TY - JOUR T1 - N-terminal variant Asp14Asn of the human p70 S6 Kinase 1 enhances translational signaling causing different effects in developing and mature neuronal cells. JF - Neurobiol Learn Mem Y1 - 2020 A1 - Venkatasubramani, Janani Priya A1 - Subramanyam, Prakash A1 - Pal, Rakhi A1 - Reddy, Bharath K A1 - Srinivasan, Durga Jeyalakshmi A1 - Chattarji, Sumantra A1 - Iossifov, Ivan A1 - Klann, Eric A1 - Bhattacharya, Aditi AB -

The ribosomal p70 S6 Kinase 1 (S6K1) has been implicated in the etiology of complex neurological diseases including autism, depression and dementia. Though no major gene disruption has been reported in humans in RPS6KB1, single nucleotide variants (SNVs) causing missense mutations have been identified, which have not been assessed for their impact on protein function. These S6K1 mutations have the potential to influence disease progression and treatment response. We mined the Simon Simplex Collection (SSC) and SPARK autism database to find inherited SNVs in S6K1 and characterized the effect of two missense SNVs, Asp14Asn (allele frequency = 0.03282%) and Glu44Gln (allele frequency = 0.0008244%), on S6K1 function in HEK293, human ES cells and primary neurons. Expressing Asp14Asn in HEK293 cells resulted in increased basal phosphorylation of downstream targets of S6K1 and increased de novo translation. This variant also showed blunted response to the specific S6K1 inhibitor, FS-115. In human embryonic cell line Shef4, Asp14Asn enhanced spontaneous neural fate specification in the absence of differentiating growth factors. In addition to enhanced translation, neurons expressing Asp14Asn exhibited impaired dendritic arborization and increased levels of phosphorylated ERK 1/2. Finally, in the SSC families tracked, Asp14Asn segregated with lower IQ scores when found in the autistic individual rather than the unaffected sibling. The Glu44Gln mutation showed a milder, but opposite phenotype in HEK cells as compared to Asp14Asn. Although the Glu44Gln mutation displayed increased neuronal translation, it had no impact on neuronal morphology. Our results provide the first characterization of naturally occurring human S6K1 variants on cognitive phenotype, neuronal morphology and maturation, underscoring again the importance of translation control in neural development and plasticity.

VL - 171 ER - TY - JOUR T1 - Optimization of Protocols for Detection of De Novo Protein Synthesis in Whole Blood Samples via Azide-Alkyne Cycloaddition. JF - J Proteome Res Y1 - 2020 A1 - Bowling, Heather L A1 - Kasper, Amanda A1 - Patole, Chhaya A1 - Venkatasubramani, Janani Priya A1 - Leventer, Sarah Parker A1 - Carmody, Erin A1 - Sharp, Kevin A1 - Berry-Kravis, Elizabeth A1 - Kirshenbaum, Kent A1 - Klann, Eric A1 - Bhattacharya, Aditi AB -

Aberrant protein synthesis and protein expression are a hallmark of many conditions ranging from cancer to Alzheimer's. Blood-based biomarkers indicative of changes in proteomes have long been held to be potentially useful with respect to disease prognosis and treatment. However, most biomarker efforts have focused on unlabeled plasma proteomics that include nonmyeloid origin proteins with no attempt to dynamically tag acute changes in proteomes. Herein we report a method for evaluating de novo protein synthesis in whole blood liquid biopsies. Using a modification of the "bioorthogonal noncanonical amino acid tagging" (BONCAT) protocol, rodent whole blood samples were incubated with l-azidohomoalanine (AHA) to allow incorporation of this selectively reactive non-natural amino acid within nascent polypeptides. Notably, failure to incubate the blood samples with EDTA prior to implementation of azide-alkyne "click" reactions resulted in the inability to detect probe incorporation. This live-labeling assay was sensitive to inhibition with anisomycin and nascent, tagged polypeptides were localized to a variety of blood cells using FUNCAT. Using labeled rodent blood, these tagged peptides could be consistently identified through standard LC/MS-MS detection of known blood proteins across a variety of experimental conditions. Furthermore, this assay could be expanded to measure de novo protein synthesis in human blood samples. Overall, we present a rapid and convenient de novo protein synthesis assay that can be used with whole blood biopsies that can quantify translational change as well as identify differentially expressed proteins that may be useful for clinical applications.

VL - 19 IS - 9 ER -