%0 Journal Article %J Neurobiol Learn Mem %D 2020 %T N-terminal variant Asp14Asn of the human p70 S6 Kinase 1 enhances translational signaling causing different effects in developing and mature neuronal cells. %A Venkatasubramani, Janani Priya %A Subramanyam, Prakash %A Pal, Rakhi %A Reddy, Bharath K %A Srinivasan, Durga Jeyalakshmi %A Chattarji, Sumantra %A Iossifov, Ivan %A Klann, Eric %A Bhattacharya, Aditi %X

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.

%B Neurobiol Learn Mem %V 171 %P 107203 %8 2020 May %G eng %R 10.1016/j.nlm.2020.107203 %0 Journal Article %J J Proteome Res %D 2020 %T Optimization of Protocols for Detection of De Novo Protein Synthesis in Whole Blood Samples via Azide-Alkyne Cycloaddition. %A Bowling, Heather L %A Kasper, Amanda %A Patole, Chhaya %A Venkatasubramani, Janani Priya %A Leventer, Sarah Parker %A Carmody, Erin %A Sharp, Kevin %A Berry-Kravis, Elizabeth %A Kirshenbaum, Kent %A Klann, Eric %A Bhattacharya, Aditi %X

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.

%B J Proteome Res %V 19 %P 3856-3866 %8 2020 Sep 04 %G eng %N 9 %R 10.1021/acs.jproteome.0c00299 %0 Journal Article %J Nat Commun %D 2019 %T Altered steady state and activity-dependent de novo protein expression in fragile X syndrome. %A Bowling, Heather %A Bhattacharya, Aditi %A Zhang, Guoan %A Alam, Danyal %A Lebowitz, Joseph Z %A Bohm-Levine, Nathaniel %A Lin, Derek %A Singha, Priyangvada %A Mamcarz, Maggie %A Puckett, Rosemary %A Zhou, Lili %A Aryal, Sameer %A Sharp, Kevin %A Kirshenbaum, Kent %A Berry-Kravis, Elizabeth %A Neubert, Thomas A %A Klann, Eric %X

Whether fragile X mental retardation protein (FMRP) target mRNAs and neuronal activity contributing to elevated basal neuronal protein synthesis in fragile X syndrome (FXS) is unclear. Our proteomic experiments reveal that the de novo translational profile in FXS model mice is altered at steady state and in response to metabotropic glutamate receptor (mGluR) stimulation, but the proteins expressed differ under these conditions. Several altered proteins, including Hexokinase 1 and Ras, also are expressed in the blood of FXS model mice and pharmacological treatments previously reported to ameliorate phenotypes modify their abundance in blood. In addition, plasma levels of Hexokinase 1 and Ras differ between FXS patients and healthy volunteers. Our data suggest that brain-based de novo proteomics in FXS model mice can be used to find altered expression of proteins in blood that could serve as disease-state biomarkers in individuals with FXS.

%B Nat Commun %V 10 %P 1710 %8 2019 Apr 12 %G eng %N 1 %R 10.1038/s41467-019-09553-8