Literature context: h sc-8017; RRID:AB_628423 Rabbit anti-p-S65-Ub Millipore
Translation of mRNAs is tightly regulated and constantly surveyed for errors. Aberrant translation can trigger co-translational protein and RNA quality control processes, impairments of which cause neurodegeneration by still poorly understood mechanism(s). Here we show that quality control of translation of mitochondrial outer membrane (MOM)-localized mRNA intersects with the turnover of damaged mitochondria, both orchestrated by the mitochondrial kinase PINK1. Mitochondrial damage causes stalled translation of complex-I 30 kDa subunit (C-I30) mRNA on MOM, triggering the recruitment of co-translational quality control factors Pelo, ABCE1, and NOT4 to the ribosome/mRNA-ribonucleoprotein complex. Damage-induced ubiquitination of ABCE1 by NOT4 generates poly-ubiquitin signals that attract autophagy receptors to MOM to initiate mitophagy. In the Drosophila PINK1 model, these factors act synergistically to restore mitophagy and neuromuscular tissue integrity. Thus ribosome-associated co-translational quality control generates an early signal to trigger mitophagy. Our results have broad therapeutic implications for the understanding and treatment of neurodegenerative diseases.
Literature context: ruz Biotechnology Cat# sc-8017, RRID:AB_628423; 1:500 dilution) and anti-FLAG
BACKGROUND: The proteome of mitochondria comprises mostly proteins that originate as precursors in the cytosol. Before import into the organelle, such proteins are exposed to cytosolic quality control mechanisms. Multiple lines of evidence indicate a significant contribution of the major cytosolic protein degradation machinery, the ubiquitin-proteasome system, to the quality control of mitochondrial proteins. Proteins that are directed to the mitochondrial intermembrane space (IMS) exemplify an entire class of mitochondrial proteins regulated by proteasomal degradation. However, little is known about how these proteins are selected for degradation. RESULTS: The present study revealed the heterogeneous cytosolic stability of IMS proteins. Using a screening approach, we found that different cytosolic factors are responsible for the degradation of specific IMS proteins, with no single common factor involved in the degradation of all IMS proteins. We found that the Cox12 protein is rapidly degraded when localized to the cytosol, thus providing a sensitive experimental model. Using Cox12, we found that lysine residues but not conserved cysteine residues are among the degron features important for protein ubiquitination. We observed the redundancy of ubiquitination components, with significant roles of Ubc4 E2 ubiquitin-conjugating enzyme and Rsp5 E3 ubiquitin ligase. The amount of ubiquitinated Cox12 was inversely related to mitochondrial import efficiency. Importantly, we found that precursor protein ubiquitination blocks its import into mitochondria. CONCLUSIONS: The present study confirms the involvement of ubiquitin-proteasome system in the quality control of mitochondrial IMS proteins in the cytosol. Notably, ubiquitination of IMS proteins prohibits their import into mitochondria. Therefore, ubiquitination directly affects the availability of precursor proteins for organelle biogenesis. Importantly, despite their structural similarities, IMS proteins are not selected for degradation in a uniform way. Instead, specific IMS proteins rely on discrete components of the ubiquitination machinery to mediate their clearance by the proteasome.
Literature context: Cruz Biotechnology Cat#sc-8017; RRID:AB_628423 Horseradish peroxidase (HRP)-co
In cystic fibrosis (CF), deletion of phenylalanine 508 (F508del) in the CFTR channel is associated with misfolding and premature degradation of the mutant protein. Among the known proteins associated with F508del-CFTR processing, the ubiquitin ligase RNF5/RMA1 is particularly interesting. We previously demonstrated that genetic suppression of RNF5 in vivo leads to an attenuation of intestinal pathological phenotypes in CF mice, validating the relevance of RNF5 as a drug target for CF. Here, we used a computational approach, based on ligand docking and virtual screening, to discover inh-02, a drug-like small molecule that inhibits RNF5. In in vitro experiments, treatment with inh-02 modulated ATG4B and paxillin, both known RNF5 targets. In immortalized and primary bronchial epithelial cells derived from CF patients homozygous for the F508del mutation, long-term incubation with inh-02 caused significant F508del-CFTR rescue. This work validates RNF5 as a drug target for CF, providing evidence to support its druggability.
Literature context: Cat.#sc-8017; RRID:AB_628423 Anti-Cleaved caspase-3 Cell Sig
Reactive oxygen species (ROS) have emerged as important signaling molecules that play crucial roles in carcinogenesis and cytotoxic responses. Nrf2 is the master regulator of ROS balance. Thus, uncovering mechanisms of Nrf2 regulation is important for the development of alternative treatment strategies for cancers. Here, we demonstrate that iASPP, a known p53 inhibitor, lowers ROS independently of p53. Mechanistically, iASPP competes with Nrf2 for Keap1 binding via a DLT motif, leading to decreased Nrf2 ubiquitination and increased Nrf2 accumulation, nuclear translocation, and antioxidative transactivation. This iASPP-Keap1-Nrf2 axis promotes cancer growth and drug resistance both in vitro and in vivo. Thus, iASPP is an antioxidative factor and represents a promising target to improve cancer treatment, regardless of p53 status.
Literature context: Cruz Biotechnology Cat# sc-8017 RRID:AB_628423 Chemicals, Peptides, and Recomb
DNA damage tolerance during eukaryotic replication is orchestrated by PCNA ubiquitination. While monoubiquitination activates mutagenic translesion synthesis, polyubiquitination activates an error-free pathway, elusive in mammals, enabling damage bypass by template switching. Fork reversal is driven in vitro by multiple enzymes, including the DNA translocase ZRANB3, shown to bind polyubiquitinated PCNA. However, whether this interaction promotes fork remodeling and template switching in vivo was unknown. Here we show that damage-induced fork reversal in mammalian cells requires PCNA ubiquitination, UBC13, and K63-linked polyubiquitin chains, previously involved in error-free damage tolerance. Fork reversal in vivo also requires ZRANB3 translocase activity and its interaction with polyubiquitinated PCNA, pinpointing ZRANB3 as a key effector of error-free DNA damage tolerance. Mutations affecting fork reversal also induced unrestrained fork progression and chromosomal breakage, suggesting fork remodeling as a global fork slowing and protection mechanism. Targeting these fork protection systems represents a promising strategy to potentiate cancer chemotherapy.
Literature context: a Cruz Biotechnology, #sc-8017, RRID:AB_628423) and 1:4000 goat anti-mouse IgG
In fission yeast, meiosis-specific transcripts are selectively eliminated during vegetative growth by the combined action of the YTH-family RNA-binding protein Mmi1 and the nuclear exosome. Upon nutritional starvation, the master regulator of meiosis Mei2 inactivates Mmi1, thereby allowing expression of the meiotic program. Here, we show that the E3 ubiquitin ligase subunit Not4/Mot2 of the evolutionarily conserved Ccr4-Not complex, which associates with Mmi1, promotes suppression of meiotic transcripts expression in mitotic cells. Our analyses suggest that Mot2 directs ubiquitination of Mei2 to preserve the activity of Mmi1 during vegetative growth. Importantly, Mot2 is not involved in the constitutive pathway of Mei2 turnover, but rather plays a regulatory role to limit its accumulation or inhibit its function. We propose that Mmi1 recruits the Ccr4-Not complex to counteract its own inhibitor Mei2, thereby locking the system in a stable state that ensures the repression of the meiotic program by Mmi1.
Literature context: sc-8017; RRID:AB_628423 Rabbit pol
Genetic studies have elucidated critical roles of Piwi proteins in germline development in animals, but whether Piwi is an actual disease gene in human infertility remains unknown. We report germline mutations in human Piwi (Hiwi) in patients with azoospermia that prevent its ubiquitination and degradation. By modeling such mutations in Piwi (Miwi) knockin mice, we demonstrate that the genetic defects are directly responsible for male infertility. Mechanistically, we show that MIWI binds the histone ubiquitin ligase RNF8 in a Piwi-interacting RNA (piRNA)-independent manner, and MIWI stabilization sequesters RNF8 in the cytoplasm of late spermatids. The resulting aberrant sperm show histone retention, abnormal morphology, and severely compromised activity, which can be functionally rescued via blocking RNF8-MIWI interaction in spermatids with an RNF8-N peptide. Collectively, our findings identify Piwi as a factor in human infertility and reveal its role in regulating the histone-to-protamine exchange during spermiogenesis.
Literature context: ubiquitin (P4D1, 1:1000; RRID; AB_628423; Santa Cruz Biotechnology), mou
Synapse formation and growth are tightly controlled processes. How synaptic growth is terminated after reaching proper size remains unclear. Here, we show that Leon, the Drosophila USP5 deubiquitinase, controls postsynaptic growth. In leon mutants, postsynaptic specializations of neuromuscular junctions are dramatically expanded, including the subsynaptic reticulum, the postsynaptic density, and the glutamate receptor cluster. Expansion of these postsynaptic features is caused by a disruption of ubiquitin homeostasis with accumulation of free ubiquitin chains and ubiquitinated substrates in the leon mutant. Accumulation of Ubiquilin (Ubqn), the ubiquitin receptor whose human homolog ubiquilin 2 is associated with familial amyotrophic lateral sclerosis, also contributes to defects in postsynaptic growth and ubiquitin homeostasis. Importantly, accumulations of postsynaptic proteins cause different aspects of postsynaptic overgrowth in leon mutants. Thus, the deubiquitinase Leon maintains ubiquitin homeostasis and proper Ubqn levels, preventing postsynaptic proteins from accumulation to confine postsynaptic growth.
Literature context: 01) and 1:1,000 Ubiquitin (Cat. sc-8017) (Santa Cruz Biotechnology); an
Heat shock protein-90 (Hsp90) is an essential molecular chaperone in eukaryotes involved in maintaining the stability and activity of numerous signalling proteins, also known as clients. Hsp90 ATPase activity is essential for its chaperone function and it is regulated by co-chaperones. Here we show that the tumour suppressor FLCN is an Hsp90 client protein and its binding partners FNIP1/FNIP2 function as co-chaperones. FNIPs decelerate the chaperone cycle, facilitating FLCN interaction with Hsp90, consequently ensuring FLCN stability. FNIPs compete with the activating co-chaperone Aha1 for binding to Hsp90, thereby providing a reciprocal regulatory mechanism for chaperoning of client proteins. Lastly, downregulation of FNIPs desensitizes cancer cells to Hsp90 inhibitors, whereas FNIPs overexpression in renal tumours compared with adjacent normal tissues correlates with enhanced binding of Hsp90 to its inhibitors. Our findings suggest that FNIPs expression can potentially serve as a predictive indicator of tumour response to Hsp90 inhibitors.
Literature context: sc-8017, RRID:AB_628423), anti-NF-
The disruption of protein quality control networks is central to pathology in Huntington's disease (HD) and other neurodegenerative disorders. The aberrant accumulation of insoluble high-molecular-weight protein complexes containing the Huntingtin (HTT) protein and SUMOylated protein corresponds to disease manifestation. We previously identified an HTT-selective E3 SUMO ligase, PIAS1, that regulates HTT accumulation and SUMO modification in cells. Here we investigated whether PIAS1 modulation in neurons alters HD-associated phenotypes in vivo. Instrastriatal injection of a PIAS1-directed miRNA significantly improved behavioral phenotypes in rapidly progressing mutant HTT (mHTT) fragment R6/2 mice. PIAS1 reduction prevented the accumulation of mHTT and SUMO- and ubiquitin-modified proteins, increased synaptophysin levels, and normalized key inflammatory markers. In contrast, PIAS1 overexpression exacerbated mHTT-associated phenotypes and aberrant protein accumulation. These results confirm the association between aberrant accumulation of expanded polyglutamine-dependent insoluble protein species and pathogenesis, and they link phenotypic benefit to reduction of these species through PIAS1 modulation.
Literature context: ouse, monoclonal, sc-8017, RRID:AB_6284231:1000 (WB)/1:500 (IHC)Anti-Î²-tu
BACKGROUND: UbcM2 is a ubiquitin-conjugating enzyme with roles in the turnover of damaged and misfolded proteins, cell cycle progression, development, and regulation of the antioxidant transcription factor, Nrf2. Recent screens have identified binding partners of the enzyme that are associated with various neurodegenerative diseases, and our previous studies have shown that UbcM2 is enriched in retina and brain. RESULTS: In the current study, we characterized UbcM2 protein expression in various structures and cell types in the murine brain. Immunofluorescence analysis of paraffin-embedded brain sections revealed that UbcM2 is ubiquitously expressed throughout the brain, is enriched in hindbrain and cortex, and is robustly expressed in neurons. In contrast, the enzyme is undetectable in most astrocytes and microglia. As dysfunction of the ubiquitin proteasome system (UPS) has been linked to many age-related neurological diseases, we compared UbcM2 expression levels in young versus aged wild-type mice and found a global decrease in expression in aged brains, with reductions of 10 % or greater in five substructures (cerebellar granule cell layer, primary motor cortex, olfactory nucleus, superior colliculus, and secondary visual cortex). CONCLUSIONS: These studies represent the first protein expression profiling of a ubiquitin-conjugating enzyme in the brain and support the notion that deficits in protein degradation and proteostasis associated with neurodegenerative diseases may be, in part, attributable to age-dependent reductions in the enzymatic machinery of the UPS.
Literature context: (sc-8017, RRID:AB_628423) and anti
Protein S-palmitoylation is a reversible post-translational modification that regulates many key biological processes, although the full extent and functions of protein S-palmitoylation remain largely unexplored. Recent developments of new chemical methods have allowed the establishment of palmitoyl-proteomes of a variety of cell lines and tissues from different species. As the amount of information generated by these high-throughput studies is increasing, the field requires centralization and comparison of this information. Here we present SwissPalm ( http://swisspalm.epfl.ch), our open, comprehensive, manually curated resource to study protein S-palmitoylation. It currently encompasses more than 5000 S-palmitoylated protein hits from seven species, and contains more than 500 specific sites of S-palmitoylation. SwissPalm also provides curated information and filters that increase the confidence in true positive hits, and integrates predictions of S-palmitoylated cysteine scores, orthologs and isoform multiple alignments. Systems analysis of the palmitoyl-proteome screens indicate that 10% or more of the human proteome is susceptible to S-palmitoylation. Moreover, ontology and pathway analyses of the human palmitoyl-proteome reveal that key biological functions involve this reversible lipid modification. Comparative analysis finally shows a strong crosstalk between S-palmitoylation and other post-translational modifications. Through the compilation of data and continuous updates, SwissPalm will provide a powerful tool to unravel the global importance of protein S-palmitoylation.