ABSTRACT
The stratum corneum (SC), the outermost epidermal layer, plays a pivotal role in skin barrier function. This review delves into the intricate process of protein degradation within the stratum corneum, elucidating the roles of specific enzymes, regulatory mechanisms and the consequent impact on various skin conditions. Protein degradation is a finely tuned process, orchestrated by a suite of proteolytic enzymes like kallikreins. These enzymes are responsible for the breakdown of corneodesmosomes and the orderly desquamation of corneocytes, a process essential for skin homeostasis. Another critical enzymatic process is the breakdown of proteins like filaggrin and the generation of amino acids and their derivatives, required in the physiological water-handling properties of the SC. Regulation of these proteolytic activities is complex, involving a balance between endogenous inhibitors and other factors like pH, hydration and environmental stressors. Dysregulation of protease activity is linked to a spectrum of skin conditions, ranging from xerosis to inflammatory diseases like atopic dermatitis and psoriasis. Aberrant protein degradation can lead to compromised skin barrier function, increased tissue water loss and heightened susceptibility to infections and allergens. Understanding the factors affecting protein degradation can inform the development of targeted skincare products. Advances in biochemistry and dermatology have paved the way for the search for active ingredients designed to modulate protease activity. Such innovations may offer promising therapeutic avenues for enhancing skin barrier function and treating skin disorders. This review underscores the significance of enzymatic protein degradation in the SC and its regulatory mechanisms. It provides insights into the pathophysiology of skin diseases and outlines the potential for novel skincare interventions. By bridging the gap between fundamental research and practical applications, this article aims to inspire further investigation for better understanding of skin physiology and innovation in the realm of skincare product development.
La couche cornée (stratum corneum, SC), la couche épidermique la plus externe, joue un rôle essentiel dans la fonction de barrière cutanée. Cette revue se penche sur le processus complexe de dégradation des protéines au sein de la couche cornée, ce qui explique les rôles des enzymes spécifiques, les mécanismes de régulation et l'impact qui en résulte sur diverses affections cutanées. La dégradation des protéines est un processus subtil, orchestré par une série d'enzymes protéolytiques telles que les kallikréines. Ces enzymes sont responsables de la décomposition des cornéodesmosomes et de la desquamation ordonnée des cornéocytes, un processus essentiel à l'homéostasie de la peau. Un autre processus enzymatique essentiel est la dégradation des protéines telles que la filaggrine et la génération d'acides aminés et de leurs dérivés, nécessaires aux propriétés physiologiques de traitement de l'eau de la SC. La régulation de ces activités protéolytiques est complexe, impliquant un équilibre entre les inhibiteurs endogènes et d'autres facteurs tels que le pH, l'hydratation et les facteurs de stress environnementaux. Le dérèglement de l'activité de la protéase est lié à un spectre d'affections cutanées, allant de la xérose à des maladies inflammatoires telles que la dermatite atopique et le psoriasis. Une dégradation aberrante des protéines peut compromettre la fonction de barrière cutanée, augmenter la perte d'eau tissulaire et augmenter la sensibilité aux infections et aux allergènes. Comprendre les facteurs affectant la dégradation des protéines peut contribuer au développement de produits de soins de la peau ciblés. Les progrès en biochimie et en dermatologie ont ouvert la voie à la recherche de principes actifs conçus pour moduler l'activité de la protéase. Ces innovations peuvent offrir des pistes thérapeutiques prometteuses pour améliorer la fonction de la barrière cutanée et traiter les troubles cutanés. Cette revue souligne l'importance de la dégradation enzymatique des protéines dans la SC et ses mécanismes de régulation. Elle fournit des informations sur la physiopathologie des maladies cutanées et souligne le potentiel de nouvelles interventions pour soins de la peau. En comblant le fossé entre la recherche fondamentale et les applications pratiques, cet article vise à inspirer des recherches supplémentaires pour mieux comprendre la physiologie de la peau et l'innovation dans le domaine du développement de produits de soins de la peau.
Subject(s)
Epidermis , Filaggrin Proteins , Humans , Epidermis/metabolism , Proteolysis , Skin/metabolism , Skin Diseases/metabolismABSTRACT
The antitumor performance of PROteolysis-TArgeting Chimeras (PROTACs) is limited by its insufficient tumor specificity and poor pharmacokinetics. These disadvantages are further compounded by tumor heterogeneity, especially the presence of cancer stem-like cells, which drive tumor growth and relapse. Herein, we design a region-confined PROTAC nanoplatform that integrates both reactive oxygen species (ROS)-activatable and hypoxia-responsive PROTAC prodrugs for the precise manipulation of bromodomain and extraterminal protein 4 expression and tumor eradication. These PROTAC nanoparticles selectively accumulate within and penetrate deep into tumors via response to matrix metalloproteinase-2. Photoactivity is then reactivated in response to the acidic intracellular milieu and the PROTAC is discharged due to the ROS generated via photodynamic therapy specifically within the normoxic microenvironment. Moreover, the latent hypoxia-responsive PROTAC prodrug is restored in hypoxic cancer stem-like cells overexpressing nitroreductase. Here, we show the ability of region-confined PROTAC nanoplatform to effectively degrade BRD4 in both normoxic and hypoxic environments, markedly hindering tumor progression in breast and head-neck tumor models.
Subject(s)
Cell Cycle Proteins , Nanoparticles , Proteolysis , Transcription Factors , Humans , Proteolysis/drug effects , Animals , Nanoparticles/chemistry , Cell Line, Tumor , Mice , Transcription Factors/metabolism , Female , Cell Cycle Proteins/metabolism , Reactive Oxygen Species/metabolism , Prodrugs/pharmacology , Prodrugs/chemistry , Photochemotherapy/methods , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Mice, Nude , Xenograft Model Antitumor Assays , Tumor Microenvironment/drug effects , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Nuclear Proteins/metabolism , Matrix Metalloproteinase 2/metabolism , Mice, Inbred BALB C , Neoplasms/drug therapy , Neoplasms/metabolism , Neoplasms/pathology , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Bromodomain Containing ProteinsABSTRACT
PROteolysis TArgeting Chimeras (PROTACs) has recently emerged as a promising technology. However, the design of rational PROTACs, especially the linker component, remains challenging due to the absence of structure-activity relationships and experimental data. Leveraging the structural characteristics of PROTACs, fragment-based drug design (FBDD) provides a feasible approach for PROTAC research. Concurrently, artificial intelligence-generated content has attracted considerable attention, with diffusion models and Transformers emerging as indispensable tools in this field. In response, we present a new diffusion model, DiffPROTACs, harnessing the power of Transformers to learn and generate new PROTAC linkers based on given ligands. To introduce the essential inductive biases required for molecular generation, we propose the O(3) equivariant graph Transformer module, which augments Transformers with graph neural networks (GNNs), using Transformers to update nodes and GNNs to update the coordinates of PROTAC atoms. DiffPROTACs effectively competes with existing models and achieves comparable performance on two traditional FBDD datasets, ZINC and GEOM. To differentiate the molecular characteristics between PROTACs and traditional small molecules, we fine-tuned the model on our self-built PROTACs dataset, achieving a 93.86% validity rate for generated PROTACs. Additionally, we provide a generated PROTAC database for further research, which can be accessed at https://bailab.siais.shanghaitech.edu.cn/service/DiffPROTACs-generated.tgz. The corresponding code is available at https://github.com/Fenglei104/DiffPROTACs and the server is at https://bailab.siais.shanghaitech.edu.cn/services/diffprotacs.
Subject(s)
Deep Learning , Proteolysis , Drug Design , Ligands , Proteolysis Targeting ChimeraABSTRACT
The role of KRAS mutation in non-small cell lung cancer (NSCLC) initiation and progression is well-established. However, "undruggable" KRAS protein poses the research of small molecule inhibitors a significant challenge. Addressing this, proteolysis-targeting chimeras (PROTACs) have become a cutting-edge treatment method, emphasizing protein degradation. A modified ethanol injection method was employed in this study to formulate liposomes encapsulating PROTAC drug LC-2 (LC-2 LPs). Precise surface modifications using cell-penetrating peptide R8 yielded R8-LC-2 liposomes (R8-LC-2 LPs). Comprehensive cellular uptake and cytotoxicity studies unveiled that R8-LC-2 LPs depended on concentration and time, showcasing the superior performance of R8-LC-2 LPs compared to normal liposomes. In vivo pharmacokinetic profiles demonstrated the capacity of DSPE-PEG2000 to prolong the circulation time of LC-2, leading to higher plasma concentrations compared to free LC-2. In vivo antitumor efficacy research underscored the remarkable ability of R8-LC-2 LPs to effectively suppress tumor growth. This study contributed to the exploration of enhanced therapeutic strategies for NSCLC, specifically focusing on the development of liposomal PROTACs targeting the "undruggable" KRAS protein. The findings provide valuable insights into the potential of this innovative approach, offering prospects for improved drug delivery and heightened antitumor efficacy.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Liposomes , Lung Neoplasms , Proteolysis , Proto-Oncogene Proteins p21(ras) , Animals , Humans , Mice , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/therapeutic use , Carcinoma, Non-Small-Cell Lung/drug therapy , Cell Line, Tumor , Cell-Penetrating Peptides , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Phosphatidylethanolamines/chemistry , Polyethylene Glycols/chemistry , Proteolysis/drug effects , Proteolysis Targeting Chimera/administration & dosage , Proteolysis Targeting Chimera/pharmacokinetics , Proteolysis Targeting Chimera/therapeutic use , Proto-Oncogene Proteins p21(ras)/genetics , RatsABSTRACT
Deposition of amyloid-ß (Aß) in the brain can impair neuronal function and contribute to cognitive decline in Alzheimer's disease (AD). Here, we found that dopamine and the dopamine precursor levodopa (also called l-DOPA) induced Aß degradation in the brain. Chemogenetic approaches in mice revealed that the activation of dopamine release from ventral tegmental area (VTA) neurons increased the abundance and activity of the Aß-degrading enzyme neprilysin and reduced the amount of Aß deposits in the prefrontal cortex in a neprilysin-dependent manner. Aged mice had less dopamine and neprilysin in the anterior cortex, a decrease that was accentuated in AD model mice. Treating AD model mice with levodopa reduced Aß deposition and improved cognitive function. These observations demonstrate that dopamine promotes brain region-specific, neprilysin-dependent degradation of Aß, suggesting that dopamine-associated strategies have the potential to treat this aspect of AD pathology.
Subject(s)
Alzheimer Disease , Amyloid beta-Peptides , Dopamine , Neprilysin , Ventral Tegmental Area , Neprilysin/metabolism , Neprilysin/genetics , Animals , Dopamine/metabolism , Amyloid beta-Peptides/metabolism , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Mice , Ventral Tegmental Area/metabolism , Ventral Tegmental Area/drug effects , Levodopa/pharmacology , Brain/metabolism , Mice, Transgenic , Disease Models, Animal , Humans , Proteolysis/drug effects , Mice, Inbred C57BL , Prefrontal Cortex/metabolism , MaleABSTRACT
Rationale: Since oncogene expression products often exhibit upregulation or abnormally activated activity, developing a technique to regulate abnormal protein levels represent a viable approach for treating tumors and protein abnormality-related diseases. Methods: We first screened out eMIATAC components with high targeted degradation efficiency and explored the mechanism by which eMIATAC induced target protein degradation, and verified the degradation efficiency of the target protein by protein imprinting and flow cytometry. Next, we recombined eMIATAC with some controllable elements to verify the regulatable degradation performance of the target protein. Subsequently, we constructed eMIATAC that can express targeted degradation of AKT1 and verified its effect on GBM cell development in vitro and in vivo. Finally, we concatenated eMIATAC with CAR sequences to construct CAR-T cells with low BATF protein levels and verified the changes in their anti-tumor efficacy. Results: we developed a system based on the endosome-microautophagy-lysosome pathway for degrading endogenous proteins: endosome-MicroAutophagy TArgeting Chimera (eMIATAC), dependent on Vps4A instead of lysosomal-associated membrane protein 2A (LAMP2A) to bind to the chaperone Hsc70 and the protein of interest (POI). The complex was then transported to the lysosome by late endosomes, where degradation occurred similarly to microautophagy. The eMIATACs demonstrated accuracy, efficiency, reversibility, and controllability in degrading the target protein EGFP. Moreover, eMIATAC exhibited excellent performance in knocking down POI when targeting endogenous proteins in vivo and in vitro. Conclusions: The eMIATACs could not only directly knock down abnormal proteins for glioma treatment but also enhance the therapeutic effect of CAR-T cell therapy for tumors by knocking down T cell exhaustion-related proteins. The newly developed eMIATAC system holds promise as a novel tool for protein knockdown strategies. By enabling direct control over endogenous protein levels, eMIATAC has the potential to revolutionize treatment for cancer and genetic diseases.
Subject(s)
Autophagy , Endosomes , Immunotherapy, Adoptive , Proteolysis , Humans , Animals , Endosomes/metabolism , Cell Line, Tumor , Mice , Immunotherapy, Adoptive/methods , Receptors, Chimeric Antigen/metabolism , Glioblastoma/therapy , Glioblastoma/metabolism , Glioblastoma/pathology , Lysosomal-Associated Membrane Protein 2/metabolism , Lysosomal-Associated Membrane Protein 2/genetics , Xenograft Model Antitumor Assays , HSC70 Heat-Shock Proteins/metabolism , Lysosomes/metabolism , T-Lymphocytes/metabolismABSTRACT
Chloroplasts are key players in photosynthesis and immunity against microbial pathogens. However, the precise and timely regulatory mechanisms governing the control of photosynthesis-associated nuclear genes (PhANGs) expression in plant immunity remain largely unknown. Here we report that TaPIR1, a Pst-induced RING-finger E3 ubiquitin ligase, negatively regulates Pst resistance by specifically interacting with TaHRP1, an atypical transcription factor histidine-rich protein. TaPIR1 ubiquitinates the lysine residues K131 and K136 in TaHRP1 to regulate its stability. TaHRP1 directly binds to the TaHRP1-binding site elements within the PhANGs promoter to activate their transcription via the histidine-rich domain of TaHRP1. PhANGs expression induces the production of chloroplast-derived ROS. Although knocking out TaHRP1 reduces Pst resistance, TaHRP1 overexpression contributes to photosynthesis, and chloroplast-derived ROS production, and improves disease resistance. TaPIR1 expression inhibits the downstream activation of TaHRP1 and TaHRP1-induced ROS accumulation in chloroplasts. Overall, we show that the TaPIR1-mediated ubiquitination and degradation of TaHRP1 alters PhANGs expression to disrupt chloroplast function, thereby increasing plant susceptibility to Pst.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Chloroplasts , Gene Expression Regulation, Plant , Photosynthesis , Reactive Oxygen Species , Ubiquitin-Protein Ligases , Ubiquitination , Chloroplasts/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/genetics , Arabidopsis/metabolism , Arabidopsis/genetics , Arabidopsis/immunology , Reactive Oxygen Species/metabolism , Plant Diseases/microbiology , Plant Diseases/immunology , Plant Diseases/genetics , Transcription Factors/metabolism , Transcription Factors/genetics , Disease Resistance/genetics , Proteolysis , Plants, Genetically Modified , Promoter Regions, Genetic/genetics , Plant Immunity/genetics , Nicotiana/metabolism , Nicotiana/geneticsABSTRACT
Compensation and intracellular storage of PD-L1 may compromise the efficacy of antibody drugs targeting the conformational blockade of PD1/PD-L1 on the cell surface. Alternative therapies aiming to reduce the overall cellular abundance of PD-L1 thus might overcome resistance to conventional immune checkpoint blockade. Here we show by bioinformatics analysis that colon adenocarcinoma (COAD) with high microsatellite instability (MSI-H) presents the most promising potential for this therapeutic intervention, and that overall PD-L1 abundance could be controlled via HSC70-mediated lysosomal degradation. Proteomic and metabolomic analyses of mice COAD with MSI-H in situ unveil a prominent acidic tumor microenvironment. To harness these properties, an artificial protein, IgP ß, is engineered using pH-responsive peptidic foldamers. This features customized peptide patterns and designed molecular function to facilitate interaction between neoplastic PD-L1 and HSC70. IgP ß effectively reduces neoplastic PD-L1 levels via HSC70-mediated lysosomal degradation, thereby persistently revitalizing the action of tumor-infiltrating CD8 + T cells. Notably, the anti-tumor effect of lysosomal-degradation-based therapy surpasses that of antibody-based immune checkpoint blockade for MSI-H COAD in multiple mouse models. The presented strategy expands the use of peptidic foldamers in discovering artificial protein drugs for targeted cancer immunotherapy.
Subject(s)
Adenocarcinoma , B7-H1 Antigen , Colonic Neoplasms , Lysosomes , Microsatellite Instability , T-Lymphocytes, Cytotoxic , Tumor Microenvironment , Lysosomes/metabolism , B7-H1 Antigen/metabolism , B7-H1 Antigen/immunology , B7-H1 Antigen/genetics , Animals , Adenocarcinoma/immunology , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Adenocarcinoma/metabolism , Colonic Neoplasms/immunology , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Colonic Neoplasms/drug therapy , Colonic Neoplasms/metabolism , Mice , Humans , Tumor Microenvironment/immunology , Tumor Microenvironment/drug effects , T-Lymphocytes, Cytotoxic/immunology , Cell Line, Tumor , Proteolysis/drug effects , Female , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Immune Checkpoint Inhibitors/pharmacology , Immune Checkpoint Inhibitors/therapeutic useABSTRACT
The accumulation of senescent cells promotes ageing and age-related diseases, but molecular mechanisms that senescent cells use to evade immune clearance and accumulate in tissues remain to be elucidated. Here we report that p16-positive senescent cells upregulate the immune checkpoint protein programmed death-ligand 1 (PD-L1) to accumulate in ageing and chronic inflammation. We show that p16-mediated inhibition of cell cycle kinases CDK4/6 induces PD-L1 stability in senescent cells via downregulation of its ubiquitin-dependent degradation. p16-expressing senescent alveolar macrophages elevate PD-L1 to promote an immunosuppressive environment that can contribute to an increased burden of senescent cells. Treatment with activating anti-PD-L1 antibodies engaging Fcγ receptors on effector cells leads to the elimination of PD-L1 and p16-positive cells. Our study uncovers a molecular mechanism of p16-dependent regulation of PD-L1 protein stability in senescent cells and reveals the potential of targeting PD-L1 to improve immunosurveillance of senescent cells and ameliorate senescence-associated inflammation.
Subject(s)
B7-H1 Antigen , Cellular Senescence , Cyclin-Dependent Kinase Inhibitor p16 , Protein Stability , Cellular Senescence/immunology , B7-H1 Antigen/metabolism , B7-H1 Antigen/genetics , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Cyclin-Dependent Kinase Inhibitor p16/genetics , Animals , Humans , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 4/genetics , Immunologic Surveillance , Mice, Inbred C57BL , Cyclin-Dependent Kinase 6/metabolism , Cyclin-Dependent Kinase 6/genetics , Mice , Proteolysis , Receptors, IgG/metabolism , Inflammation/immunology , Inflammation/metabolism , Inflammation/pathology , Inflammation/geneticsABSTRACT
Glucagon-like peptide 1 (GLP1), which is mainly processed and cleaved from proglucagon in enteroendocrine cells (EECs) of the intestinal tract, acts on the GLP1 receptor in pancreatic cells to stimulate insulin secretion and to inhibit glucagon secretion. However, GLP1 processing is not fully understood. Here, we show that reticulon 4B (Nogo-B), an endoplasmic reticulum (ER)-resident protein, interacts with the major proglucagon fragment of proglucagon to retain proglucagon on the ER, thereby inhibiting PCSK1-mediated cleavage of proglucagon in the Golgi. Intestinal Nogo-B knockout in male type 2 diabetes mellitus (T2DM) mice increases GLP1 and insulin levels and decreases glucagon levels, thereby alleviating pancreatic injury and insulin resistance. Finally, we identify aberrantly elevated Nogo-B expression and inhibited proglucagon cleavage in EECs from diabetic patients. Our study reveals the subcellular regulatory processes involving Nogo-B during GLP1 production and suggests intestinal Nogo-B as a potential therapeutic target for T2DM.
Subject(s)
Diabetes Mellitus, Type 2 , Endoplasmic Reticulum , Glucagon-Like Peptide 1 , Nogo Proteins , Proglucagon , Proprotein Convertase 1 , Animals , Humans , Male , Mice , Diabetes Mellitus, Type 2/metabolism , Diabetes Mellitus, Type 2/genetics , Endoplasmic Reticulum/metabolism , Enteroendocrine Cells/metabolism , Glucagon-Like Peptide 1/metabolism , Golgi Apparatus/metabolism , HEK293 Cells , Insulin/metabolism , Insulin Resistance , Intestines/pathology , Mice, Inbred C57BL , Mice, Knockout , Nogo Proteins/metabolism , Nogo Proteins/genetics , Proglucagon/metabolism , Proglucagon/genetics , Proprotein Convertase 1/metabolism , Proprotein Convertase 1/genetics , Protein Binding , ProteolysisABSTRACT
Mannheimia haemolytica is the main etiological bacterial agent in ruminant respiratory disease. M. haemolytica secretes leukotoxin, lipopolysaccharides, and proteases, which may be targeted to treat infections. We recently reported the purification and in vivo detection of a 110 kDa Zn metalloprotease with collagenase activity (110-Mh metalloprotease) in a sheep with mannheimiosis, and this protease may be an important virulence factor. Due to the increase in the number of multidrug-resistant strains of M. haemolytica, new alternatives to antibiotics are being explored; one option is lactoferrin (Lf), which is a multifunctional iron-binding glycoprotein from the innate immune system of mammals. Bovine apo-lactoferrin (apo-bLf) possesses many properties, and its bactericidal and bacteriostatic effects have been highlighted. The present study was conducted to investigate whether apo-bLf inhibits the secretion and proteolytic activity of the 110-Mh metalloprotease. This enzyme was purified and sublethal doses of apo-bLf were added to cultures of M. haemolytica or co-incubated with the 110-Mh metalloprotease. The collagenase activity was evaluated using zymography and azocoll assays. Our results showed that apo-bLf inhibited the secretion and activity of the 110-Mh metalloprotease. Molecular docking and overlay assays showed that apo-bLf bound near the active site of the 110-Mh metalloprotease, which affected its enzymatic activity.
Subject(s)
Lactoferrin , Mannheimia haemolytica , Metalloproteases , Proteolysis , Lactoferrin/metabolism , Lactoferrin/pharmacology , Metalloproteases/metabolism , Metalloproteases/antagonists & inhibitors , Animals , Apoproteins/metabolism , Apoproteins/chemistry , Molecular Docking Simulation , Sheep , Cattle , Collagenases/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/chemistry , Zinc/metabolismABSTRACT
Multiple myeloma (MM) is the second most common hematological tumor in adults. Immunomodulatory drugs (IMiDs), such as thalidomide and lenalidomide (Len), are effective drugs for the treatment of multiple myeloma. Len can recruit IKZF1 and IKZF3 to cereblon (CRBN), a substrate receptor of the cullin 4-RING E3 ligase (CRL4), promote their ubiquitination and degradation, and finally inhibit the proliferation of myeloma cells. However, MM patients develop resistance to IMiDs over time, leading to disease recurrence and deterioration. To explore the possible approaches that may enhance the sensitivity of IMiDs to MM, in this study, we used the proximity labeling technique TurboID and quantitative proteomics to identify Lys-63-specific deubiquitinase BRCC36 as a CRBN-interacting protein. Biochemical experiments demonstrated that BRCC36 in the BRISC complex protects CRBN from lysosomal degradation by specifically cleaving the K63-linked polyubiquitin chain on CRBN. Further studies found that a small-molecule compound SHIN1, which binds to BRISC complex subunit SHMT2, can upregulate CRBN by elevating BRCC36. The combination of SHIN1 and Len can further increase the sensitivity of MM cells to IMiDs. Therefore, this study provides the basis for the exploration of a possible strategy for the SHIN1 and Len combination treatment for MM.
Subject(s)
Adaptor Proteins, Signal Transducing , Lenalidomide , Lysosomes , Multiple Myeloma , Ubiquitin-Protein Ligases , Humans , Multiple Myeloma/pathology , Multiple Myeloma/drug therapy , Multiple Myeloma/metabolism , Lenalidomide/pharmacology , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/genetics , Lysosomes/metabolism , Lysosomes/drug effects , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Cell Line, Tumor , Ubiquitination/drug effects , Proteolysis/drug effects , Drug Resistance, Neoplasm/drug effects , Cell Proliferation/drug effects , Deubiquitinating Enzymes/metabolism , Deubiquitinating Enzymes/antagonists & inhibitorsABSTRACT
To survive extreme desiccation, seeds enter a period of quiescence that can last millennia. Seed quiescence involves the accumulation of protective storage proteins and lipids through unknown adjustments in protein homeostasis (proteostasis). Here, we show that mutation of all six type-II metacaspase (MCA-II) proteases in Arabidopsis thaliana disturbs proteostasis in seeds. MCA-II mutant seeds fail to restrict the AAA ATPase CELL DIVISION CYCLE 48 (CDC48) at the endoplasmic reticulum to discard misfolded proteins, compromising seed storability. Endoplasmic reticulum (ER) localization of CDC48 relies on the MCA-IIs-dependent cleavage of PUX10 (ubiquitination regulatory X domain-containing 10), the adaptor protein responsible for titrating CDC48 to lipid droplets. PUX10 cleavage enables the shuttling of CDC48 between lipid droplets and the ER, providing an important regulatory mechanism sustaining spatiotemporal proteolysis, lipid droplet dynamics, and protein homeostasis. In turn, the removal of the PUX10 adaptor in MCA-II mutant seeds partially restores proteostasis, CDC48 localization, and lipid droplet dynamics prolonging seed lifespan. Taken together, we uncover a proteolytic module conferring seed longevity.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Endoplasmic Reticulum , Lipid Droplets , Mutation , Seeds , Valosin Containing Protein , Arabidopsis/genetics , Arabidopsis/metabolism , Seeds/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/genetics , Endoplasmic Reticulum/metabolism , Valosin Containing Protein/metabolism , Valosin Containing Protein/genetics , Lipid Droplets/metabolism , Proteostasis , Proteolysis , Gene Expression Regulation, Plant , Longevity/physiology , Longevity/geneticsABSTRACT
Cholesterol is required to maintain the functional integrity of cellular membrane systems and signalling pathways, but its supply must be closely and dynamically regulated because excess cholesterol is toxic. Sterol regulatory element-binding protein 2 (SREBP2) and the ER-resident protein HMG-CoA reductase (HMGCR) are key regulators of cholesterol biosynthesis. Here, we assessed the mechanistic aspects of their regulation in hepatic cells. Unexpectedly, we found that the transcriptionally active fragment of SREBP2 (N-SREBP2) was produced constitutively. Moreover, in the absence of an exogenous cholesterol supply, nuclear N-SREBP2 became resistant to proteasome-mediated degradation. This resistance was paired with increased occupancy at the HMGCR promoter and HMGCR expression. Inhibiting nuclear N-SREBP2 degradation did not increase HMGCR RNA levels; this increase required cholesterol depletion. Our findings, combined with previous physiological and biophysical investigations, suggest a new model of SREBP2-mediated regulation of cholesterol biosynthesis in the organ that handles large and rapid fluctuations in the dietary supply of this key lipid. Specifically, in the nucleus, cholesterol and the ubiquitin-proteasome system provide a short-loop system that modulates the rate of cholesterol biosynthesis via regulation of nuclear N-SREBP2 turnover and HMGCR expression. Our findings have important implications for maintaining cellular cholesterol homeostasis and lowering blood cholesterol via the SREBP2-HMGCR axis.
Subject(s)
Cholesterol , Homeostasis , Hydroxymethylglutaryl CoA Reductases , Sterol Regulatory Element Binding Protein 2 , Sterol Regulatory Element Binding Protein 2/metabolism , Cholesterol/metabolism , Humans , Hydroxymethylglutaryl CoA Reductases/metabolism , Proteasome Endopeptidase Complex/metabolism , Cell Nucleus/metabolism , Promoter Regions, Genetic/genetics , Hep G2 Cells , Animals , Proteolysis/drug effectsABSTRACT
Proteolysis targeting chimeras (PROTACs) are bifunctional compounds that recruit an E3 ligase to a target protein to induce ubiquitination and degradation of the target. Rational optimization of PROTAC requires a structural model of the ternary complex. In the absence of an experimental structure, computational tools have emerged that attempt to predict PROTAC ternary complexes. Here, we systematically benchmark three commonly used tools: PRosettaC, MOE, and ICM. We find that these PROTAC-focused methods produce an array of ternary complex structures, including some that are observed experimentally, but also many that significantly deviate from the crystal structure. Molecular dynamics simulations show that PROTAC complexes may exist in a multiplicity of configurational states and question the use of experimentally observed structures as a reference for accurate predictions. The pioneering computational tools benchmarked here highlight the promises and challenges in the field and may be more valuable when guided by clear structural and biophysical data. The benchmarking data set that we provide may also be valuable for evaluating other and future computational tools for ternary complex modeling.
Subject(s)
Benchmarking , Molecular Dynamics Simulation , Proteolysis , Protein Conformation , Proteins/chemistry , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolismABSTRACT
BACKGROUND: CDK4 is highly expressed and associated with poor prognosis and decreased survival in advanced neuroblastoma (NB). Targeting CDK4 degradation presents a potentially promising therapeutic strategy compared to conventional CDK4 inhibitors. However, the autophagic degradation of the CDK4 protein and its anti-proliferation effect in NB cells has not been mentioned. RESULTS: We identified autophagy as a new pathway for the degradation of CDK4. Firstly, autophagic degradation of CDK4 is critical for NVP-BEZ235-induced G0/G1 arrest, as demonstrated by the overexpression of CDK4, autophagy inhibition, and blockade of autophagy-related genes. Secondly, we present the first evidence that p62 binds to CDK4 and then enters the autophagy-lysosome to degrade CDK4 in a CTSB-dependent manner in NVP-BEZ235 treated NB cells. Similar results regarding the interaction between p62 and CDK4 were observed in the NVP-BEZ235 treated NB xenograft mouse model. CONCLUSIONS: Autophagic degradation of CDK4 plays a pivotal role in G0/G1 cell cycle arrest in NB cells treated with NVP-BEZ235.
Subject(s)
Autophagy , Cyclin-Dependent Kinase 4 , G1 Phase Cell Cycle Checkpoints , Neuroblastoma , Cyclin-Dependent Kinase 4/metabolism , Neuroblastoma/metabolism , Neuroblastoma/pathology , Neuroblastoma/drug therapy , Neuroblastoma/genetics , Humans , Animals , Mice , Autophagy/drug effects , G1 Phase Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Xenograft Model Antitumor Assays , Quinolines/pharmacology , Resting Phase, Cell Cycle/drug effects , Cell Proliferation/drug effects , Imidazoles/pharmacology , Mice, Nude , ProteolysisABSTRACT
Sickle cell disease (SCD) is a prevalent, life-threatening condition attributable to a heritable mutation in ß-hemoglobin. Therapeutic induction of fetal hemoglobin (HbF) can ameliorate disease complications and has been intently pursued. However, safe and effective small-molecule inducers of HbF remain elusive. We report the discovery of dWIZ-1 and dWIZ-2, molecular glue degraders of the WIZ transcription factor that robustly induce HbF in erythroblasts. Phenotypic screening of a cereblon (CRBN)-biased chemical library revealed WIZ as a previously unknown repressor of HbF. WIZ degradation is mediated by recruitment of WIZ(ZF7) to CRBN by dWIZ-1, as resolved by crystallography of the ternary complex. Pharmacological degradation of WIZ was well tolerated and induced HbF in humanized mice and cynomolgus monkeys. These findings establish WIZ degradation as a globally accessible therapeutic strategy for SCD.
Subject(s)
Anemia, Sickle Cell , Antisickling Agents , Fetal Hemoglobin , Kruppel-Like Transcription Factors , Nerve Tissue Proteins , Animals , Humans , Mice , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/genetics , Anemia, Sickle Cell/drug therapy , Anemia, Sickle Cell/metabolism , Antisickling Agents/chemistry , Antisickling Agents/pharmacology , Antisickling Agents/therapeutic use , Crystallography, X-Ray , Drug Discovery , Fetal Hemoglobin/genetics , Fetal Hemoglobin/metabolism , Kruppel-Like Transcription Factors/metabolism , Macaca fascicularis , Nerve Tissue Proteins/metabolism , Proteolysis/drug effects , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Small Molecule Libraries/therapeutic use , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/geneticsABSTRACT
Machine learning (ML) systems can model quantitative structure-property relationships (QSPR) using existing experimental data and make property predictions for new molecules. With the advent of modalities such as targeted protein degraders (TPD), the applicability of QSPR models is questioned and ML usage in TPD-centric projects remains limited. Herein, ML models are developed and evaluated for TPDs' property predictions, including passive permeability, metabolic clearance, cytochrome P450 inhibition, plasma protein binding, and lipophilicity. Interestingly, performance on TPDs is comparable to that of other modalities. Predictions for glues and heterobifunctionals often yield lower and higher errors, respectively. For permeability, CYP3A4 inhibition, and human and rat microsomal clearance, misclassification errors into high and low risk categories are lower than 4% for glues and 15% for heterobifunctionals. For all modalities, misclassification errors range from 0.8% to 8.1%. Investigated transfer learning strategies improve predictions for heterobifunctionals. This is the first comprehensive evaluation of ML for the prediction of absorption, distribution, metabolism, and excretion (ADME) and physicochemical properties of TPD molecules, including heterobifunctional and molecular glue sub-modalities. Taken together, our investigations show that ML-based QSPR models are applicable to TPDs and support ML usage for TPDs' design, to potentially accelerate drug discovery.
Subject(s)
Machine Learning , Humans , Rats , Animals , Quantitative Structure-Activity Relationship , Proteolysis , Cytochrome P-450 CYP3A/metabolism , Cytochrome P-450 CYP3A/chemistry , Protein Binding , PermeabilityABSTRACT
Recently, targeted degradation has emerged as a powerful therapeutic modality. Relying on "event-driven" pharmacology, proteolysis targeting chimeras (PROTACs) can degrade targets and are superior to conventional inhibitors against undruggable proteins. Unfortunately, PROTAC discovery is limited by warhead scarcity and laborious optimization campaigns. To address these shortcomings, analogous protein-based heterobifunctional degraders, known as bioPROTACs, have been developed. Compared to small-molecule PROTACs, bioPROTACs have higher success rates and are subject to fewer design constraints. However, the membrane impermeability of proteins severely restricts bioPROTAC deployment as a generalized therapeutic modality. Here, we present an engineered bioPROTAC template able to complex with cationic and ionizable lipids via electrostatic interactions for cytosolic delivery. When delivered by biocompatible lipid nanoparticles, these modified bioPROTACs can rapidly degrade intracellular proteins, exhibiting near-complete elimination (up to 95% clearance) of targets within hours of treatment. Our bioPROTAC format can degrade proteins localized to various subcellular compartments including the mitochondria, nucleus, cytosol, and membrane. Moreover, substrate specificity can be easily reprogrammed, allowing modular design and targeting of clinically-relevant proteins such as Ras, Jnk, and Erk. In summary, this work introduces an inexpensive, flexible, and scalable platform for efficient intracellular degradation of proteins that may elude chemical inhibition.
Subject(s)
Lipids , Proteolysis , Humans , Proteolysis/drug effects , Lipids/chemistry , Nanoparticles/chemistry , Animals , Cytosol/metabolism , Drug Delivery Systems , Recombinant Proteins/metabolism , Mice , LiposomesABSTRACT
The review is focused on recent drug discovery advances based on targeted protein degradation strategies. This new area of research has exploded leading to the development of potential drugs useful in a large variety of human diseases. They first target disease relevant proteins difficult to counteract with other classical strategies and extend now to aggregates, organelles, nucleic acids or lipidic droplets. These degraders engaged either the ubiquitin-proteasome system for PROTACs and molecular glues (first generation), or the lysosomal system via endosome-lysosome degradation (LYTACs) and autophagy-lysosome degradation (ATTEC, AUTAC, AUTOTAC) (following generations of degraders). PROTACs have expanded from the orthodox heterobifunctional ones to new derivatives such as homo-PROTACs, pro-PROTACs, CLIPTACs, HaloPROTACs, PHOTOTACs, Bac-PROTACs, AbTACs, ARN-PROTACs. The small molecular-weight molecular glues induce the formation of new ternary complexes which implicate the targeted protein and an ubiquitin ligase E3 allowing the protein ubiquinitation followed by its proteasomal degradation. Lysosomal degraders (LYTAC, ATTEC, AUTAC, AUTOTAC) specifically recognize extracellular and membrane proteins or dysfunctional organelles and transport them into lysosomes where they are degraded. They overcome the limitations observed with proteasomal degradations induced by PROTAC and molecular glues and demonstrate their potential to treat human diseases, especially neurodegenerative ones. Pharmaceutical companies are engaged at the world level to develop these new potential drugs targeting cancers, immuno-inflammatory and neurodegenerative diseases as well as a variety of other ones. Efficiency and risks for these novel therapeutic strategies are discussed.
Title: Induction de proximité et dégradation de cibles thérapeutiques par les nouveaux dégradeurs : quels concepts, quels développements, quel futur ? Abstract: La recherche dans le domaine de la dégradation ciblée des protéines s'est considérablement développée conduisant à l'élaboration de nouveaux outils chimiques à visée thérapeutique, les dégradeurs, potentiellement utiles dans diverses pathologies. Une grande variété d'objets à dégrader appartenant à divers compartiments intra- ou extracellulaires (protéines, complexes ou agrégats, organelles, acides nucléiques, gouttelettes lipidiques) a été ciblée à l'aide de ligands déjà existants, d'autres restent à découvrir. Les molécules de première génération, PROTAC et colles moléculaires, utilisent le système ubiquitine-protéasome pour détruire spécifiquement des protéines pathogéniques, certaines considérées jusqu'à présent comme inaccessibles en tant que cibles thérapeutiques. Au cours des cinq dernières années, ont été développés de nouveaux types de PROTAC hétéro-bifonctionnels comme les homo-PROTAC, pro-PROTAC, CLIPTAC, HaloPROTAC, PHOTOTAC, Bac-PROTAC, mais aussi des PROTAC macromoléculaires comme les AbTAC et ARN-PROTAC. Du fait de la grande diversité des substrats dégradés par les lysosomes, de nouveaux dégradeurs impliquant deux voies distinctes ont été ensuite produits : les chimères LYTAC pour la voie endosome-lysosome et les chimères ATTEC, AUTAC et AUTOTAC pour la voie autophagie-lysosome, augmentant ainsi considérablement le champ d'action des dégradeurs. Ces nouvelles molécules reconnaissent spécifiquement des protéines et/ou des organelles et permettent leur transport dans les lysosomes où ils sont dégradés. Les succès obtenus, que ce soit par dégradation protéasomale ou lysosomale pour plusieurs dizaines de dégradeurs (preuves de concepts et études cliniques en cours), expliquent l'intérêt quasi mondial des industries pharmaceutiques pour ces nouvelles molécules. Les challenges posés par leur développement et leur utilisation en clinique sont discutés.