Ubiquitin C-terminal Hydrolase-1 (UCHL1) is a deubiquitinating enzyme, which plays a key role in Parkinson’s disease (PD). It is one of the most important proteins, which constitute Lewy body in PD patient. However, how this well folded highly soluble protein presents in this proteinaceous aggregate is still unclear. We report here that UCHL1 undergoes S-nitrosylation in vitro and rotenone induced PD mouse model. The preferential nitrosylation in the Cys 90, Cys 152 and Cys 220 has been observed which alters the catalytic activity and structural stability. We show here that nitrosylation induces structural instability and produces amorphous aggregate, which provides a nucleation to the native α-synuclein for faster aggregation. Our findings provide a new link between UCHL1-nitrosylation and PD pathology.
Carboplatin is a chemotherapeutic drug, used for the treatment of different types of cancers, particularly solid tumors. Carboplatin, like other platinum containing drugs, exerts its cytotoxic effect through DNA binding via cross-linking. It forms interstrand and intrastrand cross-linking with DNA. Intrastrand crosslinking is dominant and believed to be conferring antitumoral efficacy of the drug. This cross-linking results in alteration of DNA winding and bending, which hampers DNA replication and transcription and finally leads to cell death. In the present work, we studied the interaction of carboplatin with calfthymus DNA in buffer solution under physiological conditions. Different concentrations of carboplatin were incubated with a constant DNA concentration to form carboplatin-DNA complexes. These complexes were studied with Fourier Transform Infrared (FTIR) spectroscopy and circular dichroism (CD) spectroscopy to understand the binding modes of carboplatin with DNA and its effect on DNA conformation. The results showed that carboplatin binds to DNA through direct interaction of platin-DNA bases (guanine, thymine, adenine and cytosine), with a small perturbation of phosphate group of DNA backbone, while DNA remains in the B-conformation. DNA aggregation was also observed at higher drug concentrations.
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 β-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.
Amsacrine is an anilinoacridine derivative anticancer drug, used to treat a wide variety of malignancies. In cells, amsacrine poisons topoisomerase 2 by stabilizing DNA-drug-enzyme ternary complex. Presence of amsacrine increases the steady-state concentration of these ternary complexes which in turn hampers DNA replication and results in subsequent cell death. Due to reversible binding and rapid slip-out of amsacrine from DNA duplex, structural data is not available on amsacrine-DNA complexes. In the present work, we designed five oligonucleotide duplexes, differing in their minor groove widths and hydration pattern, and examined their binding with amsacrine using attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. Complexes of amsacrine with calf thymus DNA were also evaluated for a comparison. Our results demonstrate for the first time that amsacrine is not a simple intercalator; rather mixed type of DNA binding (intercalation and minor groove) takes place between amsacrine and DNA. Further, this binding is highly sensitive towards the geometries and hydration patterns of different minor grooves present in the DNA. This study shows that ligand binding to DNA could be very sensitive to DNA base composition and DNA groove structures. Results demonstrated here could have implication for understanding cytotoxic mechanism of aminoacridine based anticancer drugs and provide directions to modify these drugs for better efficacy and few side-effects.
α-Synuclein, a major constituent of proteinaceous inclusions named Lewy body, has been shown to be released and taken up by cells, which may facilitate its progressive pathological spreading and neuronal cell death in Parkinson's disease. However, the pathophysiological effect and signaling cascade initiated by extracellular α-synuclein in cellular milieu are not well understood. Herein we have investigated the perturbations induced by low molecular weight α-synuclein and different types of α-synuclein oligomers in the neuroblastoma SH-SY5Y cells. Atomic force microscopy studies have revealed formation of nanopores and enhanced roughness in the cell surface leading to membrane disruption. The damaged membrane allows altered ionic homeostasis leading to activation of nitric oxide synthase (NOS) machinery releasing burst of nitric oxide. The elevated levels of nitric oxide induces S-nitrosylation of key proteins like Actin, DJ-1, HSP70 UCHL1, Parkin, and GAPDH that alter cytoskeletal network, protein folding machinery, ubiquitin proteasome system inducing apoptosis.
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