N-terminal variant Asp14Asn of the human p70 S6 Kinase 1 enhances translational signaling causing different effects in developing and mature neuronal cells.

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.

Engineered fusion protein-loaded gold nanocarriers for targeted co-delivery of doxorubicin and erbB2-siRNA in human epidermal growth factor receptor-2+ ovarian cancer.

Designed recombinant proteins comprising functional domains offer selective targeting of cancer cells for the efficient delivery of therapeutic agents. The efficacy of these carriers can be further enhanced by conjugating engineered proteins to nanoparticle surfaces. However, recombinant protein-loaded nanoparticle-based drug delivery systems are not well addressed for ovarian cancer therapy. In the present study, using a combinatorial approach, we designed and fabricated a drug delivery system by combining gold nanoparticles (AuNPs) with an engineered bi-functional recombinant fusion protein TRAF(C) (TR), loaded with an anticancer drug, namely doxorubicin (DX), and erbB2-siRNA (si), to mediate target specific delivery into SK-OV-3, a model human ovarian cancer cell line over expressing HER2 receptors (i.e. human epidermal growth factor receptor-2). The nanoparticle-based targeted drug delivery system, designated as TDDS (Au-TR-DX-si), was found to be stable and homogenous as revealed by physicochemical and biochemical studies in vitro. In addition, TDDS was functional upon evaluation in vivo. Intraperitoneal administration of TDDS at 2.5 mg kg-1 of DX and 0.25 mg kg-1 of erbB2 siRNA into SK-OV-3 xenograft nude mice, revealed target specific uptake and consequent gene silencing resulting in significant tumor suppression. We attribute these results to specific co-delivery of erbB2 siRNA and DX mediated by TDDS into SK-OV-3 cells via HER2 receptors. Additionally, the biodistribution of TDDS, as quantitated by ICP-OES, confirmed tumor-specific accumulation of AuNPs primarily in tumor tissues, which firmly establishes the efficacy of the nanomedicine-based combinatorial approach for the treatment of ovarian cancer in a non-toxic manner. Based on these findings, we strongly believe that the nanomedicine-based combinatorial approach can be developed as a universal strategy for treatment of HER2+ ovarian cancers.