Dataset Information

Initial Kinetic Characterization of Sterile Alpha and Toll/Interleukin Receptor Motif-Containing Protein 1.

ABSTRACT: Sterile alpha and toll/interleukin receptor (TIR) motif-containing protein 1 (SARM1) plays a pivotal role in triggering the neurodegenerative processes that underlie peripheral neuropathies, traumatic brain injury, and neurodegenerative diseases. Importantly, SARM1 knockdown or knockout prevents degeneration, thereby demonstrating that SARM1 is a promising therapeutic target. Recently, SARM1 was shown to promote neurodegeneration via its ability to hydrolyze NAD⁺, forming nicotinamide and ADP ribose (ADPR). Herein, we describe the initial kinetic characterization of full-length SARM1, as well as the truncated constructs corresponding to the SAM^1-2TIR and TIR domains, highlighting the distinct challenges that have complicated efforts to characterize this enzyme. Moreover, we show that bacterially expressed full-length SARM1 (k_cat/K_M = 6000 ± 2000 M^-1 s^-1) is at least as active as the TIR domain alone (k_cat/K_M = 1500 ± 300 M^-1 s^-1). Finally, we show that the SARM1 hydrolyzes NAD⁺ via an ordered uni-bi reaction in which nicotinamide is released prior to ADPR.

SUBMITTER: Loring HS

PROVIDER: S-EPMC7085114 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Initial Kinetic Characterization of Sterile Alpha and Toll/Interleukin Receptor Motif-Containing Protein 1.

Loring Heather S HS Icso Janneke D JD Nemmara Venkatesh V VV Thompson Paul R PR

Biochemistry 20200217 8

Sterile alpha and toll/interleukin receptor (TIR) motif-containing protein 1 (SARM1) plays a pivotal role in triggering the neurodegenerative processes that underlie peripheral neuropathies, traumatic brain injury, and neurodegenerative diseases. Importantly, SARM1 knockdown or knockout prevents degeneration, thereby demonstrating that SARM1 is a promising therapeutic target. Recently, SARM1 was shown to promote neurodegeneration via its ability to hydrolyze NAD<sup>+</sup>, forming nicotinamide ...[more]

PMID: 32049506

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Project description:Abstract Introduction: SAMD9 variants are associated with colon, breast and lung tumors. SAMD9 mutations result in adrenal hypoplasia, suggesting its importance in adrenal development. We investigated the contribution of abnormal expression of SAMD9 and its homologous, SAMD9L, to the pathogenesis of pediatric adrenocortical tumors (pACT) Objective: To evaluate the involvement of SAMD9 and SAMD9L in normal human adrenal cortex development and adrenocortical tumorigenesis, as well as to evaluate their association with tumor presentation and patient outcome. Methods: pACT samples (n= 72), normal pediatric adrenal cortices (n= 11), and normal fetal and post-natal adrenals (20 weeks of gestation to 10 years of age; n= 51) were enrolled. Protein expression of SAMD9 (immunohistochemistry) and SAMD9/SAMD9L mRNA levels were evaluated (qPCR). The associations between SAMD9/SAMD9L expression in pACT with tumor presentation (P53 p.R337H genotype and metastasis occurrence) and patient outcome (Overall (OS) and Disease-Free Survival) were analyzed. In silico, publicly available data from pediatric patients with ACT available in the Gene Expression Omnibus were used to evaluate the aforementioned associations with SAMD9 (GSE76021) and SAMD9L (GSE76019) mRNA levels. In vitro, SAMD9 subcellular localization pattern was investigated in the ACT cell line NCI-H295R (immunofluorescence). Results: Nuclear and cytoplasmic SAMD9 expression was observed throughout all different phases of adrenal development evaluated. However, in pACT samples, 26% presented nuclear and 86% cytoplasmic immunostaining. In line, NCI-H295R cells presented mostly cytoplasmic SAMD9 immunostaining under basal conditions. No differential expression was observed between SAMD9 or SAMD9L mRNA levels in pACT and in normal pediatric adrenals. Moreover, no association between SAMD9 immunostaining, SAMD9 or SAMD9L mRNA levels, tumor presentation and patient outcome were observed in our cohort, nor in the in silico evaluation. Conclusion: SAMD9 is nuclear and cytoplasmic expressed during adrenal cortex adrenal development and its loss of function is associated with adrenal hypoplasia. On the other hand, SAMD9 is mostly cytoplasmic expressed in pACT and immortalized NCI-H295R cells. No differential expression of SAMD9 nor its counterpart SAMD9L mRNA were associated with tumor presentation and pediatric patient outcome.

Dataset Information

Initial Kinetic Characterization of Sterile Alpha and Toll/Interleukin Receptor Motif-Containing Protein 1.

Publications

Initial Kinetic Characterization of Sterile Alpha and Toll/Interleukin Receptor Motif-Containing Protein 1.

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