Industry perspective on the nonclinical safety assessment of heterobifunctional degraders.

Targeted protein degraders (TPDs), which act through the ubiquitin proteasome system (UPS), are one of the newest small-molecule drug modalities. Since the initiation of the first clinical trial in 2019, investigating the use of ARV-110 in patients with cancer, the field has rapidly expanded. Recently, some theoretical absorption, distribution, metabolism, and excretion (ADME) and safety challenges have been posed for the modality. Using these theoretical concerns as a framework, the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ Consortium) Protein Degrader Working Group (WG) conducted two surveys to benchmark current preclinical practices for TPDs. Conceptually, the safety assessment of TPDs is the same as for standard small molecules; however, the techniques used, assay conditions/study endpoints, and timing of assessments might need to be modified to address differences in mode of action of the class.

Invited review: Muscle protein breakdown and its assessment in periparturient dairy cows.

Mobilization of body reserves including fat, protein, and glycogen is necessary to overcome phases of negative nutrient balance typical for high-yielding dairy cows during the periparturient period. Skeletal muscle, the largest internal organ in mammals, plays a crucial role in maintaining metabolic homeostasis. However, unlike in liver and adipose tissue, the metabolic and regulatory role of skeletal muscle in the adaptation of dairy cows to the physiological needs of pregnancy and lactation has not been studied extensively. The functional integrity and quality of skeletal muscle are maintained through a constant turnover of protein, resulting from both protein breakdown and protein synthesis. Thus, muscle protein breakdown (MPB) and synthesis are intimately connected and tightly controlled to ensure proper protein homeostasis. Understanding the regulation of MPB, the catabolic component of muscle turnover, and its assessment are therefore important considerations to provide information about the timing and extent of tissue mobilization in periparturient dairy cows. Based on animal models and human studies, it is now evident that MPB occurs via the integration of 3 main systems: autophagy-lysosomal, calpain Ca2+-dependent cysteine proteases, and the ubiquitin-proteasome system. These 3 main systems are interconnected and do not work separately, and the regulation is complex. The ubiquitin-proteasomal system is the most well-known cellular proteolytic system and plays a fundamental role in muscle physiology. Complete degradation of a protein often requires a combination of the systems, depending on the physiological situation. Determination of MPB in dairy cows is technically challenging, resulting in a relative dearth of information. The methods for assessing MPB can be divided into either direct or indirect measurements, both having their strengths and limitations. Available information on the direct measures of MPB primarily comes from stable isotopic tracer methods and those of indirect measurements from assessing expression and activity measures of the components of the 3 MPB systems in muscle biopsy samples. Other indirect approaches (i.e., potential indicators of MPB), including ultrasound imaging and measuring metabolites from muscle degradation (i.e., 3-methylhistidine and creatinine), seem to be applicable methods and can provide useful information about the extent and timing of MPB. This review presents our current understanding, including methodological considerations, of the process of MPB in periparturient dairy cows.

Proteomic Assessment of Extracellular Vesicles from Canine Tissue Explants as a Pipeline to Identify Molecular Targets in Osteosarcoma: PSMD14/Rpn11 as a Proof of Principle.

Osteosarcoma (OS) is a highly malignant bone tumour that has seen little improvement in treatment modalities in the past 30 years. Understanding what molecules contribute to OS biology could aid in the discovery of novel therapies. Extracellular vesicles (EVs) serve as a mode of cell-to-cell communication and have the potential to uncover novel protein signatures. In our research, we developed a novel pipeline to isolate, characterize, and profile EVs from normal bone and osteosarcoma tissue explants from canine OS patients. Proteomic analysis of vesicle preparations revealed a protein signature related to protein metabolism. One molecule of interest, PSMD14/Rpn11, was explored further given its prognostic potential in human and canine OS, and its targetability with the drug capzimin. In vitro experiments demonstrated that capzimin induces apoptosis and reduces clonogenic survival, proliferation, and migration in two metastatic canine OS cell lines. Capzimin also reduces the viability of metastatic human OS cells cultured under 3D conditions that mimic the growth of OS cells at secondary sites. This unique pipeline can improve our understanding of OS biology and identify new prognostic markers and molecular targets for both canine and human OS patients.

Functional In Vitro Assessment of VEGFA/NOTCH2 Signaling Pathway and pRB Proteasomal Degradation and the Clinical Relevance of Mucolipin TRPML2 Overexpression in Glioblastoma Patients.

Glioblastoma (GBM) is the most malignant glioma with an extremely poor prognosis. It is characterized by high vascularization and its growth depends on the formation of new blood vessels. We have previously demonstrated that TRPML2 mucolipin channel expression increases with the glioma pathological grade. Herein by ddPCR and Western blot we found that the silencing of TRPML2 inhibits expression of the VEGFA/Notch2 angiogenic pathway. Moreover, the VEGFA/Notch2 expression increased in T98 and U251 cells stimulated with the TRPML2 agonist, ML2-SA1, or by enforced-TRPML2 levels. In addition, changes in TRPML2 expression or ML2-SA1-induced stimulation, affected Notch2 activation and VEGFA release. An increased invasion capability, associated with a reduced VEGF/VEGFR2 expression and increased vimentin and CD44 epithelial-mesenchymal transition markers in siTRPML2, but not in enforced-TRPML2 or ML2-SA1-stimulated glioma cells, was demonstrated. Furthermore, an increased sensitivity to Doxorubicin cytotoxicity was demonstrated in siTRPML2, whereas ML2-SA1-treated GBM cells were more resistant. The role of proteasome in Cathepsin B-dependent and -independent pRB degradation in siTRPML2 compared with siGLO cells was studied. Finally, through Kaplan-Meier analysis, we found that high TRPML2 mRNA expression strongly correlates with short survival in GBM patients, supporting TRPML2 as a negative prognostic factor in GBM patients.

Deubiquitylating enzymes and drug discovery: emerging opportunities.

More than a decade after a Nobel Prize was awarded for the discovery of the ubiquitin-proteasome system and clinical approval of proteasome and ubiquitin E3 ligase inhibitors, first-generation deubiquitylating enzyme (DUB) inhibitors are now approaching clinical trials. However, although our knowledge of the physiological and pathophysiological roles of DUBs has evolved tremendously, the clinical development of selective DUB inhibitors has been challenging. In this Review, we discuss these issues and highlight recent advances in our understanding of DUB enzymology and biology as well as technological improvements that have contributed to the current interest in DUBs as therapeutic targets in diseases ranging from oncology to neurodegeneration.

Quantitative assessment of the degradation of aggregated TDP-43 mediated by the ubiquitin proteasome system and macroautophagy.

Amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions are neurodegenerative disorders that share the cytosolic deposition of TDP-43 (TAR DNA-binding protein 43) in the CNS. TDP-43 is well known as being actively degraded by both the proteasome and macroautophagy. The well-documented decrease in the efficiency of these clearance systems in aging and neurodegeneration, as well as the genetic evidence that many of the familial forms of TDP-43 proteinopathies involve genes that are associated with them, suggest that a failure of these protein degradation systems is a major factor that contributes to the onset of TDP-43-associated disorders. Here, we inserted preformed human TDP-43 aggregates in the cytosol of murine NSC34 and N2a cells in diffuse form and observed their degradation under conditions in which exogenous TDP-43 is not expressed and endogenous nuclear TDP-43 is not recruited, thereby allowing a time zero to be established in TDP-43 degradation and to observe its disposal kinetically and analytically. TDP-43 degradation was observed in the absence and presence of selective inhibitors and small interfering RNAs against the proteasome and autophagy. We found that cytosolic diffuse aggregates of TDP-43 can be distinguished in 3 different classes on the basis of their vulnerability to degradation, which contributed to the definition-with previous reports-of a total of 6 distinct classes of misfolded TDP-43 species that range from soluble monomer to undegradable macroaggregates. We also found that the proteasome and macroautophagy-degradable pools of TDP-43 are fully distinguishable, rather than in equilibrium between them on the time scale required for degradation, and that a significant crosstalk exists between the 2 degradation processes.-Cascella, R., Fani, G., Capitini, C., Rusmini, P., Poletti, A., Cecchi, C., Chiti, F. Quantitative assessment of the degradation of aggregated TDP-43 mediated by the ubiquitin proteasome system and macroautophagy.

Proteomic data reveal a physiological basis for costs and benefits associated with thermal acclimation.

Physiological adaptation through acclimation is one way to cope with temperature changes. Biochemical studies on acclimation responses in ectotherms have so far mainly investigated consequences of short-term acclimation at the adult stage and focussed on adaptive responses. Here, we assessed the consequences of rearing Drosophila melanogasterat low (12°C), benign (25°C) and high (31°C) temperatures. We assessed cold and heat tolerance and obtained detailed proteomic profiles of flies from the three temperatures. The proteomic profiles provided a holistic understanding of the underlying biology associated with both adaptive and non-adaptive temperature responses. Results show strong benefits and costs across tolerances: rearing at low temperature increased adult cold tolerance and decreased adult heat tolerance and vice versa with development at high temperatures. In the proteomic analysis, we were able to identify and quantify a large number of proteins compared with previous studies on ectotherms (1440 proteins across all replicates and rearing regimes), enabling us to extend the proteomic approach using enrichment analyses. This gave us detailed information on individual proteins, as well as pathways affected by rearing temperature, pinpointing potential mechanisms responsible for the strong costs and benefits of rearing temperature on functional phenotypes. Several well-known heat shock proteins, as well as proteins not previously associated with thermal stress, were among the differentially expressed proteins. Upregulation of proteasome proteins was found to be an important adaptive process at high-stress rearing temperatures, and occurs at the expense of downregulation of basal metabolic functions.

Assessment of cytokine-modulated proteasome activity.

This chapter presents two methods for assessment of proteasome function. The first is a modification of the standard fluorogenic peptide cleavage assay which takes into account the effect of ATP on proteasome activity. This method is described in both its macro and high throughput micro-assay forms. The second is the Proteasome Constitutive Immuno-Subunit (active site) ELISA or ProCISE method. ProCISE is a modification of active site directed probe analysis and allows for convenient differentiation between active constitutive and immuno-subunits. While the utility of measuring proteasome activity and its relationship to cytokine action and inflammation are clear, the assessment and interpretation is not always straightforward. Therefore, we also discuss the pitfalls of the standard fluorogenic assay, particularly in the interpretation of results obtained, and the advantages of the newer, ProCISE assay.

MG132 ameliorates kidney lesions by inhibiting the degradation of Smad7 in streptozotocin-induced diabetic nephropathy.

BACKGROUND: Smad7 is the main negative regulatory protein in the transforming growth factor-ß (TGF-ß) downstream signaling pathway, which plays an important role in diabetic nephropathy (DN) and may be related to the ubiquitin proteasome pathway (UPP). AIM: We investigated the role of UPP in regulating TGF-ß/SMAD signaling and explored the therapeutic effect of the ubiquitin proteasome inhibitor MG132 on DN. METHODS: Wistar rats were randomly divided into a diabetes group and a normal control group. Rats in the diabetes group were injected intraperitoneally with streptozotocin. Diabetic rats were then randomly divided into a diabetic nephropathy group (DN group), an MG132 high concentration (MH) group, and an MG132 low concentration (ML) group. After 8 weeks of treatment, 24-hour urinary microalbumin (UAlb), urinary protein/urinary creatinine (Up/Ucr) values, ALT, AST, Bcr, kidney damage, TGF-ß, Smad7, fibronectin (FN), and Smurf2 were detected. RESULTS: The body mass and Smad7 protein expression decreased in DN group, but kidney weight, kidney weight index, UAlb, Up/Ucr, FN and Smurf2 mRNA expression, and TGF-ß protein expression increased. However, these changes diminished following treatment with MG132, and a more pronounced effect was evident in MH group compared to ML group. CONCLUSION: MG132 alleviates kidney damage by inhibiting Smad7 ubiquitin degradation and TGF-ß activation in DN.

Assessment of the risk of blastomere biopsy during preimplantation genetic diagnosis in a mouse model: reducing female ovary function with an increase in age by proteomics method.

Preimplantation genetic diagnosis (PGD) is important for screening genetic and chromosome mutations in embryos so that the efficiency of assisted reproductive treatment can be increased and birth defects can be decreased; however, some studies have reported a risk from this technology as well as other assisted reproductive technologies. We have developed a blastomere biopsy mouse model to assess the potential effects of blastomere biopsy that was one key procedure in PGD on the fertility of female mice at different ages. We showed that female fertility was decreased in the biopsied mouse model with an increase in age. Moreover, the ovarian weight, serum hormone levels, and the number of primordial, primary, preantral, and antral stage follicles were also decreased in the middle-aged biopsied mouse model. To elucidate the underlying molecular mechanism, we did proteomics analysis on ovarian tissues from puberty biopsied and nonbiopsied mice of the 23 differentially expressed proteins that were screened for in both groups, 3 proteins (PSMB8, ALDH1A1, and HSPA4) were selected and identified by Western blotting and quantitative RT-PCR methods, which showed the 3 proteins to regulate 12 cellular pathways. Furthermore, these three proteins were shown to be located in ovarian tissues, and the dynamic changes of expression profiling in middle-aged biopsied and nonbiopsied mice were demonstrated. The present study showed that blastomere biopsy technology impairs fertility when mice are middle-aged, which possibly resulted in abnormal expression profiling of PSMB8, ALDH1A1, and HSPA4 proteins. Thus, additional studies should be performed to assess the overall risk of blastomere biopsies during PGD procedures.

The ATP costs and time required to degrade ubiquitinated proteins by the 26 S proteasome.

The degradation of ubiquitinated proteins by 26 S proteasomes requires ATP hydrolysis. To investigate if the six proteasomal ATPases function independently or in a cyclic manner, as proposed recently, we used yeast mutants that prevent ATP binding to Rpt3, Rpt5, or Rpt6. Although proteasomes contain six ATPase subunits, each of these single mutations caused a 66% reduction in basal ATP hydrolysis, and each blocked completely the 2-3-fold stimulation of ATPase activity induced by ubiquitinated substrates. Therefore, the ATPase subunits must function in a ordered manner, in which each is required for the stimulation of ATPase activity by substrates. Although ATP is essential for multiple steps in proteasome function, when the rate of ATP hydrolysis was reduced incrementally, the degradation of Ub5-DHFR (where Ub is ubiquitin and DHFR is dihydrofolate reductase) decreased exactly in parallel. This direct proportionality implies that a specific number of ATPs is consumed in degrading a ubiquitinated protein. When the ubiquitinated DHFR was more tightly folded (upon addition of the ligand folate), the rate of ATP hydrolysis was unchanged, but the time to degrade a Ub5-DHFR molecule (∼13 s) and the energy expenditure (50-80 ATPs/Ub5-DHFR) both increased by 2-fold. With a mutation in the ATPase C terminus that reduced gate opening into the 20 S proteasome, the energy costs and time required for conjugate degradation also increased. Thus, different ubiquitin conjugates activate similarly the ATPase subunit cycle that drives proteolysis, but polypeptide structure determines the time required for degradation and thus the energy cost.

Quantitative assessment of the influence of PSMA6 variant (rs1048990) on coronary artery disease risk.

The proteasome system is a proteolytic pathway that regulates the expression of genes involved in inflammation. Recently, an association of a functional sequence variation, -8C/G, in the human proteasome subunit a type 6 gene (PSMA6) with the susceptibility to coronary artery disease (CAD) was reported. After that, several validation studies have been conducted among various ethnic populations, but the results have been inconsistent. To investigate this inconsistency and derive a more precise estimation of the relationship, a meta-analysis of 15,991 cases and 16,784 controls from 10 case-control studies was performed. Potential sources of heterogeneity including ethnicity, sample size and HWE status of study were also assessed. In a combined analysis, the summary per-allele OR for CAD of the -8C/G polymorphism was 1.09 (95 % CI: 1.02-1.16; P = 0.006). In the subgroup analysis by ethnicity, significantly increased risks were found in East Asians for the polymorphism; while no significant associations were found among Caucasians and other ethnic population in all genetic models. When restricted to studies concerning myocardial infarction patients, significant associations were detected in all genetic models. Furthermore, significant difference of PSMA6 mRNA expression was found between genotypes. In conclusion, this meta-analysis suggests that G allele of PSMA6-8C/G polymorphism is a risk factor associated with increased CAD susceptibility, but these associations vary in different ethnic populations.

An economical method for production of (2)H, (13)CH3-threonine for solution NMR studies of large protein complexes: application to the 670 kDa proteasome.

NMR studies of very high molecular weight protein complexes have been greatly facilitated through the development of labeling strategies whereby (13)CH(3) methyl groups are introduced into highly deuterated proteins. Robust and cost-effective labeling methods are well established for all methyl containing amino acids with the exception of Thr. Here we describe an inexpensive biosynthetic strategy for the production of L-[α-(2)H; ß-(2)H;γ-(13)C]-Thr that can then be directly added during protein expression to produce highly deuterated proteins with Thr methyl group probes of structure and dynamics. These reporters are particularly valuable, because unlike other methyl containing amino acids, Thr residues are localized predominantly to the surfaces of proteins, have unique hydrogen bonding capabilities, have a higher propensity to be found at protein nucleic acid interfaces and can play important roles in signaling pathways through phosphorylation. The utility of the labeling methodology is demonstrated with an application to the 670 kDa proteasome core particle, where high quality Thr (13)C,(1)H correlation spectra are obtained that could not be generated from samples prepared with commercially available U-[(13)C,(1)H]-Thr.

Assessment of proteasome impairment and accumulation/aggregation of ubiquitinated proteins in neuronal cultures.

The ubiquitin/proteasome pathway (UPP) is the major proteolytic quality control system in cells and involves tightly regulated removal of unwanted proteins and retention of those that are essential. In addition to its function in normal protein degradation, the UPP plays a critical role in the quality control process by degrading mutated or abnormally folded proteins. The proteolytic component of the UPP is a multiprotein complex known as the proteasome. Many factors, including the aging process, can cause proteasome impairment leading to formation of abnormal ubiquitin-protein aggregates that are found in most progressive neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. In this chapter, we describe protocols to measure proteasome activity, evaluate its state of assembly, and assess the accumulation and aggregation of ubiquitinated proteins in two types of neuronal cultures: human neuroblastoma cells and rat primary cortical cultures. These protocols can be used with different types of neuronal cultures to estimate proteasome activity and the levels and aggregation of ubiquitinated proteins. In addition, they can be used to identify compounds potentially capable of preventing a decline in proteasome activity and formation of ubiquitin-protein aggregates associated with neurodegeneration.

Ubiquitin-mediated regulation of TNFR1 signaling.

Ubiquitin ligase enzymes promote substrate protein ubiquitination, a post-translational modification whereby the 76-amino acid protein ubiquitin is covalently bound to substrate proteins. Ubiquitination may target substrates for proteasomal degradation or regulate substrate function in a degradation-independent manner. Ubiquitination is reversible, and this is achieved by de-ubiquitinase enzymes [Jackson PK, Eldridge AG, Freed E, et al. The lore of the RINGs: substrate recognition and catalysis by ubiquitin ligases. Trends Cell Biol 2000;10(October (10)):429-39]. The first identified target of ubiquitination in the Tumor Necrosis Factor Receptor 1 (TNFR1) signaling cascade was Inhibitor of NF-kappaB (I-kappaB), which sequesters Nuclear Factors at kappa-chain promoters in B-cells (NF-kappaB) transcription factors in the cytosol. Following TNF-alpha stimulation, I-kappaB is ubiquitinated and subsequently degraded by the proteasome, permitting NF-kappaB transcriptional activity [Glickman MH, Ciechanover A. The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev 2002;82(April (2)):373-428]. Since this seminal finding, it is now evident that nearly every step of TNFR1 signaling is regulated by ubiquitination. In this review, we will summarize the ubiquitin/proteasome system and discuss the ubiquitin-mediated regulation of TNFR1 signaling.

Assessment of the direct and indirect effects of MPP+ and dopamine on the human proteasome: implications for Parkinson's disease aetiology.

Mitochondrial impairment, glutathione depletion and oxidative stress have been implicated in the pathogenesis of Parkinson's disease (PD), linked recently to proteasomal dysfunction. Our study analysed how these factors influence the various activities of the proteasome in human SH-SY5Y neuroblastoma cells treated with the PD mimetics MPP+ (a complex 1 inhibitor) or dopamine. Treatment with these toxins led to dose- and time-dependent reductions in ATP and glutathione and also chymotrypsin-like and post-acidic like activities; trypsin-like activity was unaffected. Antioxidants blocked the effects of dopamine, but not MPP+, suggesting that oxidative stress was more important in the dopamine-mediated effects. With MPP+, ATP depletion was a prerequisite for loss of proteasomal activity. Thus in a dopaminergic neuron with complex 1 dysfunction both oxidative stress and ATP depletion will contribute independently to loss of proteasomal function. We show for the first time that addition of MPP+ or dopamine to purified samples of the human 20S proteasome also reduced proteasomal activities; with dopamine being most damaging. As with toxin-treated cells, chymotrypsin-like activity was most sensitive and trypsin-like activity the least sensitive. The observed differential sensitivity of the various proteasomal activities to PD mimetics is novel and its significance needs further study in human cells.