Ubiquitin-proteasome System-mediated Degradation Research Articles

A Novel BCR-ABL1 Degrader, Ubx-362, Can Overcome Resistance By BCR-ABL1 Kinase Domain Mutations in Chronic Myeloid Leukemia

Background Chronic Myeloid Leukemia (CML) is a clonal hematopoietic stem cell disorder characterized by the presence of the BCR::ABL1 fusion gene. Tyrosine kinase inhibitors (TKIs) have revolutionized CML treatment, providing remarkable responses in patients. However, resistance mutations within the BCR-ABL1 kinase domain can emerge, leading to treatment failure and disease progression. Method To overcome TKI resistance, we developed a novel BCR-ABL1 degrader (UBX-362), which is a proteolysis-targeting chimera (PROTAC), and investigated its therapeutic potential using several Ba/F3 cell lines expressing various BCR-ABL1 mutations. UBX-362 is designed to target BCR-ABL1 through the ubiquitin-proteasome system-mediated degradation. Proliferation assays (IC 50) were performed using the Cell Titer-Glo assay, and western blotting was conducted to measure the degradation levels of the BCR-ABL1 protein in the tested cell lines. For the in vivo xenograft mice experiments, 5 × 10 6 K562 cells were subcutaneously injected into Balb/c nude mice. UBX-362 will be orally administered at doses of 5 or 20 mg/kg, and dasatinib or ponatinib will be administered at a dose of 20 mg/kg once daily for 21 days. Results We identified potent and orally available PROTACs, among which UBX-362 exhibited superior degradation activity against both wild-type (WT) and mutant BCR-ABL1 proteins in various Ba/F3 BCR-ABL1 kinase domain mutant-expressing cell lines. In vitro experiments showed that UBX-362 effectively degrades WT and various mutant BCR-ABL1 proteins. The levels of WT BCR-ABL1 protein were measured by western blotting, yielding a half-maximal degradation concentration of UBX-362 at ~1.78 nM. Furthermore, the DC 50 of UBX-362 against E255K, V299L, F317L, and F359C mutant BCR-ABL1 proteins were in the single-digit nM range (6.395nM, 7.679nM, 4.635nM, and 5.688nM, respectively). While UBX-362's anti-proliferative activity (~2.562nM) was not found to be superior to dasatinib (~0.378nM), it still demonstrated a strong anti-proliferative effect in the CML cell line K562 and various BCR-ABL1 mutant-expressing Ba/F3 cell lines. Specifically, the IC 50 of UBX-362 was 2.477nM in the Ba/F3 WT cell line. An in vivo anti-cancer efficacy study is currently underway in the K562 xenograft mouse model to compare its effectiveness with dasatinib or ponatinib. Conclusion The findings from this pre-clinical study show the possibility of clinical development of a BCR-ABL1 degrader. And, UBX-362 can overcome resistance in CML patients with BCR-ABL1 mutations and can be a good candidate for further clinical development.

Novel Orally Available Degrader to Target Lck in T-Cell Acute Lymphoblastic Leukemia

Background T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive blood cancer that develops from abnormal growth of immature T-cell progenitors and constitutes about 15% of all pediatric ALL cases. Despite an overall survival rate of 80% in pediatric T-ALL patients, 20% of patients eventually experience relapse or succumb to refractory disease. Therefore, novel therapies for refractory/relapsed T-ALL patients are urgently needed, and they are currently being developed in clinical and preclinical studies. Based on recent literature, the over-activation of preTCR-LCK signaling has been revealed as a highly effective therapeutic target in T-ALL, with LCK being identified as a therapeutic vulnerability in ~44% of T-ALL cases. The LCK inhibitor, dasatinib, exhibited an anti-leukemia effect in a subset of T-ALL cases but was transient in most cases. Therefore, the development of new agents capable of sustaining inhibition of LCK signaling is needed. Method To validate the effects of the LCK degrader UBX-363, both in vitro and in vivo tests were conducted using dasatinib-sensitive HSB-2 cells (which have higher LCK activity compared to other T-ALL cell lines) and dasatinib-insensitive CCRF-CEM cells. UBX-363 is designed to target LCK through ubiquitin-proteasome system-mediated degradation. Cell proliferation was assessed using detection methods such as the CellTiter-Glo assay, and the degradation activity of the LCK protein in the tested cell lines was evaluated by immunoblotting. To examine the in vivo antitumor effects of UBX-363 or dasatinib, we performed HSB-2 and CCRF-CEM xenograft murine models. CB17/SCID mice were subcutaneously inoculated with 1x10 7 HSB-2 and 5x10 6 CCRF-CEM cells. UBX-363 was orally administered at doses of 0.2, 1, or 5 mg/kg (for HSB-2) and 2.5, 5, or 10 mg/kg (for CCRF-CEM), while dasatinib was orally administered at doses of 5 mg/kg (for HBS-2) and 10 mg/kg (for CCRF-CEM) once daily for 12 days. Results UBX-363 showed superior degradation activity against LCK proteins in the subnanomolar or single-digit nanomolar range of half-maximal degradation concentration in HSB-2 and CCRF-CEM cell lines (0.478 nM and 1.502 nM, respectively). In comparison to the LCK inhibitors dasatinib (~0.307 nM) and saracatinib (~17.41 nM), UBX-363 (~0.121 nM) demonstrated slightly superior anti-proliferative efficacy in HSB-2 cells. In CCRF-CEM cells, UBX-363 exhibited prominent anti-proliferative activity at lower treatment concentrations compared to the other LCK inhibitors (UBX-363: 11.12 nM, dasatinib: 3483 nM, saracatinib: 4383 nM). Furthermore, UBX-363 demonstrated faster tumor regression than dasatinib in the HSB-2 xenograft models. In the CCRF-CEM xenograft model, UBX-363 effectively suppressed tumor growth in a dose-dependent manner, whereas dasatinib showed no inhibitory effect on tumor growth. After 12 days of UBX-363 dosing on CCRF-CEM xenograft mice, the levels of LCK protein in tumor tissue significantly reduced. The degradation activity of UBX-363 on LCK showed about 100% reduction at dosages of 5 mg/kg and 10 mg/kg on day 12. Conclusion UBX-363 demonstrated strong anti-proliferative and anti-tumor effects, as well as degradation activity of LCK, in both in vitro and in vivo models of T-ALL. These results highlight UBX-363 as a promising therapeutic agent for LCK-activated T-ALL. In the future, we will conduct additional research to explore the advantages of this degrader, such as its ability to disrupt scaffolding of LCK, over LCK small molecule inhibitors.

OGT/HIF-2α axis promotes the progression of clear cell renal cell carcinoma and regulates its sensitivity to ferroptosis

O-GlcNAc transferase (OGT) acts in the development of various cancers, but its role in clear cell renal cell carcinoma (ccRCC) remains unclear. In this study, we found that OGT was upregulated in ccRCC and this upregulation was associated with a worse survival. Moreover, OGT promoted the proliferation, clone formation, and invasion of VHL-mutated ccRCC cells. Mechanistically, OGT increased the protein level of hypoxia-inducible factor-2α (HIF-2α) (the main driver of the clear cell phenotype) by repressing ubiquitin‒proteasome system-mediated degradation. Interestingly, the OGT/HIF-2α axis conferred ccRCC a high sensitivity to ferroptosis. In conclusion, OGT promotes the progression of VHL-mutated ccRCC by inhibiting the degradation of HIF-2α, and agents that can modulate the OGT/HIF-2α axis may exert therapeutic effects on mutated VHL ccRCC.

Oncogenic Activities of Tribbles1 (TRIB1) Pseudokinase Overexpressed in GBM are Mediated by Protein-Protein Interactions

Glioblastoma (GBM) is the most aggressive form of glioma with a low 5-year survival rate. The current treatments are inadequate and crippled by therapy resistance. Therefore, there is an unmet need to identify druggable therapeutic targets in GBM. In this study we identified TRIB1, a Ser/Thr pseudokinase that acts as a scaffold to initiate Ubiquitin Proteasome System-mediated degradation of its substrates. We and others have found that TRIB1 activates the canonical MAPK and Akt signaling cascades. Previous reports also suggest that TRIB1 contributes to chemotherapy resistance in various cancers. Therefore, we evaluated oncogenic roles of TRIB1 in GBM cells and its contribution to therapy resistance. Patient-centered reverse translational approach was utilized to identify novel therapeutic targets. To this end, TRIB1 was identified by statistical association (Cox regression analysis) of the patient-derived gene expression profiling data publicly available from TCGA GBM cohort. TRIB1 was functionally validated in vitro by generating stable overexpression cell lines (patient-derived) by antibiotic selection. Conditional knockdown of TRIB1 was achieved by doxycycline induction. Protein-protein interactions were evaluated by co-immunoprecipitation. Protein levels were detected by western blotting. Changes in tumor volume and overall survival (OS) were calculated. The mRNA profiling of TCGA GBM cohort revealed that increased TRIB1 gene expression was associated with worse OS of GBM patients [HR = 1.3 (1.0-1.5); P = 0.019]. The same analyses in our institutional cohort revealed a similar association. Mice bearing TRIB1 transgene overexpressing tumors had the increased tumor volume and shorter OS compared to empty vector control at the end of experiment. Overexpression of TRIB1 increased the phosphorylation/activation of ERK and Akt in patient-derived primary cell lines. Akt but not ERK activation was decreased after TRIB1 knockdown. TRIB1 bound directly to ERK and Akt in these cells. TRIB1 also formed a complex with p53, COP1 and HDAC1 in patient-derived primary cell lines. This protein-protein interaction was independent of TP53 mutation status. Our data suggest that TRIB1 overexpressed in GBM executes various oncogenic functions through interaction with different proteins. Activating ERK signaling, can induce cell proliferation. Similarly, by activating Akt it can cause prosurvival effects. Finally, by associating with HDAC1 and COP1, TRIB1 can modulate p53 function. All these protein-protein interactions ultimately contribute to chemoradiotherapy resistance in GBM cells. We are currently developing small molecule inhibitors targeting the above-mentioned interactions of TRIB1 to overcome therapeutic resistance.

Balanced activities of Hsp70 and the ubiquitin proteasome system underlie cellular protein homeostasis.

To counteract proteotoxic stress and cellular aging, protein quality control (PQC) systems rely on the refolding, degradation and sequestration of misfolded proteins. In Saccharomyces cerevisiae the Hsp70 chaperone system plays a central role in protein refolding, while degradation is predominantly executed by the ubiquitin proteasome system (UPS). The sequestrases Hsp42 and Btn2 deposit misfolded proteins in cytosolic and nuclear inclusions, thereby restricting the accessibility of misfolded proteins to Hsp70 and preventing the exhaustion of limited Hsp70 resources. Therefore, in yeast, sequestrase mutants show negative genetic interactions with double mutants lacking the Hsp70 co-chaperone Fes1 and the Hsp104 disaggregase (fes1Δ hsp104Δ, ΔΔ) and suffering from low Hsp70 capacity. Growth of ΔΔbtn2Δ mutants is highly temperature-sensitive and results in proteostasis breakdown at non-permissive temperatures. Here, we probed for the role of the ubiquitin proteasome system in maintaining protein homeostasis in ΔΔbtn2Δ cells, which are affected in two major protein quality control branches. We show that ΔΔbtn2Δ cells induce expression of diverse stress-related pathways including the ubiquitin proteasome system to counteract the proteostasis defects. Ubiquitin proteasome system dependent degradation of the stringent Hsp70 substrate firefly Luciferase in the mutant cells mirrors such compensatory activities of the protein quality control system. Surprisingly however, the enhanced ubiquitin proteasome system activity does not improve but aggravates the growth defects of ΔΔbtn2Δ cells. Reducing ubiquitin proteasome system activity in the mutant by lowering the levels of functional 26S proteasomes improved growth, increased refolding yield of the Luciferase reporter and attenuated global stress responses. Our findings indicate that an imbalance between Hsp70-dependent refolding, sequestration and ubiquitin proteasome system-mediated degradation activities strongly affects protein homeostasis of Hsp70 capacity mutants and contributes to their severe growth phenotypes.

TRIBBLES1 (TRIB1) Pseudokinase Induces Treatment Resistance in GBM by Promoting Survival of Tumor Cells through the Akt Pathway

<h3>Purpose/Objective(s)</h3> Glioblastoma (GBM; WHO grade IV) is the most aggressive form of glioma with a dismal 5-year survival rate. The current therapies are largely ineffective and therapy resistance is commonly encountered. Therefore, there is an urgent need to identify druggable therapeutic targets in GBM. In this study we identified TRIB1, a Ser/Thr pseudokinase that functions as a scaffold to initiate Ubiquitin Proteasome System-mediated degradation of target proteins in the cell. It has also been shown to activate the MAPK and Akt signaling cascades in various cell types. Reports also suggest that TRIB1 contributes to chemotherapy resistance in various cancers. Therefore, we evaluated the role of TRIB1 in GBM cell proliferation/ survival and its contribution to therapy resistance. <h3>Materials/Methods</h3> We utilized patient-centered reverse translational approach to identify novel therapeutic targets. To this end, TRIB1 was identified by statistical association (Cox regression analysis) of the patient-derived mRNA profiling data publicly available from TCGA GBM cohort. TRIB1 was functionally validated <i>in vitro</i> by generating stable overexpression cell lines by antibiotic selection. TRIB1 was conditionally knocked down by doxycycline induction. Co-immunoprecipitation was performed to evaluate protein-protein interactions. Protein levels were detected by western blotting. Site-directed mutagenesis was performed to mutate specific amino acid. Patient derived primary cell lines overexpressing the wild type and mutant (W337A) Trib1 transgenes were injected intracranially to create tumors in nude mice. Changes in tumor volume and overall survival (OS) were determined. <h3>Results</h3> The mRNA profiling of TCGA GBM cohort revealed that increased <i>TRIB1</i> gene expression was associated with worse OS of GBM patients (HR=1.3 (1.0-1.5); P=0.019). Mice bearing TRIB1 transgene overexpressing tumors had highest tumor volume and lowest OS whereas mice bearing TRIB1-W337A tumors had the highest OS and lower tumor volume when measured. We also observed that overexpression of TRIB1 caused a decrease in apoptosis of primary GBM cell lines after RT and TMZ treatments <i>in vitro</i>. Overexpression of TRIB1 also increased the phosphorylation/activation of Akt in patient derived primary cell lines. Akt activation was similarly decreased after TRIB1 knockdown. TRIB1 bound directly to Akt in these cells. Following PI3K inhibition, Akt phosphorylation was detected to a greater level in TRIB1 overexpressing cells. <h3>Conclusion</h3> Our data suggest that heightened expression of TRIB1 in GBM cells contributes to RT/TMZ resistance by activating the prosurvival Akt signaling cascades. Therefore, targeting TRIB1 mediated Akt activation could provide a therapeutic benefit over targeting Akt directly, which may reduce Akt's oncogenic signal without risking the toxicity associated with available Akt- specific inhibitors.

Targeting TRIBBLES1 (TRIB1) Pseudokinase as a New Therapeutic Approach in GBM

Glioblastoma (GBM) (WHO grade IV) is the most aggressive form of glioma with a 5-year survival rate of 5%. The current therapeutic regimen involves maximal safe surgical resection followed by radiation therapy and concurrent and adjuvant TMZ (Stupp protocol). Due to dismal survival, there is an urgent need to identify druggable therapeutic targets in GBM for drug development. In this study we identified TRIB1, a Ser/Thr pseudokinase that acts as a scaffold protein to initiate Ubiquitin Proteasome System-mediated degradation of target proteins in the cell. It has also been shown to activate MAPK and Akt signaling in various cell types. Reports suggest that TRIB1 also contributes towards chemotherapy resistance in NSCLC and CRC.We utilized a patient-centered reverse translational approach to identify novel therapeutic targets. TRIB1 was identified by statistical association (Cox regression analysis) and logic-based network analyses of the patient-derived methylation data generated using EPIC methylation array. TRIB1 was functionally validated in vitro by overexpression and knockdown approaches. Stable cell lines were generated by puromycin selection and cell sorting. Site-directed mutagenesis was used to create specific amino acid mutations. Co-immunoprecipitation was performed to identify protein-protein interaction. Protein levels were detected by western blotting.The global methylation analysis on an institutional GBM cohort revealed that TRIB1 promoter methylation was associated with better OS of GBM patients (HR = 0.81 (0.62-1.05); P = 0.11). We also observed that overexpression of TRIB1 caused a decrease in apoptosis of patient derived GBM cell lines after radiation and TMZ treatment. Overexpression of TRIB1 also increased the phosphorylation of ERK in patient derived cell lines. TRIB1 directly bound to MEK1 in these cell lines and the disruption of its biding by site-directed mutation led to reduced TRIB1-MEK1 interaction.Our data suggest that TRIB1 is a potential therapeutic target for GBM therapy as targeting TRIB1 could promote treatment sensitizing glioma cells to RT/TMZ. Targeting MEK1 binding site on TRIB1 could also reduce MAPK oncogenic signaling in these cells thereby providing an additional treatment strategy for GBM.K. Singh: None. C. Han: None. J.L. Fleming: None. J. McElroy: None. E.H. Bell: None. P. Robe: None. S.J. Haque: None. A. Chakravarti: None.

Targeting TRIBBLES1 (TRIB1) Pseudokinase in GBM: A New Therapeutic Strategy

GBM (WHO grade IV) is the most aggressive form of glioma with a 5-year survival rate of 5%. The current therapeutic regimen involves radiation therapy and concurrent and adjuvant TMZ (Stupp protocol). Due to dismal survival outcomes, there is an urgent need to identify druggable therapeutic targets in GBM for drug development. In this study we identified TRIB1, a Ser/Thr pseudokinase that acts as a scaffold protein to initiate Ubiquitin Proteasome System-mediated degradation of target proteins in the cell. TRIB1 mRNA and protein have been found upregulated in several cancers (e.g. NSCLC, prostate etc.). It has also been shown to contribute towards chemotherapy resistance in NSCLC and CRC. It has also been observed that COP1, a TRIB1 associated E3-ligase is also upregulated in glioma cells. We utilized a patient-centered reverse translational approach to identify novel therapeutic targets. TRIB1 was identified by statistical association (Cox regression analysis) and logic-based network analyses of the patient-derived methylation data generated using EPIC methylation array. TRIB1 was functionally validated in vitro by overexpression and knockdown approaches. For knockdown of TRIB1, a doxycycline inducible system was used. Stable cell lines were generated by puromycin selection and cell sorting. Protein levels were detected by western blotting. The global methylation analysis on a Utrecht GBM cohort revealed that TRIB1 promoter methylation was associated with better OS of GBM patients (HR = 0.81 (0.62-1.05); p = 0.11). We also observed that overexpression of TRIB1 caused a decrease in apoptosis of PDX GBM cell lines after radiation exposure and TMZ treatment. Overexpression of TRIB1 also increased the phosphorylation of ERK and Akt in PDX cell lines. However, in TRIB1 knockdown cell lines only phosphorylation of Akt was decreased. It was also observed that TRIB1 levels were upregulated in p53-mutant cell lines suggesting a correlation between TRIB1 and p53. TRIB1 is a potential therapeutic target for GBM therapy as targeting TRIB1 could promote radio-sensitivity of p53-mutant glioma cells by enhancing TMZ action. Targeting TRIB1 would also reduce oncogenic signaling in these cells; overall providing an additional treatment strategy for GBM.

CSN6 aggravates Ang II-induced cardiomyocyte hypertrophy via inhibiting SIRT2

The constitutive photomorphogenic 9 (COP9) signalosome complex subunit 6 (COPS6/CSN6) is crucial for structural integrity of the COP9 signalosome complex. CSN6 participates in various aspects of cancer progression, but its role in hypertrophic cardiomyopathy is not clear. Here, we found that the expression of CSN6 was increased in Angiotensin II (Ang II)-induced hypertrophic mice hearts and neonatal rat cardiomyocytes (NRCMs). Inhibition of CSN6 decreased the cardiomyocyte size and fetal genes’ expression in Ang II-induced hypertrophic NRCMs, while overexpression of CSN6 aggravated Ang II-induced myocardial hypertrophy. Moreover, we demonstrated that the pro-hypertrophic function of CSN6 was mediated by SIRT2, which acts as a cardioprotective factor in pathological cardiac hypertrophy. CSN6 inhibited the expression of SIRT2, and re-expression of SIRT2 attenuated the myocardial hypertrophy caused by CSN6 overexpression. Further investigation discovered that CSN6 suppressed the expression of SIRT2 via up-regulating Nkx2.2, a transcription suppressor of SIRT2. Mechanistically, CSN6 blocked the ubiquitin proteasome system-mediated degradation of Nkx2.2 protein by interacting with it and inhibiting its ubiquitination directly in cardiomyocytes. Finally, our data showed that CSN6 was partially dependent on the stabilization of Nkx2.2 protein to inhibit SIRT2 and promote myocardial hypertrophy. Overall, our study identified CSN6 as a pro-hypertrophic deubiquitinase, and CSN6 inhibition may be a potential treatment strategy for heart failure.

CSN5 upregulates glycolysis to promote hepatocellular carcinoma metastasis via stabilizing the HK2 protein

Aerobic glycolysis promotes metastasis and correlates with poorer clinical outcomes in hepatocellular carcinoma (HCC), but the controllers and mechanisms of abnormally activated glycolysis remain unclear. Herein, we demonstrated that the fifth component of the constitutive photomorphogenic 9 (COP9) signalosome complex (COPS5/CSN5) was a controller of glycolysis. For the first time, we found that CSN5 could influence the expression of glycolytic metabolism-associated proteins, especially hexokinase 2 (HK2), a glycolytic rate-limiting enzyme. In addition, we found that CSN5 was associated with HK2 overexpression in HCC tissues. Silencing CSN5 expression caused a decrease in the level of the HK2 protein, glucose uptake, glycolysis capacity and the production of glycolytic intermediates in HCC cells. Re-expression of HK2 rescued the decreased glycolytic flux induced by CSN5 knockdown, whereas inhibition of HK2 alleviated CSN5-enhanced glycolysis. Functionally, CSN5 regulated HCC cell invasion and metastasis via HK2-mediated glycolysis. Mechanistically, we demonstrated that CSN5 attenuated the ubiquitin-proteasome system-mediated degradation of HK2 through its deubiquitinase function. Inhibition of CSN5 kinase activity by curcumin decreased HK2 protein expression and glycolysis, repressed the metastasis of HCC cells in vitro and in vivo, and prolonged the survival time of tumor-bearing nude mice. Overall, our study identified CSN5 as a controller of glycolysis, and it may be a potential treatment target for HCC.

Involvement of Parkin in the ubiquitin proteasome system-mediated degradation of N-type voltage-gated Ca2+ channels.

N-type calcium (CaV2.2) channels are widely expressed in the brain and the peripheral nervous system, where they play important roles in the regulation of transmitter release. Although CaV2.2 channel expression levels are precisely regulated, presently little is known regarding the molecules that mediate its synthesis and degradation. Previously, by using a combination of biochemical and functional analyses, we showed that the complex formed by the light chain 1 of the microtubule-associated protein 1B (LC1-MAP1B) and the ubiquitin-proteasome system (UPS) E2 enzyme UBE2L3, may interact with the CaV2.2 channels promoting ubiquitin-mediated degradation. The present report aims to gain further insights into the possible mechanism of degradation of the neuronal CaV2.2 channel by the UPS. First, we identified the enzymes UBE3A and Parkin, members of the UPS E3 ubiquitin ligase family, as novel CaV2.2 channel binding partners, although evidence to support a direct protein-protein interaction is not yet available. Immunoprecipitation assays confirmed the interaction between UBE3A and Parkin with CaV2.2 channels heterologously expressed in HEK-293 cells and in neural tissues. Parkin, but not UBE3A, overexpression led to a reduced CaV2.2 protein level and decreased current density. Electrophysiological recordings performed in the presence of MG132 prevented the actions of Parkin suggesting enhanced channel proteasomal degradation. Together these results unveil a novel functional coupling between Parkin and the CaV2.2 channels and provide a novel insight into the basic mechanisms of CaV channels protein quality control and functional expression.

Proteolytic degradation and potential role of onconeural protein cdr2 in neurodegeneration.

Cerebellar degeneration-related protein 2 (cdr2) is expressed in the central nervous system, and its ectopic expression in tumor cells of patients with gynecological malignancies elicits immune responses by cdr2-specific autoantibodies and T lymphocytes, leading to neurological symptoms. However, little is known about the regulation and function of cdr2 in neurodegenerative diseases. Because we found that cdr2 is highly expressed in the midbrain, we investigated the role of cdr2 in experimental models of Parkinson's disease (PD). We found that cdr2 levels were significantly reduced after stereotaxic injection of 1-methyl-4-phenylpyridinium (MPP+) into the striatum. cdr2 levels were also decreased in the brains of post-mortem PD patients. Using primary cultures of mesencephalic neurons and MN9D cells, we confirmed that MPP+ reduces cdr2 in tyrosine hydroxylase-positive dopaminergic neuronal cells. The MPP+-induced decrease of cdr2 was primarily caused by calpain- and ubiquitin proteasome system-mediated degradation, and cotreatment with pharmacological inhibitors of these enzymes or overexpression of calcium-binding protein rendered cells less vulnerable to MPP+-mediated cytotoxicity. Consequently, overexpression of cdr2 rescued cells from MPP+-induced cytotoxicity, whereas knockdown of cdr2 accelerated toxicity. Collectively, our findings provide insights into the novel regulatory mechanism and potentially protective role of onconeural protein during dopaminergic neurodegeneration.

Impact of Concanavalin-A-Mediated Cytoskeleton Disruption on Low-Density Lipoprotein Receptor-Related Protein-1 Internalization and Cell Surface Expression in Glioblastomas.

The low-density lipoprotein receptor-related protein 1 (LRP-1) is a multiligand endocytic receptor, which plays a pivotal role in controlling cytoskeleton dynamics during cancer cell migration. Its rapid endocytosis further allows efficient clearance of extracellular ligands. Concanavalin-A (ConA) is a lectin used to trigger in vitro physiological cellular processes, including cytokines secretion, nitric oxide production, and T-lymphocytes activation. Given that ConA exerts part of its effects through cytoskeleton remodeling, we questioned whether it affected LRP-1 expression, intracellular trafficking, and cell surface function in grade IV U87 glioblastoma cells. Using flow cytometry and confocal microscopy, we found that loss of the cell surface 600-kDa mature form of LRP-1 occurs upon ConA treatment. Consequently, internalization of the physiological α2-macroglobulin and the synthetic angiopep-2 ligands of LRP-1 was also decreased. Silencing of known mediators of ConA, such as the membrane type-1 matrix metalloproteinase, and the Toll-like receptors (TLR)-2 and TLR-6 was unable to rescue ConA-mediated LRP-1 expression decrease, implying that the loss of LRP-1 was independent of cell surface relayed signaling. The ConA-mediated reduction in LRP-1 expression was emulated by the actin cytoskeleton-disrupting agent cytochalasin-D, but not by the microtubule inhibitor nocodazole, and required both lysosomal- and ubiquitin-proteasome system-mediated degradation. Our study implies that actin cytoskeleton integrity is required for proper LRP-1 cell surface functions and that impaired trafficking leads to specialized compartmentation and degradation. Our data also strengthen the biomarker role of cell surface LRP-1 functions in the vectorized transport of therapeutic angiopep bioconjugates into brain cancer cells.

2‐Methoxyestradiol confers neuroprotection and inhibits a maladaptive HIF‐1α response after traumatic brain injury in mice

HIF-1α is pivotal for cellular homeostasis in response to cerebral ischemia. Pharmacological inhibition of HIF-1α may reduce secondary brain damage by targeting post-translational mechanisms associated with its proteasomal degradation and nuclear translocation. This study examined the neuroprotective effects of 2-methoxyestradiol (2ME2), the involved HIF-1α-dependent response, and alternative splicing in exon 14 of HIF-1α (HIF-1α∆Ex14) after traumatic brain injury (TBI) in mice. Intraperitoneal 2ME2 administration 30 min after TBI caused a dose-dependent reduction in secondary brain damage after 24 h. 2ME2 was physiologically tolerated, showed no effects on immune cell brain migration, and mitigated trauma-induced brain expression of neuropathologically relevant HIF-1α target genes encoding for Plasminogen activator inhibitor 1 and tumor necrosis factor alpha. Moreover, TBI-induced expression of pro-apoptotic BNIP3 was attenuated by 2ME2 treatment. Alternatively, spliced HIF-1α∆Ex14 was substantially up-regulated from 6 to 48 h after TBI. In vitro, nuclear location and gene transcription activity of HIF-1α∆Ex14 were impaired compared to full-length HIF-1α, but no effects on nuclear translocation of the transcriptional complex partner HIF-1β were observed. This study demonstrates that 2ME2 confers neuroprotection after TBI. While the role of alternatively spliced HIF-1α∆Ex14 remains elusive, the in vivo data provide evidence that inhibition of a maladaptive HIF-1α-dependent response contributes to the neuroprotective effects of 2ME2. We examined neuroprotective effects of 2-methoxyestradiol (2ME2) and the hypoxia-inducible factor 1-α (HIF-1α) response following traumatic brain injury in mice. Early 2ME2 administration reduced the secondary brain damage and neuronal HIF-1α probably involving ubiquitin proteasome system-mediated degradation. The up-regulation of neuropathological HIF-1α target genes and pro-apoptotic BNIP3 protein was attenuated. We propose that the inhibition of a maladaptive HIF-1α response may contribute to 2ME2-mediated neuroprotection.

SlNAC1, a stress‐related transcription factor, is fine‐tuned on both the transcriptional and the post‐translational level

The plant-specific NAC (NAM, ATAF1,2, CUC2) transcription factors play significant roles in diverse physiological processes. In this study, we determined the regulation of a stress-related tomato (Solanum lycopersicum) NAC1 (SlNAC1) transcription factor at both the transcriptional and the post-translational level. The SlNAC1 protein was found to be stable in the presence of proteasome-specific inhibitor MG132 or MG115 and ubiquitinated in plant cells, suggesting that the SlNAC1 is subject to the ubiquitin-proteasome system-mediated degradation. Deletion analysis identified a short segment of 10 amino acids (aa261-270) that was required for ubiquitin-proteasome system-mediated degradation, among which two leucine residues (L268 and L269) were critical for the protein instability of SlNAC1. Fusion of the degron (SlNAC1(191-270) ) containing these 10 amino acids to green fluorescent protein was found to be sufficient to trigger the degradation of the fusion protein. In addition, the SlNAC1 gene is strongly upregulated during Pseudomonas infection, while repression of the NAC1 ortholog in Nicotiana benthamiana resulted in enhanced susceptibility to Pseudomonas bacteria. These results suggest that rapid upregulation of the NAC1 gene resulting in more protein production is likely one of the strategies plants use to defend themselves against pathogen infection.

Prefoldin Subunits Are Protected from Ubiquitin-Proteasome System-mediated Degradation by Forming Complex with Other Constituent Subunits

The molecular chaperone prefoldin (PFD) is a complex comprised of six different subunits, PFD1-PFD6, and delivers newly synthesized unfolded proteins to cytosolic chaperonin TRiC/CCT to facilitate the folding of proteins. PFD subunits also have functions different from the function of the PFD complex. We previously identified MM-1α/PFD5 as a novel c-Myc-binding protein and found that MM-1α suppresses transformation activity of c-Myc. However, it remains unclear how cells regulate protein levels of individual subunits and what mechanisms alter the ratio of their activities between subunits and their complex. In this study, we found that knockdown of one subunit decreased protein levels of other subunits and that transfection of five subunits other than MM-1α into cells increased the level of endogenous MM-1α. We also found that treatment of cells with MG132, a proteasome inhibitor, increased the level of transfected/overexpressed MM-1α but not that of endogenous MM-1α, indicating that overexpressed MM-1α, but not endogenous MM-1α, was degraded by the ubiquitin proteasome system (UPS). Experiments using other PFD subunits showed that the UPS degraded a monomer of PFD subunits, though extents of degradation varied among subunits. Furthermore, the level of one subunit was increased after co-transfection with the respective subunit, indicating that there are specific combinations between subunits to be stabilized. These results suggest mutual regulation of protein levels among PFD subunits and show how individual subunits form the PFD complex without degradation.

224. Pyruvate Dehydrogenase Complex Deficiency Caused by the Ubiquitin-Proteasome System-Mediated Degradation in E1β Subunit

We describe a new case of pyruvate dehydrogenase (PDH) complex deficiency associated with the PDH E1|[beta]| subunit (PDHB) gene. The patient was a 5 year old girl with severe developmental delay, microcephaly and agenesis of the corpus callosum. She had mild hyperlactatemia and moderately elevated lactate levels in her cerebrospinal fluid. Her cultured skin fibroblasts demonstrated a 55% reduction in PDH activity and markedly decreased immunoreactivity for PDHB protein, compared to the activity, measured in fibroblasts from healthy controls. The sequence of the total cDNA rorresponding to the patient's E1|[alpha]| and PDHB gene revealed no pathological mutation. The relative expression level of PDHB mRNA (determined by quantitative polymerase chain reaction, qPCR) and the rate of transcription and translation of the PDHB gene were normal. In contrast, the rate of degradation of the patient's E1|[beta]| protein was decreased by the proteosome inhibitor MG132. These data indicate that PDH complex deficiency in this patient is due to enhanced ubiquitination of the E1|[beta]| protein, leading to its accelerated destruction.