Biology subjects

Sciaky, N.

Publications and source records attributed to Sciaky, N..

3 recordsLinked to original sources

bioRxiv2024

Quantitative proteomic mass spectrometry of protein kinases to determine dynamic heterogeneity of the human kinome

The kinome is a dynamic system of kinases regulating signaling networks in cells and dysfunction of protein kinases contributes to many diseases. Regulation of the protein expression of kinases alters cellular responses to environmental changes and perturbations. We configured a library of 672 proteotypic peptides to quantify >300 kinases in a single LC-MS experiment using ten micrograms protein from human tissues including biopsies. This enables absolute quantitation of kinase protein abundance at attomole-femtomole expression levels, requiring no kinase enrichment and less than ten micrograms of starting protein from flash-frozen and formalin fixed paraffin embedded tissues. Breast cancer biopsies, organoids, and cell lines were analyzed using the SureQuant method, demonstrating the heterogeneity of kinase protein expression across and within breast cancer clinical subtypes. Kinome quantitation was coupled with nanoscale phosphoproteomics, providing a feasible method for novel clinical diagnosis and understanding of patient kinome responses to treatment.

biochemistry↗

bioRxiv2023

A suite of engineered mice for interrogating psychedelic drug actions

Psychedelic drugs like lysergic acid diethylamide (LSD) and psilocybin have emerged as potentially transformative therapeutics for many neuropsychiatric diseases, including depression, anxiety, post-traumatic stress disorder, migraine, and cluster headaches. LSD and psilocybin exert their psychedelic effects via activation of the 5-hydroxytryptamine 2A receptor (HTR2A). Here we provide a suite of engineered mice useful for clarifying the role of HTR2A and HTR2A-expressing neurons in psychedelic drug actions. We first generated Htr2a-EGFP-CT-IRES-CreERT2 mice (CT:C-terminus) to independently identify both HTR2A-EGFP-CT receptors and HTR2A-containing cells thereby providing a detailed anatomical map of HTR2A and identifying cell types that express HTR2A. We also generated a humanized Htr2a mouse line and an additional constitutive Htr2A-Cre mouse line. Psychedelics induced a variety of known behavioral changes in our mice validating their utility for behavioral studies. Finally, electrophysiology studies revealed that extracellular 5-HT elicited a HTR2A-mediated robust increase in firing of genetically-identified pyramidal neurons--consistent with a plasma membrane localization and mode of action. These mouse lines represent invaluable tools for elucidating the molecular, cellular, pharmacological, physiological, behavioral, and other actions of psychedelic drugs in vivo.

neuroscience↗

bioRxiv2017

Proteomic Analysis Defines Kinase Taxonomies Specific For Subtypes Of Breast Cancer

Multiplexed small molecule inhibitors covalently bound to Sepharose beads (MIBs) were used to capture functional kinases in luminal, HER2-enriched and triple negative, basal-like and claudin-low breast cancer cell lines and tumors. Kinase MIB-binding profiles at baseline without perturbation proteomically distinguished the four breast cancer subtypes. Kinases lacking defined functions in breast cancer were highly represented in the MIB-binding taxonomies. We show that these understudied kinases, whose disease associations and pharmacology are generally unexplored, are integrated in kinase signaling subnetworks with kinases that have been previously well characterized in breast cancer. Computationally it was possible to define subtypes using profiles of less than 50 of the more than 300 kinases bound to MIBs that included understudied as well as metabolic and lipid kinases. Furthermore, analysis of MIB-binding profiles established potential functional annotations for these understudied kinases. Thus, comprehensive MIBs-based capture of kinases provides a unique proteomics-based method for integration of poorly characterized kinases of the understudied kinome into functional subnetworks in breast cancer cells and tumors that is not possible using genomic strategies. The MIB-binding profiles readily defined subtype-selective differential adaptive kinome reprogramming in response to targeted kinase inhibition, demonstrating how MIB profiles can be used in determining dynamic kinome changes that result in subtype selective phenotypic state changes.

cancer biology↗