PTEN status determines therapeutic vulnerability to celastrol in cholangiocarcinoma.

BACKGROUND: A balanced protein homeostasis network helps cholangiocarcinoma (CCA) maintain their oncogenic growth, and disrupting proteostasis therapeutically will induce proteotoxic stress. Phosphatase and tensin homolog (PTEN) have been reported to be involved in proteostasis, and PTEN-associated pathways are commonly altered in CCA. Celastrol, a triterpene from plants, exhibits cytotoxic effects in various types of cancer. However, the underlying mechanisms remain unclear. PURPOSE: We investigated the therapeutic effect of celastrol in CCA and identified the molecular characteristics of tumors that were sensitive to celastrol. The target of celastrol was explored. We then evaluated the candidate combination therapeutic strategy to increase the effectiveness of celastrol in celastrol-insensitive CCA tumors. METHODS: Various CCA cells were categorized as either celastrol-sensitive or celastrol-insensitive based on their response to celastrol. The molecular characteristics of cells from different groups were determined by RNA-seq. PTEN status and its role in proteasome activity in CCA cells were investigated. The CMAP analysis, molecular docking, and functional assay were performed to explore the effect of celastrol on proteasome activities. The correlation between PTEN status and clinical outcomes, as well as proteasomal activity, were measured in CCA patients. The synergistic therapeutic effect of autophagy inhibitors on celastrol-insensitive CCA cells were measured. RESULTS: Diverse responses to celastrol were observed in CCA cells. PTEN expression varied among different CCA cells, and its status could impact cell sensitivity to celastrol: PTENhigh tumor cells were resistant to celastrol, while PTENlow cells were more sensitive. Celastrol induced proteasomal dysregulation in CCA cells by directly targeting PSMB5. Cells with low PTEN status transcriptionally promoted proteasome subunit expression in an AKT-dependent manner, making these cells more reliant on proteasomal activities to maintain proteostasis. This caused the PTENlow CCA cells sensitive to celastrol. A negative correlation was found between PTEN levels and the proteasome signature in CCA patients. Moreover, celastrol treatment could induce autophagy in PTENhigh CCA cells. Disrupting the autophagic pathway in PTENhigh CCA cells enhanced the cytotoxic effect of celastrol. CONCLUSION: PTEN status in CCA cells determines their sensitivity to celastrol, and autophagy inhibitors could enhance the anti-tumor effect in PTENhigh CCA.

Bortezomib Inhibits Open Configurations of the 20S Proteasome.

Bortezomib, a small dipeptide-like molecule, is a proteasome inhibitor used widely in the treatment of myeloma and lymphoma. This molecule reacts with threonine side chains near the center of the 20S proteasome and disrupts proteostasis by blocking enzymatic sites that are responsible for protein degradation. In this work, we use novel mass-spectrometry-based techniques to examine the influence of bortezomib on the structures and stabilities of the 20S core particle. These studies indicate that bortezomib binding dramatically favors compact 20S structures (in which the axial gate is closed) over larger structures (in which the axial gate is open)─suppressing gate opening by factors of at least ∼400 to 1300 over the temperature range that is studied. Thus, bortezomib may also restrict degradation in the 20S proteasome by preventing substrates from entering the catalytic pore. That bortezomib influences structures at the entrance region of the pore at such a long distance (∼65 to 75 Å) from its binding sites raises a number of interesting biophysical issues.

HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity.

Proteasomes degrade intracellular proteins to generate antigenic peptides that are recognized by the adaptive immune system and promote anticancer immunity. However, tumors subvert the antigen presentation machinery to escape immunosurveillance. We hypothesized that proteasome activation could concomitantly increase antigen abundance and diversity in multiple myeloma cells. High-throughput screens revealed that histone deacetylase 6 (HDAC6) inhibitors activated proteasomes to unmask neoantigens and amplify the tumor-specific antigenic landscape. Treatment of patient CD138+ cells with HDAC6 inhibitors significantly promoted the antimyeloma activity of autologous CD8+ T cells. Pharmacologic blockade and genetic ablation of the HDAC6 ubiquitin-binding domain released HR23B, which shuttles ubiquitinylated cargo to proteasomes, while silencing HDAC6 or HR23B in multiple myeloma cells abolished the effect of HDAC6 inhibitors on proteasomes, antigen presentation, and T-cell cytotoxicity. Taken together, our results demonstrate the paradigm-shifting translational impact of proteasome activators to expand the myeloma immunopeptidome and have revealed novel, actionable antigenic targets for T cell-directed immunotherapy. SIGNIFICANCE: The elimination of therapy-resistant tumor cells remains a major challenge in the treatment of multiple myeloma. Our study identifies and functionally validates agents that amplify MHC class I-presented antigens and pave the way for the development of proteasome activators as immune adjuvants to enhance immunotherapeutic responses in patients with multiple myeloma.

Acetaminophen induces mitochondrial apoptosis through proteasome dysfunctions.

Acetaminophen is a known antipyretic and non-opioid analgesic for mild pain and fever. Numerous studies uncover their hidden chemotherapeutics applications, including chronic cancer pain management. Acetaminophen also represents an anti-proliferative effect in some cancer cells. Few studies also suggest that the use of Acetaminophen can trigger apoptosis and impede cellular growth. However, Acetaminophen's molecular potential and precise mechanism against improper cellular proliferation and use as an effective anti-proliferative agent still need to be better understood. Here, our current findings show that Acetaminophen induces proteasomal dysfunctions, resulting in aberrant protein accumulation and mitochondrial abnormalities, and consequently induces cell apoptosis. We observed that the Acetaminophen treatment leads to improper aggregation of ubiquitylated expanded polyglutamine proteins, which may be due to the dysfunctions of proteasome activities. Our in-silico analysis suggests the interaction of Acetaminophen and proteasome. Furthermore, we demonstrated the accumulation of proteasome substrates and the depletion of proteasome activities after treating Acetaminophen in cells. Acetaminophen induces proteasome dysfunctions and mitochondrial abnormalities, leading to pro-apoptotic morphological changes and apoptosis successively. These results suggest that Acetaminophen can induce cell death and may retain a promising anti-proliferative effect. These observations can open new possible molecular strategies in the near future for developing and designing specific and effective proteasome inhibitors, which can be helpful in conjugation with other anti-tumor drugs for their better efficiency.

Protein quality control systems in the endoplasmic reticulum and the cytosol coordinately prevent alachlor-induced proteotoxic stress in Saccharomyces cerevisiae.

Alachlor, a widely used chloroacetanilide herbicide for controlling annual grasses in crops, has been reported to rapidly trigger protein denaturation and aggregation in the eukaryotic model organism Saccharomyces cerevisiae. Therefore, this study aimed to uncover cellular mechanisms involved in preventing alachlor-induced proteotoxicity. The findings reveal that the ubiquitin-proteasome system (UPS) plays a crucial role in eliminating alachlor-denatured proteins by tagging them with polyubiquitin for subsequent proteasomal degradation. Exposure to alachlor rapidly induced an inhibition of proteasome activity by 90 % within 30 min. The molecular docking analysis suggests that this inhibition likely results from the binding of alachlor to ß subunits within the catalytic core of the proteasome. Notably, our data suggest that nascent proteins in the endoplasmic reticulum (ER) are the primary targets of alachlor. Consequently, the unfolded protein response (UPR), responsible for coping with aberrant proteins in the ER, becomes activated within 1 h of alachlor treatment, leading to the splicing of HAC1 mRNA into the active transcription activator Hac1p and the upregulation of UPR gene expression. These findings underscore the critical roles of the protein quality control systems UPS and UPR in mitigating alachlor-induced proteotoxicity by degrading alachlor-denatured proteins and enhancing the protein folding capacity of the ER.

CP41, a novel curcumin analogue, induces apoptosis in endometrial cancer cells by activating the H3F3A/ proteasome-MAPK signaling pathway and enhancing oxidative stress.

AIMS: Curcumin is a natural compound and has good antitumor properties, but its clinical use is limited by its low bioavailability. We constructed the derivative CP41 (3,5-bis(2-chlorobenzylidene)-1-piperidin-4-one) by enhancing the bioavailability of curcumin while retaining its antitumor properties. MAIN METHODS: CCK-8 (Cell Counting Kit-8) was used to detect the effect of CP41 on cell proliferation; Western blotting, immunofluorescence, immunoprecipitation, quantitative PCR and enzyme-linked immunosorbent assay were used to evaluate the expression of subcutaneous tumor-related molecules in cells and mice. KEY FINDINGS: Our results showed that CP41 inhibited the proliferation of endometrial cancer cells by suppressing the proliferation of AN3CA and HEC-1-B cells. We found that CP41 significantly increased H3F3A and inhibited proteasome activity, which activated MAPK signaling and led to apoptosis. Further experiments showed that H3F3A is a potential target of CP41. Correlation analysis showed that H3F3A was positively correlated with the sensitivity to chemotherapeutic agents in endometrial cancer. CP41 significantly induced reactive oxygen species (ROS) levels and activated endoplasmic reticulum stress, which led to apoptosis. The safety profile of CP41 was also evaluated, and CP41 did not cause significant drug toxicity in mice. SIGNIFICANCE: CP41 showed stronger antitumor potency than curcumin, and its antitumor activity may be achieved by inducing ROS and activating H3F3A-mediated apoptosis.

The stem cell-supporting small molecule UM171 triggers Cul3-KBTBD4-mediated degradation of ELM2 domain-harboring proteins.

To chemically modulate the ubiquitin-proteasome system for the degradation of specific target proteins is currently emerging as an alternative therapeutic modality. Earlier, we discovered such properties of the stem cell-supporting small molecule UM171 and identified that members of the CoREST complex (RCOR1 and LSD1) are targeted for degradation. UM171 supports the in vitro propagation of hematopoietic stem cells by transiently perturbing the differentiation-promoting effects of CoREST. Here, we employed global proteomics to map the UM171-targeted proteome and identified the additional target proteins, namely RCOR3, RREB1, ZNF217, and MIER2. Further, we discovered that critical elements recognized by Cul3KBTBD4 ligase in the presence of UM171 are located within the EGL-27 and MTA1 homology 2 (ELM2) domain of the substrate proteins. Subsequent experiments identified conserved amino acid sites in the N-terminus of the ELM2 domain that are essential for UM171-mediated degradation. Overall, our findings provide a detailed account on the ELM2 degrome targeted by UM171 and identify critical sites required for UM171-mediated degradation of specific substrates. Given the target profile, our results are highly relevant in a clinical context and point towards new therapeutic applications for UM171.

New trends in synthetic drugs and natural products targeting 20S proteasomes in cancers.

Cancer is a global health challenge that remains to be a field of extensive research aiming to find new anticancer therapeutics. The 20S proteasome complex is one of the targets of anticancerdrugs, as it is correlated with several cancer types. Herein, we aim to discuss the 20S proteasome subunits and investigatethe currently studied proteasome inhibitors targeting the catalytically active proteasome subunits. In this review, we summarize the proteindegradation mechanism of the 20S proteasome complex and compareit with the 26S proteasome complex. Afterwards, the localization of the 20S proteasome is summarized as well as its use as a diagnosticandprognostic marker. The FDA-approved proteasome inhibitors (PIs) under clinical trials are summarized and their current limited use in solid tumors is also reviewed in addition to the expression of theß5 subunit in differentcell lines. The review discusses in-silico analysis of the active subunit of the 20S proteasome complex. For development of new proteasome inhibitor drugs, the natural products inhibiting the 20S proteasome are summarized, as well as novel methodologies and challenges for the natural product discovery and current information about the biosynthetic gene clusters encoding them. We herein briefly summarize some resistancemechanismsto the proteasomeinhibitors. Additionally, we focus on the three main classes of proteasome inhibitors: 1] boronic acid, 2] beta-lactone and 3] epoxide inhibitor classes, as well as other PI classes, and their IC50 values and their structure-activity relationship (SAR). Lastly,we summarize several future prospects of developing new proteasome inhibitors towards the treatment of tumors, especially solid tumors.

Discovery of Potent, Selective, and In Vivo Efficacious AKT Kinase Protein Degraders via Structure-Activity Relationship Studies.

We recently reported a potent, selective, and in vivo efficacious AKT degrader, MS21, which is a von Hippel-Lindau (VHL)-recruiting proteolysis targeting chimera (PROTAC) based on the AKT inhibitor AZD5363. However, no structure-activity relationship (SAR) studies that resulted in this discovery have been reported. Herein, we present our SAR studies that led to the discovery of MS21, another VHL-recruiting AKT degrader, MS143 (compound 20) with similar potency as MS21, and a novel cereblon (CRBN)-recruiting PROTAC, MS5033 (compound 35). Compounds 20 and 35 induced rapid and robust AKT degradation in a concentration- and time-dependent manner via hijacking the ubiquitin-proteasome system. Compound 20 suppressed cell growth more effectively than AZD5363 in multiple cancer cell lines. Furthermore, 20 and 35 displayed good plasma exposure levels in mice and are suitable for in vivo efficacy studies. Lastly, compound 20 effectively suppressed tumor growth in vivo in a xenograft model without apparent toxicity.

Novel and conventional inhibitors of canonical autophagy differently affect LC3-associated phagocytosis.

In autophagy, LC3-positive autophagophores fuse and encapsulate the autophagic cargo in a double-membrane structure. In contrast, lipidated LC3 (LC3-II) is directly formed at the phagosomal membrane in LC3-associated phagocytosis (LAP). In this study, we dissected the effects of autophagy inhibitors on LAP. SAR405, an inhibitor of VPS34, reduced levels of LC3-II and inhibited LAP. In contrast, the inhibitors of endosomal acidification bafilomycin A1 and chloroquine increased levels of LC3-II, due to reduced degradation in acidic lysosomes. However, while bafilomycin A1 inhibited LAP, chloroquine did not. Finally, EACC, which inhibits the fusion of autophagosomes with lysosomes, promoted LC3 degradation possibly by the proteasome. Targeting LAP with small molecule inhibitors is important given its emerging role in infectious and autoimmune diseases.

Ethanol attenuates presentation of cytotoxic T-lymphocyte epitopes on hepatocytes of HBV-infected humanized mice.

BACKGROUND AND AIMS: Approximately 3.5% of the global population is chronically infected with Hepatitis B Virus (HBV), which puts them at high risk of end-stage liver disease, with the risk of persistent infection potentiated by alcohol consumption. However, the mechanisms underlying the effects of alcohol on HBV persistence remain unclear. Here, we aimed to establish in vivo/ex vivo evidence that alcohol suppresses HBV peptides-major histocompatibility complex (MHC) class I antigen display on primary human hepatocytes (PHH), which diminishes the recognition and clearance of HBV-infected hepatocytes by cytotoxic T-lymphocytes (CTLs). METHODS: We used fumarylacetoacetate hydrolase (Fah)-/-, Rag2-/-, common cytokine receptor gamma chain knock-out (FRG-KO) humanized mice transplanted with human leukocyte antigen-A2 (HLA-A2)-positive hepatocytes. The mice were HBV-infected and fed control and alcohol diets. Isolated hepatocytes were exposed ex vivo to HBV 18-27-HLA-A2-restricted CTLs to quantify cytotoxicity. For mechanistic studies, we measured proteasome activities, unfolded protein response (UPR), and endoplasmic reticulum (ER) stress in hepatocytes from HBV-infected humanized mouse livers. RESULTS AND CONCLUSIONS: We found that alcohol feeding attenuated HBV core 18-27-HLA-A2 complex presentation on infected hepatocytes due to the suppression of proteasome function and ER stress induction, which diminished both the processing of HBV peptides and trafficking of HBV-MHC class I complexes to the hepatocyte surface. This alcohol-mediated decrease in MHC class I-restricted antigen presentation of the CTL epitope on target hepatocytes reduced the CTL-specific elimination of infected cells, potentially leading to HBV-infection persistence, which promotes end-stage liver disease outcomes.

Carfilzomib alleviated osteoporosis by targeting PSME1/2 to activate Wnt/ß-catenin signaling.

Osteoporosis (OP) is characterized by decreased bone mineral density and impaired bone strength. Carfilzomib (CFZ) is a new-generation proteasome inhibitor and has been found to affect bone metabolism. However, the effect and mechanism of CFZ on OP has not been investigated systematically. In this study, we found that protein levels of proteasome activator subunit 1/2 (PSME1/2) increased in OP, and accumulated mostly in osteoblasts and osteoclasts. Treatment with PSME1/2 recombinant protein inhibited osteogenesis and promoted osteoclast formation in vitro. Also, PSME1/2 inhibited the expression of ß-catenin protein, resulting in limitation of Wnt/ß-catenin signaling. CFZ inhibited PSME1 and PSME2 proteasome activities and increased ß-catenin protein level, resulting in the translocation of ß-catenin to the nucleus and activation of canonical Wnt/ß-catenin signaling, further promoting osteogenesis and inhibiting osteoclastic differentiation. In vivo, we conducted ovariectomy (OVX) to create a model of OVX-induced postmenopausal OP in mice. When analyzed by micro-CT scanning, enhancement of bone mineral density, bone volume, trabecular number, and thickness was seen in the CFZ-treated mice. Also, we noticed increased osteogenesis and decreased osteoclastogenesis, diminished expression of PSME1 and PSME2 and activated Wnt/ß-catenin signaling in bone sections from OP mice treated with CFZ. Overall, our data indicated that PSME1/2 may serve as new targets for the treatment of OP, and targeting PSME1/2 with CFZ provides a candidate therapeutic molecule for postmenopausal OP.

Ursolic acid ameliorates amyloid ß-induced pathological symptoms in Caenorhabditis elegans by activating the proteasome.

BACKGROUND: Amyloid ß induces pathological symptoms in various neurodegenerative disorders. It is the hallmark of these neurodegenerative disorders, such as Alzheimer's disease, and is reported to induce neurotoxicity leading to neuronal impairment. The continuous development of neurodegenerative disease accompanies pathological changes in amyloid ß deposition in the brain. After amyloid ß accumulates, the inadequate clearance of amyloid ß further accelerates the development of events in the pathological cascade. In eukaryotes, the proteasome is responsible for the degradation of misfolded and damaged proteins to maintain proteostasis. Therefore, screening candidates that preserve proteasomal activity may promote amyloid ß homeostasis, which is expected to provide new therapeutic opportunities for these neurodegenerative diseases. Ursolic acid, a natural triterpenoid, has prominent pharmacological antioxidant, anti-inflammatory, neuroprotective, and nontoxic activities. Here, we explored the protective effects of ursolic acid on amyloid ß-induced pathological symptoms. METHODS: This study investigated the therapeutic potential of ursolic acid and its underlying molecular mechanisms using a Caenorhabditis elegans transgenic pathological model. RESULTS: In our study, ursolic acid successfully repressed amyloid ß-induced paralysis and hypersensitivity to serotonin in Caenorhabditis elegans. The levels of amyloid ß monomers, oligomers, and deposits were decreased after treatment with ursolic acid in transgenic nematodes overexpressing human amyloid ß; however, ursolic acid did not affect exogenous transgene transcription and expression levels. Ursolic acid transcriptionally enhanced the ubiquitin-proteasome system and augmented proteasome activity in vivo. However, the proteasome inhibitor MG132 abolished the therapeutic effect of ursolic acid on behavioral paralysis, and Parkinson's disease-related-1 was required for the therapeutic effect of ursolic acid. CONCLUSIONS: Our study revealed that ursolic acid prevented amyloid ß-induced proteotoxic stress, specifically by reducing the amount of amyloid ß and increasing proteasome activity in vivo. Furthermore, the therapeutic effect of ursolic acid on transgenic nematodes expressing amyloid ß depended on the increased activity of the proteasome. This work provides an essential supplement to the information on the pharmacological mechanism of ursolic acid.

Microbial proteasomes as drug targets.

Proteasomes are compartmentalized, ATP-dependent, N-terminal nucleophile hydrolases that play essentials roles in intracellular protein turnover. They are present in all 3 kingdoms. Pharmacological inhibition of proteasomes is detrimental to cell viability. Proteasome inhibitor rugs revolutionize the treatment of multiple myeloma. Proteasomes in pathogenic microbes such as Mycobacterium tuberculosis (Mtb), Plasmodium falciparum (Pf), and other parasites and worms have been validated as therapeutic targets. Starting with Mtb proteasome, efforts in developing inhibitors selective for microbial proteasomes have made great progress lately. In this review, we describe the strategies and pharmacophores that have been used in developing proteasome inhibitors with potency and selectivity that spare human proteasomes and highlight the development of clinical proteasome inhibitor candidates for treatment of leishmaniasis and Chagas disease. Finally, we discuss the future challenges and therapeutical potentials of the microbial proteasome inhibitors.

Fragment-Sized and Bidentate (Immuno)Proteasome Inhibitors Derived from Cysteine and Threonine Targeting Warheads.

Constitutive- and immunoproteasomes are part of the ubiquitin-proteasome system (UPS), which is responsible for the protein homeostasis. Selective inhibition of the immunoproteasome offers opportunities for the treatment of numerous diseases, including inflammation, autoimmune diseases, and hematologic malignancies. Although several inhibitors have been reported, selective nonpeptidic inhibitors are sparse. Here, we describe two series of compounds that target both proteasomes. First, benzoxazole-2-carbonitriles as fragment-sized covalent immunoproteasome inhibitors are reported. Systematic substituent scans around the fragment core of benzoxazole-2-carbonitrile led to compounds with single digit micromolar inhibition of the ß5i subunit. Experimental and computational reactivity studies revealed that the substituents do not affect the covalent reactivity of the carbonitrile warhead, but mainly influence the non-covalent recognition. Considering the small size of the inhibitors, this finding emphasizes the importance of the non-covalent recognition step in the covalent mechanism of action. As a follow-up series, bidentate inhibitors are disclosed, in which electrophilic heterocyclic fragments, i.e., 2-vinylthiazole, benzoxazole-2-carbonitrile, and benzimidazole-2-carbonitrile were linked to threonine-targeting (R)-boroleucine moieties. These compounds were designed to bind both the Thr1 and ß5i-subunit-specific residue Cys48. However, inhibitory activities against (immuno)proteasome subunits showed that bidentate compounds inhibit the ß5, ß5i, ß1, and ß1i subunits with submicromolar to low-micromolar IC50 values. Inhibitory assays against unrelated enzymes showed that compounds from both series are selective for proteasomes. The presented nonpeptidic and covalent derivatives are suitable hit compounds for the development of either ß5i-selective immunoproteasome inhibitors or compounds targeting multiple subunits of both proteasomes.

A Promising Intracellular Protein-Degradation Strategy: TRIMbody-Away Technique Based on Nanobody Fragment.

Most recently, a technology termed TRIM-Away has allowed acute and rapid destruction of endogenous target proteins in cultured cells using specific antibodies and endogenous/exogenous tripartite motif 21 (TRIM21). However, the relatively large size of the full-size mAbs (150 kDa) results in correspondingly low tissue penetration and inaccessibility of some sterically hindered epitopes, which limits the target protein degradation. In addition, exogenous introduction of TRIM21 may cause side effects for treated cells. To tackle these limitations, we sought to replace full-size mAbs with the smaller format of antibodies, a nanobody (VHH, 15 kDa), and construct a new type of fusion protein named TRIMbody by fusing the nanobody and RBCC motif of TRIM21. Next, we introduced enhanced green fluorescent protein (EGFP) as a model substrate and generated αEGFP TRIMbody using a bispecific anti-EGFP (αEGFP) nanobody. Remarkably, inducible expression of αEGFP TRIMbody could specifically degrade intracellular EGFP in HEK293T cells in a time-dependent manner. By treating cells with inhibitors, we found that intracellular EGFP degradation by αEGFP TRIMbody relies on both ubiquitin-proteasome and autophagy-lysosome pathways. Taken together, these results suggested that TRIMbody-Away technology could be utilized to specifically degrade intracellular protein and could expand the potential applications of degrader technologies.

Pharmacological Inhibition of the VCP/Proteasome Axis Rescues Photoreceptor Degeneration in RHOP23H Rat Retinal Explants.

Rhodopsin (RHO) misfolding mutations are a common cause of the blinding disease autosomal dominant retinitis pigmentosa (adRP). The most prevalent mutation, RHOP23H, results in its misfolding and retention in the endoplasmic reticulum (ER). Under homeostatic conditions, misfolded proteins are selectively identified, retained at the ER, and cleared via ER-associated degradation (ERAD). Overload of these degradation processes for a prolonged period leads to imbalanced proteostasis and may eventually result in cell death. ERAD of misfolded proteins, such as RHOP23H, includes the subsequent steps of protein recognition, targeting for ERAD, retrotranslocation, and proteasomal degradation. In the present study, we investigated and compared pharmacological modulation of ERAD at these four different major steps. We show that inhibition of the VCP/proteasome activity favors cell survival and suppresses P23H-mediated retinal degeneration in RHOP23H rat retinal explants. We suggest targeting this activity as a therapeutic approach for patients with currently untreatable adRP.

Proteasomal Degradation of Zn-Dependent Hdacs: The E3-Ligases Implicated and the Designed Protacs That Enable Degradation.

Protein degradation by the Ubiquitin-Proteasome System is one of the main mechanisms of the regulation of cellular proteostasis, and the E3 ligases are the key effectors for the protein recognition and degradation. Many E3 ligases have key roles in cell cycle regulation, acting as checkpoints and checkpoint regulators. One of the many important proteins involved in the regulation of the cell cycle are the members of the Histone Deacetylase (HDAC) family. The importance of zinc dependent HDACs in the regulation of chromatin packing and, therefore, gene expression, has made them targets for the design and synthesis of HDAC inhibitors. However, achieving potency and selectivity has proven to be a challenge due to the homology between the zinc dependent HDACs. PROteolysis TArgeting Chimaera (PROTAC) design has been demonstrated to be a useful strategy to inhibit and selectively degrade protein targets. In this review, we attempt to summarize the E3 ligases that naturally ubiquitinate HDACs, analyze their structure, and list the known ligands that can bind to these E3 ligases and be used for PROTAC design, as well as the already described HDAC-targeted PROTACs.

Proteasome inhibition by bortezomib parallels a reduction in head and neck cancer cells growth, and an increase in tumor-infiltrating immune cells.

Head and neck cancer (HNC) has frequently an aggressive course for the development of resistance to standard chemotherapy. Thus, the use of innovative therapeutic drugs is being assessed. Bortezomib is a proteasome inhibitor with anticancer effects. In vitro antitumoral activity of Bortezomib was investigated employing human tongue (SCC-15, CAL-27), pharynx (FaDu), salivary gland (A-253) cancer cell lines and a murine cell line (SALTO-5) originated from a salivary gland adenocarcinoma arising in BALB-neuT male mice transgenic for the oncogene neu. Bortezomib inhibited cell proliferation, triggered apoptosis, modulated the expression and activation of pro-survival signaling transduction pathways proteins activated by ErbB receptors and inhibited proteasome activity in vitro. Intraperitoneal administration of Bortezomib delayed tumor growth of SALTO-5 cells transplanted in BALB-neuT mice, protracted mice survival and adjusted tumor microenvironment by increasing tumor-infiltrating immune cells (CD4+ and CD8+ T cells, B lymphocytes, macrophages, and Natural Killer cells) and by decreasing vessels density. In addition, Bortezomib modified the expression of proteasome structural subunits in transplanted SALTO-5 cells. Our findings further support the use of Bortezomib for the treatment of HNC and reveal its ineffectiveness in counteracting the activation of deregulated specific signaling pathways in HNC cell lines when resistance to proteasome inhibition is developed.