Structural analysis and conformational dynamics of SOCS1 gene mutations involved in diffuse large B-cell lymphoma.

OBJECTIVES: The SOCS1 gene is frequently mutated in primary Diffuse Large B-Cell Lymphoma (DLBCL) patients and is associated with a reduced survival rate. Using various computational techniques, the current study aims to identify Single Nucleotide Polymorphisms (SNPs) in the SOCS1 gene that are associated with the mortality rate of DLBCL patients. This study also evaluates the effects of SNPs on the structural instability of the SOCS1 protein in DLBCL patient. METHODS: The cBioPortal webserver was used for mutations and determining how the SNP mutations affect the SOCS1 protein with various algorithms (PolyPhen-2.0, Provean, PhD-SNPg, SNPs&GO, SIFT, FATHMM, Predict SNP and SNAP). Five webservers (I-Mutant 2.0, MUpro, mCSM, DUET and SDM) were used for protein instability and the conserved status and were also predicted through different tools (ConSurf, Expasy, SOMPA). Lastly, MD simulations were run on the two chosen mutations (S116N and V128G) using GROMACS 5.0.1 to study how the mutations change the structure of SOCS1. RESULTS: Among the 93 SOCS1 mutations detected in DLBCL patients, nine mutations were found to have a detrimental effect (damaging/deleterious/pathogenic/altered) on the SOCS1 protein. All the nine selected mutations are in the conserved region and four are on the extended strand site, four on the random coil site and one on the alpha helix position of the secondary protein structure. After anticipating the structural effects of these nine mutations, two were chosen (S116N and V128G) based on mutational frequency, location within the protein, structural effect (primary, secondary and tertiary) on stability and conservation status within the SOCS1 protein. The simulation of a 50 ns time interval revealed that the Rg value of S116N (2.17 nm) is higher than that of WT (1.98 nm), indicating a loss of structural compactness. In the case of the RMSD value, this mutated type (V128G) shows more deviation (1.54 nm) in comparison to the wild-type (2.14 nm) and another mutant type (S116N) (2.12 nm). The average RMSF values of wild-type and mutant types (V128G and S116N) were 0.88 nm, 0.49 nm, and 0.93 nm, respectively. The RMSF result shows that the mutant V128G structure is more stable than the wild-type and mutant S116N structures. CONCLUSION: Based on all these computational predictions, this study finds that certain mutations, particularly S116N, have a destabilising and robust effect on the SOCS1 protein. These results can be used to learn more about the importance of SOCS1 mutations in DLBCL patients and to develop new ways to treat DLBCL.

Early loss of endogenous NAD+ following rotenone treatment leads to mitochondrial dysfunction and Sarm1 induction that is ameliorated by PARP inhibition.

Sarm1 is an evolutionary conserved innate immune adaptor protein that has emerged as a primary regulator of programmed axonal degeneration over the past decade. In vitro structural insights have revealed that although Sarm1 induces energy depletion by breaking down nicotinamide adenine dinucleotide+ (NAD+ ), it is also allosterically inhibited by NAD+ . However, how NAD+ levels modulate the activation of intracellular Sarm1 has not been elucidated so far. This study focuses on understanding the events leading to Sarm1 activation in both neuronal and non-neuronal cells using the mitochondrial complex I inhibitor rotenone. Here, we report the regulation of rotenone-induced cell death by loss of NAD+ that may act as a 'biological trigger' of Sarm1 activation. Our study revealed that early loss of endogenous NAD+ levels arising due to PARP1 hyperactivation preceded Sarm1 induction following rotenone treatment. Interestingly, replenishing NAD+ levels by the PARP inhibitor, PJ34 restored mitochondrial complex I activity and also prevented subsequent Sarm1 activation in rotenone-treated cells. These cellular data were further validated in Drosophila melanogaster where a significant reduction in rotenone-mediated loss of locomotor abilities, and reduced dSarm expression was observed in the flies following PARP inhibition. Taken together, these observations not only uncover a novel regulation of Sarm1 induction by endogenous NAD+ levels but also point towards an important understanding on how PARP inhibitors could be repurposed in the treatment of mitochondrial complex I deficiency disorders.

Development of Low-Grade Serous Ovarian Carcinoma from Benign Ovarian Serous Cystadenoma Cells.

Despite the knowledge about numerous genetic mutations essential for the progression of low-grade serous ovarian carcinoma (LGSOC), the specific combination of mutations required remains unclear. Here, we aimed to recognize the oncogenic mutations responsible for the stepwise development of LGSOC using immortalized HOVs-cyst-1 cells, developed from ovarian serous cystadenoma cells, and immortalized via cyclin D1, CDK4R24C, and hTERT gene transfection. Furthermore, oncogenic mutations, KRAS and PIK3CA, were individually and simultaneously introduced in immortalized HOV-cyst-1 cells. Cell functions were subsequently analyzed via in vitro assays. KRAS or PIK3CA double mutant HOV-cyst-1 cells exhibited higher cell proliferation and migration capacity than the wild-type cells, or those with either a KRAS or a PIK3CA mutation, indicating that these mutations play a causative role in LGSOC tumorigenesis. Moreover, KRAS and PIK3CA double mutants gained tumorigenic potential in nude mice, whereas the cells with a single mutant exhibited no signs of tumorigenicity. Furthermore, the transformation of HOV-cyst-1 cells with KRAS and PIK3CA mutants resulted in the development of tumors that were grossly and histologically similar to human LGSOCs. These findings suggest that simultaneous activation of the KRAS/ERK and PIK3CA/AKT signaling pathways is essential for LGSOC development.

High Frequency of PIK3CA Mutations in Low-Grade Serous Ovarian Carcinomas of Japanese Patients.

The frequency of KRAS/BRAF mutations associated with low-grade serous ovarian carcinoma (LGSC)/serous borderline tumors (SBTs) in Japan is unknown. We aimed to identify genetic variations in KRAS, BRAF, PIK3CA, and ERBB2 in LGSC/SBT/serous cystadenomas (SCAs) in a Japanese population. We performed a mutation analysis (by Sanger sequencing) of 33 cases of LGSC/SBT/SCA and 4 cases of LGSC with synchronous SBTs using microdissected paraffin-embedded sections. Immunohistochemistry of p53 and ARID1A was also performed. The frequency of oncogenic mutations in PIK3CA was 60.0% (6/10) in LGSCs, 63.6% (7/11) in SBTs, and 8.3% (1/12) in SCAs. All cases harbored wild-type KRAS. The frequency of BRAF mutations was 20.0% (2/10) in LGSCs, whereas all SBTs and SCAs harbored the wild-type allele. The frequency of ERBB2 mutations was 30.0% (3/10) in LGSCs, 0.0% (0/11) in SBTs, and 16.7% (2/12) in SCAs. ARID1A staining was positive in all cases. p53 staining was positive in 0% (0/10) LGSCs, 9.1% (1/11) SBTs, and 0.0% (0/12) SCAs. One LGSC case had two PIK3CA mutations (G1633A and G3149A) in both LGSC and SBT lesions, but a BRAF mutation was detected only in an LGSC lesion. These results suggest that, compared with the values in Western populations (16-54%), the KRAS mutation frequency in LGSCs/SBTs is lower and that of PIK3CA mutations in LGSCs/SBTs is much higher in Japanese populations. Therefore, the main carcinogenesis signaling pathways may be different between Japanese and Western LGSCs. Molecular therapies targeting the PIK3CA/AKT pathway may be effective in LGSCs in Japan.

Sarm1 induction and accompanying inflammatory response mediates age-dependent susceptibility to rotenone-induced neurotoxicity.

Aging is a complex biological process and environmental risk factors like pesticide exposure have been implicated in the increased incidence of age-related neurodegenerative diseases like Parkinson's disease (PD) but the etiology remains unknown. There is also lack of a proper animal model system to study the progressive effect of these environmental toxins on age-associated neurodegeneration. In this study, we established a drosophila model of aging to study the age-dependent vulnerability to the environmental toxin rotenone that has been implicated in sporadic cases of PD. We demonstrate that age plays a determining role in the increased susceptibility to chronic rotenone exposure that is accompanied by severe locomotor deficits, decreased lifespan and loss of dopaminergic (DA) neurons. Chronic low dose exposure to rotenone results in the rapid induction of the neurodegenerative molecule SARM1/dSarm. Further, the age-dependent dSarm induction is accompanied by a heightened inflammatory response (increased expression of Eiger and Relish) that is independent of reactive oxygen species (ROS) generation in the observed rotenone-induced neurotoxicity. dSarm induction and subsequent locomotor deficits is reversed in the presence of the anti-inflammatory molecule resveratrol. Thus, dSarm and heightened inflammatory responses may play a crucial role in age-dependent vulnerability to the pesticide rotenone thus making it an attractive target to help develop cost-effective therapeutic strategies to prevent ongoing dopaminergic neuronal loss as seen in PD.