RIG-I recognizes metabolite-capped RNAs as signaling ligands.

The innate immune receptor RIG-I recognizes 5'-triphosphate double-stranded RNAs (5' PPP dsRNA) as pathogenic RNAs. Such RNA-ends are present in viral genomes and replication intermediates, and they activate the RIG-I signaling pathway to produce a potent interferon response essential for viral clearance. Endogenous mRNAs cap the 5' PPP-end with m7G and methylate the 2'-O-ribose to evade RIG-I, preventing aberrant immune responses deleterious to the cell. Recent studies have identified RNAs in cells capped with metabolites such as NAD+, FAD and dephosphoCoA. Whether RIG-I recognizes these metabolite-capped RNAs has not been investigated. Here, we describe a strategy to make metabolite-capped RNAs free from 5' PPP dsRNA contamination, using in vitro transcription initiated with metabolites. Mechanistic studies show that metabolite-capped RNAs have a high affinity for RIG-I, stimulating the ATPase activity at comparable levels to 5' PPP dsRNA. Cellular signaling assays show that the metabolite-capped RNAs potently stimulate the innate antiviral immune response. This demonstrates that RIG-I can tolerate diphosphate-linked, capped RNAs with bulky groups at the 5' RNA end. This novel class of RNAs that stimulate RIG-I signaling may have cellular roles in activating the interferon response and may be exploited with proper functionalities for RIG-I-related RNA therapeutics.

Amyloid aggregates of the deubiquitinase OTUB1 are neurotoxic, suggesting that they contribute to the development of Parkinson's disease.

Parkinson's disease (PD) is a multifactorial malady and the second most common neurodegenerative disorder, characterized by loss of dopaminergic neurons in the midbrain. A hallmark of PD pathology is the formation of intracellular protein inclusions, termed Lewy bodies (LBs). Recent MS studies have shown that OTU deubiquitinase ubiquitin aldehyde-binding 1 (OTUB1), a deubiquitinating enzyme of the OTU family, is enriched together with α-synuclein in LBs from individuals with PD and is also present in amyloid plaques associated with Alzheimer's disease. In the present study, using mammalian cell cultures and a PD mouse model, along with CD spectroscopy, atomic force microscopy, immunofluorescence-based imaging, and various biochemical assays, we demonstrate that after heat-induced protein aggregation, OTUB1 reacts strongly with both anti-A11 and anti-osteocalcin antibodies, detecting oligomeric, prefibrillar structures or fibrillar species of amyloidogenic proteins, respectively. Further, recombinant OTUB1 exhibited high thioflavin-T and Congo red binding and increased ß-sheet formation upon heat induction. The oligomeric OTUB1 aggregates were highly cytotoxic, characteristic of many amyloid proteins. OTUB1 formed inclusions in neuronal cells and co-localized with thioflavin S and with α-synuclein during rotenone-induced stress. It also co-localized with the disease-associated variant pS129-α-synuclein in rotenone-exposed mouse brains. Interestingly, OTUB1 aggregates were also associated with severe cytoskeleton damage, rapid internalization inside the neuronal cells, and mitochondrial damage, all of which contribute to neurotoxicity. In conclusion, the results of our study indicate that OTUB1 may contribute to LB pathology through its amyloidogenic properties.

Ubiquitin recognition of BAP1: understanding its enzymatic function.

BRCA1-associated protein 1 (BAP1) is a nuclear localizing UCH, having tumor suppressor activity and is widely involved in many crucial cellular processes. BAP1 has garnered attention for its links with cancer, however, the molecular mechanism in the regulation of cancer by BAP1 has not been established. Amongst the four UCHs, only BAP1 and UCHL5 are able to hydrolyze small and large ubiquitin adducts but UCHL5 hydrolyzes only when it is present in the PA700 complex of the proteasome. The ability of BAP1 to cleave large ubiquitin derivatives is because of its relatively longer active-site crossover loop than other UCHs. The mechanism of ubiquitin recognition has not been studied for BAP1. The comparative enzymatic analysis of ubiquitin C-terminal hydrolase L1 (UCHL1), ubiquitin C-terminal hydrolase L3 (UCHL3), ubiquitin C-terminal hydrolase L5 (UCHL5N), and BAP1N has confirmed that enzymatically BAP1 is similar to UCHL5, which corroborates with the bioinformatics analysis done earlier. We have undertaken extensive mutational approaches to gain mechanistic insight into BAP1-ubiquitin interaction. Based on the homology-modeled BAP1 structure, we have identified a few BAP1 residues which possibly play a crucial role in ubiquitin interaction of which a few mutations have been identified in many cancers. Our comparative thermodynamic analysis reveals that BAP1-ubiquitin interaction is majorly driven by entropy factor which is unique amongst UCHs. Our study sheds light on BAP1 interaction with ubiquitin, which will be useful in understanding its enzymatic function.

Cancer associated missense mutations in BAP1 catalytic domain induce amyloidogenic aggregation: A new insight in enzymatic inactivation.

BRCA1 associated protein 1 (BAP1) is a nuclear deubiquitinase that regulates tumor suppressor activity and widely involves many cellular processes ranging from cell cycle regulation to gluconeogenesis. Impairment of enzymatic activity and nuclear localization induce abnormal cell proliferation. It is considered to be an important driver gene, which undergoes frequent mutations in several cancers. However the role of mutation and oncogenic gain of function of BAP1 are poorly understood. Here, we investigated cellular localization, enzymatic activity and structural changes for four missense mutants of the catalytic domain of BAP1, which are prevalent in different types of cancer. These mutations triggered cytoplasmic/perinuclear accumulation in BAP1 deficient cells, which has been observed in proteins that undergo aggregation in cellular condition. Amyloidogenic activity of mutant BAP1 was revealed from its reactivity towards anti oligomeric antibody in HEK293T cells. We have also noted structural destabilization in the catalytic domain mutants, which eventually produced beta amyloid structure as indicated in atomic force microscopy study. The cancer associated mutants up-regulate heat shock response and activates transcription of genes normally co-repressed by BAP1. Overall, our results unambiguously demonstrate that structural destabilization and subsequent aggregation abrogate its cellular mechanism leading to adverse outcome.

Deubiquitinating enzymes in cellular signaling and disease regulation.

Protein post-translational modification by ubiquitin represents a complex signaling system that regulates many cellular events including proteostasis to intercellular communications. Deubiquitinating enzymes (DUBs) that specifically disassemble Ub-chains or regulate ubiquitin homeostasis reside as a central component in ubiquitin signaling. Human genome encodes almost 100 DUBs and majority of them are not well characterized. Considerable progress has been made in the understanding of enzymatic mechanism; however, their cellular substrate specificity and regulation are largely unknown. Involvement of DUBs in disease regulation has been depicted since its discovery and several attempts have been made for evaluating DUBs as a drug target. In this review, we have updated briefly a new insight of DUBs activity, their cellular role, disease regulation, and therapeutic potential.