The 26S proteasome degrades the majority of cellular proteins and affects all aspects of cellular life. Therefore, the 26S proteasome abundance, proper assembly, and activity in different life contexts need to be precisely controlled. Impaired proteasome activity is considered a causative factor in several serious disorders. Recent advances in proteasome biology have revealed that the proteasome can be activated by different factors or small molecules. Thus, activated ubiquitin-dependent proteasome degradation has effects such as extending the lifespan in different models, preventing the accumulation of protein aggregates, and reducing their negative impact on cells. Increased 26S proteasome-mediated degradation reduces proteotoxic stress and can potentially improve the efficacy of engineered degraders, such as PROTACs, particularly in situations characterized by proteasome malfunction. Here, emerging ideas and recent insights into the pharmacological activation of the proteasome at the transcriptional and posttranslational levels are summarized.
- Publication type
- Journal Article MeSH
- Review MeSH
BACKGROUND: Renal cell carcinoma (RCC) is a disease typified by anomalies in cell metabolism. The function of mitochondria, including subunits of mitochondrial respiratory complex II (CII), in particular SDHB, are often affected. Here we investigated the state and function of CII in RCC patients. METHODS: We evaluated tumour tissue as well as the adjacent healthy kidney tissue of 78 patients with RCC of different histotypes, focusing on their mitochondrial function. As clear cell RCC (ccRCC) is by far the most frequent histotype of RCC, we focused on these patients, which were grouped based on the pathological WHO/ISUP grading system to low- and high-grade patients, indicative of prognosis. We also evaluated mitochondrial function in organoids derived from tumour tissue of 7 patients. RESULTS: ccRCC tumours were characterized by mutated von Hippel-Lindau gene and high expression of carbonic anhydrase IX. We found low levels of mitochondrial DNA, protein and function, together with CII function in ccRCC tumour tissue, but not in other RCC types and non-tumour tissues. Mitochondrial content increased in high-grade tumours, while the function of CII remained low. Tumour organoids from ccRCC patients recapitulated molecular characteristics of RCC tissue. CONCLUSIONS: Our findings suggest that the state of CII, epitomized by its assembly and SDHB levels, deteriorates with the progressive severity of ccRCC. These observations hold the potential for stratification of patients with worse prognosis and may guide the exploration of targeted therapeutic interventions.
- MeSH
- Antigens, Neoplasm MeSH
- Adult MeSH
- Carbonic Anhydrase IX metabolism genetics MeSH
- Carcinoma, Renal Cell * pathology metabolism genetics MeSH
- Middle Aged MeSH
- Humans MeSH
- DNA, Mitochondrial genetics metabolism MeSH
- Mitochondria * metabolism pathology genetics MeSH
- Mutation MeSH
- Von Hippel-Lindau Tumor Suppressor Protein genetics metabolism MeSH
- Kidney Neoplasms * pathology metabolism genetics MeSH
- Electron Transport Complex II * metabolism genetics MeSH
- Aged MeSH
- Succinate Dehydrogenase genetics metabolism MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
Mitochondria are key to cellular energetics, metabolism, and signaling. Their dysfunction is linked to devastating diseases, including mitochondrial disorders, diabetes, neurodegenerative diseases, cardiac disorders, and cancer. Here, we present a knockout mouse model lacking the complex IV assembly factor SMIM20/MITRAC7. SMIM20-/- mice display cardiac pathology with reduced heart weight and cardiac output. Heart mitochondria present with reduced levels of complex IV associated with increased complex I activity, have altered fatty acid oxidation, and display elevated levels of ROS production. Interestingly, mutant mouse ventricular myocytes show unphysiological Ca2+ handling, which can be attributed to the increase in mitochondrial ROS production. Our study presents an example of a tissue-specific phenotype in the context of OXPHOS dysfunction. Moreover, our data suggest a link between complex IV dysfunction and Ca2+ handling at the endoplasmic reticulum through ROS signaling.
- MeSH
- Endoplasmic Reticulum metabolism MeSH
- Myocytes, Cardiac metabolism MeSH
- Membrane Proteins * metabolism genetics MeSH
- Mitochondrial Proteins * metabolism genetics MeSH
- Myocardium * metabolism MeSH
- Mice, Inbred C57BL MeSH
- Mice, Knockout MeSH
- Mice MeSH
- Oxidative Phosphorylation MeSH
- Zebrafish Proteins MeSH
- Reactive Oxygen Species metabolism MeSH
- Electron Transport Complex IV * metabolism MeSH
- Mitochondria, Heart metabolism MeSH
- Calcium metabolism MeSH
- Calcium Signaling * MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Tumor suppressor p53 is a key player in the cell response to DNA damage that suffers by frequent inactivating aberrations. Some of them disturb p53 oligomerization and influence cell decision between proliferation, growth arrest and apoptosis. Active p53 resides mostly in the nucleus, degradation occurs in the cytoplasm. Acute myeloid leukemia (AML)-related mutation of NPM (NPMmut) induces massive mislocalization of p53 to the cytoplasm, which might be related to leukemia initiation. Since both proteins interact and execute their function as oligomers, we investigated the role of perturbed p53 oligomerization in the p53 mislocalization process in live cells by FLIM (fluorescence lifetime imaging microscopy), fluorescence anisotropy imaging (FAIM), fluorescence cross-correlation spectroscopy (FCCS) and immunochemical methods. On a set of fluorescently labeled p53 variants, monomeric R337G and L344P, dimeric L344A, and multimeric D352G and A353S, we correlated their cellular localization, oligomerization and interaction with NPMmut. Interplay between nuclear export signal (NES) and nuclear localization signal (NLS) of p53 was investigated as well. While NLS was found critical for the nuclear p53 localization, NES plays less significant role. We observed cytoplasmic translocation only for multimeric A353S variant with sufficient stability and strong interaction with NPMmut. Less stable multimer D352G and L344A dimer were not translocated, monomeric p53 variants always resided in the nucleus independently of the presence of NPMmut and NES intactness. Oligomeric state of NPMmut is not required for p53 translocation, which happens also in the presence of the nonoligomerizing NPMmut variant. The prominent structural and functional role of the R337 residue is shown.
- MeSH
- Leukemia, Myeloid, Acute * genetics metabolism MeSH
- Cell Nucleus metabolism MeSH
- Cytoplasm metabolism MeSH
- Nuclear Localization Signals metabolism MeSH
- Nuclear Proteins * genetics metabolism MeSH
- Humans MeSH
- Protein Multimerization MeSH
- Mutation * MeSH
- Cell Line, Tumor MeSH
- Tumor Suppressor Protein p53 * metabolism genetics chemistry MeSH
- Nucleophosmin MeSH
- Nuclear Export Signals MeSH
- Protein Transport MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Nucleotide excision repair (NER) is crucial for repairing bulky lesions and crosslinks in DNA caused by exogenous and endogenous genotoxins. The number of studies that have considered DNA repair as a biomarker is limited, and therefore one of the primary objectives of the European COST Action hCOMET (CA15132) was to assemble and analyse a pooled database of studies with data on NER activity. The database comprised 738 individuals, gathered from 5 laboratories that ran population studies using the comet-based in vitro DNA repair assay. NER activity data in peripheral blood mononuclear cells were normalized and correlated with various host-related factors, including sex, age, body mass index (BMI), and smoking habits. This multifaceted analysis uncovered significantly higher NER activity in female participants compared to males (1.08 ± 0.74 vs. 0.92 ± 0.71; P = .002). Higher NER activity was seen in older subjects (>30 years), and the effect of age was most pronounced in the oldest females, particularly those over 70 years (P = .001). Females with a normal BMI (<25 kg/m2) exhibited the highest levels of NER, whereas the lowest NER was observed in overweight males (BMI ≥ 25 kg/m2). No independent effect of smoking was found. After stratification by sex and BMI, higher NER was observed in smoking males (P = .017). The biological implication of higher or lower repair capacity remains unclear; the inclusion of DNA repair as a biomarker in molecular epidemiological trials should elucidate the link between health and disease status.
- MeSH
- Adult MeSH
- Excision Repair MeSH
- Body Mass Index MeSH
- Comet Assay MeSH
- Smoking MeSH
- Leukocytes, Mononuclear metabolism MeSH
- Middle Aged MeSH
- Humans MeSH
- DNA Repair * MeSH
- DNA Damage MeSH
- Aged MeSH
- Sex Factors MeSH
- Age Factors MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
Quantitative genomic mapping of DNA damage may provide insights into the underlying mechanisms of damage and repair. Sequencing based approaches are bound to the limitations of PCR amplification bias and read length which hamper both the accurate quantitation of damage events and the ability to map them to structurally complex genomic regions. Optical Genome mapping in arrays of parallel nanochannels allows physical extension and genetic profiling of millions of long genomic DNA fragments, and has matured to clinical utility for characterization of complex structural aberrations in cancer genomes. Here we present a new mapping modality, Repair-Assisted Damage Detection - Optical Genome Mapping (RADD-OGM), a method for single-molecule level mapping of DNA damage on a genome-wide scale. Leveraging ultra-long reads to assemble the complex structure of a sarcoma cell-line genome, we mapped the genomic distribution of oxidative DNA damage, identifying regions more susceptible to DNA oxidation. We also investigated DNA repair by allowing cells to repair chemically induced DNA damage, pinpointing locations of concentrated repair activity, and highlighting variations in repair efficiency. Our results showcase the potential of the method for toxicogenomic studies, mapping the effect of DNA damaging agents such as drugs and radiation, as well as following specific DNA repair pathways by selective induction of DNA damage. The facile integration with optical genome mapping enables performing such analyses even in highly rearranged genomes such as those common in many cancers, a challenging task for sequencing-based approaches.
- MeSH
- Bromates toxicity MeSH
- Humans MeSH
- Chromosome Mapping * instrumentation methods MeSH
- Microfluidic Analytical Techniques * instrumentation methods MeSH
- Cell Line, Tumor MeSH
- Nanotechnology * instrumentation methods MeSH
- DNA Repair genetics MeSH
- Oxidative Stress drug effects genetics MeSH
- DNA Damage * genetics MeSH
- Gene Expression Regulation MeSH
- Gene Expression Profiling MeSH
- Toxicogenetics * instrumentation methods MeSH
- DNA Copy Number Variations MeSH
- Single Molecule Imaging * instrumentation methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Activation of the Wnt/β-catenin pathway crucially depends on the polymerization of dishevelled 2 (DVL2) into biomolecular condensates. However, given the low affinity of known DVL2 self-interaction sites and its low cellular concentration, it is unclear how polymers can form. Here, we detect oligomeric DVL2 complexes at endogenous protein levels in human cell lines, using a biochemical ultracentrifugation assay. We identify a low-complexity region (LCR4) in the C-terminus whose deletion and fusion decreased and increased the complexes, respectively. Notably, LCR4-induced complexes correlated with the formation of microscopically visible multimeric condensates. Adjacent to LCR4, we mapped a conserved domain (CD2) promoting condensates only. Molecularly, LCR4 and CD2 mediated DVL2 self-interaction via aggregating residues and phenylalanine stickers, respectively. Point mutations inactivating these interaction sites impaired Wnt pathway activation by DVL2. Our study discovers DVL2 complexes with functional importance for Wnt/β-catenin signaling. Moreover, we provide evidence that DVL2 condensates form in two steps by pre-oligomerization via high-affinity interaction sites, such as LCR4, and subsequent condensation via low-affinity interaction sites, such as CD2.
- MeSH
- beta Catenin metabolism genetics MeSH
- HEK293 Cells MeSH
- Humans MeSH
- Protein Multimerization MeSH
- Dishevelled Proteins * metabolism genetics MeSH
- Wnt Signaling Pathway * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Histones are positively charged proteins found in the chromatin of eukaryotic cells. They regulate gene expression and are required for the organization and packaging of DNA within the nucleus. Histones are extremely conserved, allowing for transcription, replication, and repair. This review delves into their complex structure and function in DNA assembly, their role in nucleosome assembly, and the higher-order chromatin structures they generate. We look at the five different types of histone proteins: H1, H2A, H2B, H3, H4, and their variations. These histones bind with DNA to produce nucleosomes, the basic units of chromatin that are essential for compacting DNA and controlling its accessibility. Their dynamic control of chromatin accessibility has important implications for genomic stability and cellular activities. We elucidate regulatory mechanisms in both normal and pathological situations by investigating their structural features, diverse interaction mechanisms, and chromatin impact. In addition, we discuss the functions of histone post-translational modifications (PTMs) and their significance in various disorders. These alterations, which include methylation, acetylation, phosphorylation, and ubiquitination, are crucial in regulating histone function and chromatin dynamics. We specifically describe and explore the role of changed histones in the evolution of cancer, neurological disorders, sepsis, autoimmune illnesses, and inflammatory conditions. This comprehensive review emphasizes histone's critical role in genomic integrity and their potential as therapeutic targets in various diseases.
- MeSH
- Chromatin metabolism genetics chemistry MeSH
- DNA * metabolism chemistry MeSH
- Genome MeSH
- Histones * metabolism chemistry genetics MeSH
- Humans MeSH
- Neoplasms genetics metabolism MeSH
- Protein Processing, Post-Translational MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
The cytokine TNF can trigger highly proinflammatory RIPK1-dependent cell death. Here, we show that the two adapter proteins, TANK and AZI2, suppress TNF-induced cell death by regulating the activation of TBK1 kinase. Mice lacking either TANK or AZI2 do not show an overt phenotype. Conversely, animals deficient in both adapters are born in a sub-Mendelian ratio and suffer from severe multi-organ inflammation, excessive antibody production, male sterility, and early mortality, which can be rescued by TNFR1 deficiency and significantly improved by expressing a kinase-dead form of RIPK1. Mechanistically, TANK and AZI2 both recruit TBK1 to the TNF receptor signaling complex, but with distinct kinetics due to interaction with different complex components. While TANK binds directly to the adapter NEMO, AZI2 is recruited later via deubiquitinase A20. In summary, our data show that TANK and AZI2 cooperatively sustain TBK1 activity during different stages of TNF receptor assembly to protect against autoinflammation.
- MeSH
- Adaptor Proteins, Signal Transducing * metabolism genetics MeSH
- Cell Death MeSH
- Endopeptidases MeSH
- Intracellular Signaling Peptides and Proteins metabolism genetics MeSH
- Humans MeSH
- Mice, Inbred C57BL MeSH
- Mice, Knockout * MeSH
- Mice MeSH
- Protein Serine-Threonine Kinases * metabolism genetics MeSH
- Receptors, Tumor Necrosis Factor, Type I * metabolism genetics MeSH
- Receptor-Interacting Protein Serine-Threonine Kinases * metabolism genetics MeSH
- Signal Transduction MeSH
- Tumor Necrosis Factor-alpha * metabolism MeSH
- Tumor Necrosis Factor alpha-Induced Protein 3 metabolism genetics MeSH
- Inflammation metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
MICAL proteins play a crucial role in cellular dynamics by binding and disassembling actin filaments, impacting processes like axon guidance, cytokinesis, and cell morphology. Their cellular activity is tightly controlled, as dysregulation can lead to detrimental effects on cellular morphology. Although previous studies have suggested that MICALs are autoinhibited, and require Rab proteins to become active, the detailed molecular mechanisms remained unclear. Here, we report the cryo-EM structure of human MICAL1 at a nominal resolution of 3.1 Å. Structural analyses, alongside biochemical and functional studies, show that MICAL1 autoinhibition is mediated by an intramolecular interaction between its N-terminal catalytic and C-terminal coiled-coil domains, blocking F-actin interaction. Moreover, we demonstrate that allosteric changes in the coiled-coil domain and the binding of the tripartite assembly of CH-L2α1-LIM domains to the coiled-coil domain are crucial for MICAL activation and autoinhibition. These mechanisms appear to be evolutionarily conserved, suggesting a potential universality across the MICAL family.
- MeSH
- Actins metabolism chemistry MeSH
- Allosteric Regulation MeSH
- Calponins MeSH
- Cryoelectron Microscopy * MeSH
- Humans MeSH
- Actin Cytoskeleton metabolism ultrastructure MeSH
- Microfilament Proteins metabolism chemistry ultrastructure MeSH
- Models, Molecular MeSH
- Mixed Function Oxygenases MeSH
- Protein Domains MeSH
- LIM Domain Proteins metabolism chemistry genetics MeSH
- Protein Binding * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
