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A human sterile alpha motif domain polymerizome.

ABSTRACT: The sterile alpha motif (SAM) domain is one of the most common protein modules found in eukaryotic genomes. Many SAM domains have been shown to form helical polymer structures suggesting that SAM modules can be used to create large protein complexes in the cell. Because many polymeric SAM domains form heterogenous and insoluble aggregates that are experimentally intractable when isolated, it is likely that many polymeric SAM domains have gone uncharacterized. We, therefore, developed a method to maintain polymeric SAM domains in a soluble form that allowed rapid screening for potential SAM polymers. SAM domains were expressed as fusions to a super-negatively charged green fluorescent protein (negGFP). The negGFP imparts three useful properties to the SAM domains: (1) the charge helps to maintain solubility; (2) the charge leads to reliable migration toward the cathode on native gels; and (3) the fluorescence emission allows visualization in crude extracts. Using the negGFP-SAM fusions, we screened a large library of human SAM domains for polymerization using a native gel screen. A selected set of hSAM domains were then purified and examined for true polymer formation by electron microscopy. In this manner, we identified a set of new potential SAM polymers: ANKS3, Atherin, BicaudalC1, Caskin1, Caskin2, Kazrin, L3MBTL3, L3MBTL4, LBP, LiprinB1, LiprinB2, SAMD8, SAMD9, and STIM2. While further characterization will be necessary to verify that the SAM domains identified here truly form polymers, our results provide a much stronger working hypothesis for a large number of proteins that was possible from sequence analysis alone.

SUBMITTER: Knight MJ

PROVIDER: S-EPMC3218363 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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A human sterile alpha motif domain polymerizome.

Knight Mary Jane MJ Leettola Catherine C Gingery Mari M Li Hao H Bowie James U JU

Protein science : a publication of the Protein Society 20110818 10

The sterile alpha motif (SAM) domain is one of the most common protein modules found in eukaryotic genomes. Many SAM domains have been shown to form helical polymer structures suggesting that SAM modules can be used to create large protein complexes in the cell. Because many polymeric SAM domains form heterogenous and insoluble aggregates that are experimentally intractable when isolated, it is likely that many polymeric SAM domains have gone uncharacterized. We, therefore, developed a method to ...[more]

PMID: 21805519

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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

A human sterile alpha motif domain polymerizome.

Publications

A human sterile alpha motif domain polymerizome.

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