agents that produced a significant decrease and five that produced a significant increase in activated E1 were identified. These studies suggest that E1 is a sensitive redox sensor that can be modulated by exposure to environmental agents and can regulate downstream cellular processes.

Infection Tunes Stability of Key Molecules in Lung Diseases

efficiently fulfill a vast range of life processes. In the processes, proteins are dynamic in responding to the environmental changes. If a given protein is needed, de novo synthesis from mRNA will be augmented. Of note, a less emphasized but probably more efficacious way is to enhance the protein stability through preventing from degradation. If redundant, proteins will be promptly dumped out by degradation. Regulation of protein stability via degradation plays an important role in a vast range of life processes. Most proteins destined for turnover are via ubiquitin proteasomal/lysosomal degradation machinery. Protein turnover is not a randomly happened event but highly regulated and is a substrate-specific signal transduction cascade. Firstly, targeted proteins are posttranslational modified for recognition by the ubiquitin proteasomal/lysosomal degradation machinery. A well-studied prototype is phosphorylation that may directly affects ubiquitin proteasome system recognition of the substrate. Other posttranslational modifications, such as acetylation, methylation, or palmitoylation are important modifications involved in ubiquitination process as well. Secondly, the destined protein is ubiquitinated. Ubiquitination covalently adds a ubiquitin or polyubiquitin moieties to a lysine residue within the protein substrate. The process of ubiquitination is an enzymatic cascade involving E1 ubiquitin activation enzyme, E2 ubiquitin conjugation enzyme, and E3 ubiquitin ligase. Among the ubiquitin enzymes, E3 ubiquitin ligase recognizes the substrate that is substrate specific. Strikingly, the ubiquitin can be removed from the ubiquitinated protein by a family of enzymes called de ubiquitin enzymes. Finally, the ubiquitinated protein is then escorted into proteasome or lysosome for degradation. Bacterial infection fine-turns proteins involving in cell surface sensors, signal mediator proteins, and effect or proteins. For example, endotoxin lipopolysaccharide treatment or bacterial infection regulates IL22R, IL33, kinases, and TRAF proteins in lung epithelial cells. Epigenetic enzymes that control essential life process such as DNA replication and gene transcription are also deregulated in these models. Enzymes involved in active lung surfactant or other phospholipids synthesis, metabolism, and energy production are also regulated in this system. Understanding the molecular mechanism(s) of protein turnover in infectious lung disease has shed light on the development of new pharmaceutical therapeutic approaches. Successful results have been achieved to screen small molecules against novel molecular targets including E3 ubiquitin ligases in infectious lung diseases.

β-Hydroxy-β-methylbutyrate (HMB) normalizes dexamethasone-induced autophagy-lysosomal pathway in skeletal muscle.

Dexamethasone-induced muscle atrophy is due to an increase in protein breakdown and a decrease in protein synthesis, associated with an over-stimulation of the autophagy-lysosomal pathway. These effects are mediated by alterations in IGF-1 and PI3K/Akt signaling. In this study, we have investigated the effects of β-Hydroxy-β-methylbutyrate (HMB) on the regulation of autophagy and proteosomal systems. Rats were treated during 21 days with dexamethasone as a model of muscle atrophy. Co-administration of HMB attenuated the effects promoted by dexamethasone. HMB ameliorated the loss in body weight, lean mass and the reduction of the muscle fiber cross-sectional area (shrinkage) in gastrocnemius muscle. Consequently, HMB produced an improvement in muscle strength in the dexamethasone-treated rats. To elucidate the molecular mechanisms responsible for these effects, rat L6 myotubes were used. In these cells, HMB significantly attenuated lysosomal proteolysis induced by dexamethasone by normalizing the changes observed in autophagosome formation, LC3 II, p62 and Bnip3 expression after dexamethasone treatment. HMB effects were mediated by an increase in FoxO3a phosphorylation and concomitant decrease in FoxO transcriptional activity. The HMB effect was due to the restoration of Akt signaling diminished by dexamethasone treatment. Moreover, HMB was also involved in the regulation of the activity of ubiquitin and expression of MurF1 and Atrogin-1, components of the proteasome system that are activated or up-regulated by dexamethasone. In conclusion, in vivo and in vitro studies suggest that HMB exerts protective effects against dexamethasone-induced muscle atrophy by normalizing the Akt/FoxO axis that controls autophagy and ubiquitin proteolysis.

Src phosphorylation converts Mdm2 from a ubiquitinating to a neddylating E3 ligase

Murine double minute-2 protein (Mdm2) is a multifaceted phosphorylated protein that plays a role in regulating numerous proteins including the tumor suppressor protein p53. Mdm2 binds to and is involved in conjugating either ubiquitin or Nedd8 (Neural precursor cell expressed, developmentally down-regulated 8) to p53. Although regulation of the E3 ubiquitin activity of Mdm2 has been investigated, regulation of the neddylating activity of Mdm2 remains to be defined. Here we show that activated c-Src kinase phosphorylates Y281 and Y302 of Mdm2, resulting in an increase in Mdm2 stability and its association with Ubc12, the E2 enzyme of the neddylating complex. Mdm2-dependent Nedd8 conjugation of p53 results in transcriptionally inactive p53, a process that is reversed with a small molecule inhibitor to either Src or Ubc12. Thus, our studies reveal how Mdm2 may neutralize and elevate p53 in actively proliferating cells and also provides a rationale for using therapies that target the Nedd8 pathway in wild-type p53 tumors.

Frataxin inactivation leads to steroid deficiency in flies and human ovarian cells

Friedreich ataxia (FA), the most common inherited autosomal-recessive ataxia in Caucasians, is characterized by progressive degeneration of the central and peripheral nervous system, hypertrophic cardiomyopathy and increased incidence of diabetes. FA is caused by a GAA repeat expansion in the first intron of the gene encoding frataxin, an evolutionarily conserved mitochondrial protein, which results in decreased gene expression. Ubiquitous inactivation of the fly frataxin ortholog dfh blocks the transition from larval to pupal stages. In this study, we show that this phenotype is due to ecdysteroid deficiency and that feeding larvae with the 20-hydroxyecdysone steroid hormone rescues this developmental blockage. In mammals, adrenodoxin, the ferredoxin FDX1, is an Fe-S-containing protein essential for the synthesis of various steroid hormones. We show here that the two fly ferredoxins, Fdxh and Fdxh2 (encoded by CG1319), are also involved in steroidogenesis. This provides a potent mechanism by which frataxin, known to be involved in Fe-S cluster biosynthesis, could affect steroidogenesis through reduced ferredoxin activity. Finally, we show that frataxin inactivation decreases progesterone synthesis in human KGN ovarian granulosa cells. Thus, the involvement of frataxin in steroid synthesis appears to be a conserved function of the protein from flies to human and our data suggest that steroidogenesis could be affected in FA patients.

Long-lived species have improved proteostasis compared to phylogenetically-related shorter-lived species

Our previous studies have shown that the liver from Naked Mole Rats (NMRs), a long-lived rodent, has increased proteasome activity and lower levels of protein ubiquitination compared to mice. This suggests that protein quality control might play a role in assuring species longevity. To determine whether enhanced proteostasis is a common mechanism in the evolution of other long-lived species, here we evaluated the major players in protein quality control including autophagy, proteasome activity, and heat shock proteins (HSPs), using skin fibroblasts from three phylogenetically-distinct pairs of short- and long-lived mammals: rodents, marsupials, and bats. Our results indicate that in all cases, macroautophagy was significantly enhanced in the longer-lived species, both at basal level and after induction by serum starvation. Similarly, basal levels of most HSPs were elevated in all the longer-lived species. Proteasome activity was found to be increased in the long-lived rodent and marsupial but not in bats. These observations suggest that long-lived species may have superior mechanisms to ensure protein quality, and support the idea that protein homeostasis might play an important role in promoting longevity.

Analysis of a temperature-sensitive mutation in Uba1: Effects of the click reaction on subsequent immunolabeling of proteins involved in DNA replication

In our previous study, a Met-to-Ile substitution at amino acid 256 in the catalytic domain of Uba1 was determined in temperature-sensitive CHO-K1 mutant tsTM3 cells, which exhibited chromosomal instability and cell-cycle arrest in the S to G2 phases with decreased DNA synthesis at the nonpermissive temperature, 39°C. Mutant cells were also characterized by a significant decrease of Uba1 in the nucleus with decreased ubiquitination activity at 39°C. Defects of ubiquitination activity in the nucleus resulted in an inappropriate balance between Cdt1 and geminin, a licensing factor of DNA replication and its inhibitor. In the present study, we found that the Cu(I)-catalyzed [3+2] cycloaddition (click) reaction inhibits the subsequent indirect immunolabeling of Cdt1 but allows for the detection of PCNA with nascent DNA. Using a procedure without the click reaction, we also demonstrated that Cdt1 remained close to active replication sites in tsTM3 cells at the nonpermissive temperature. Analysis of genome replication by DNA fiber spreading revealed that DNA synthesis continues for at least 10h after incubation at 39°C, suggesting that impaired ubiquitination in the nucleus, caused by the defect of Uba1, affected DNA replication only after a long delay.