Functional Analysis of Keratin-Associated Proteins in Intestinal Epithelia Heat-Shock Protein Chaperoning and Kinase Rescue

Anastasia Mashukova, Radia Forteza, Pedro J. Salas

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

A growing body of evidence from several laboratories points at nonmechanical functions of keratin intermediate filaments (IF), such as control of apoptosis, modulation of signaling, or regulation of innate immunity, among others. While these functions are generally assigned to the ability of IF to scaffold other proteins, direct mechanistic causal relationships between filamentous keratins and the observed effects of keratin knockout or mutations are still missing. We have proposed that the scaffolding of chaperones such as Hsp70/40 may be key to understand some IF nonmechanical functions if unique features or specificity of the chaperoning activity in the IF scaffold can be demonstrated. The same criteria of uniqueness could be applied to other biochemical functions of the IF scaffold. Here, we describe a subcellular fractionation technique based on established methods of keratin purification. The resulting keratin-enriched fraction contains several proteins tightly associated with the IF scaffold, including Hsp70/40 chaperones. Being nondenaturing, this fractionation method enables direct testing of chaperoning and other enzymatic activities associated with IF, as well as supplementation experiments to determine the need for soluble (cytosolic) proteins. This method also permits to analyze inhibitory activity of cytosolic proteins at independently characterized physiological concentrations. When used as complementary approaches to knockout, knockdown, or site-directed mutagenesis, these techniques are expected to shed light on molecular mechanisms involved in the effects of IF loss of function.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Pages139-154
Number of pages16
DOIs
StatePublished - 2016

Publication series

NameMethods in Enzymology
Volume569
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • aPKC
  • Bag-1
  • Hsc70
  • Hsp40
  • Hsp70
  • In vitro chaperoning assay
  • Intermediate filament
  • Polarity complex
  • Subcellular fractionation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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    Mashukova, A., Forteza, R., & Salas, P. J. (2016). Functional Analysis of Keratin-Associated Proteins in Intestinal Epithelia Heat-Shock Protein Chaperoning and Kinase Rescue. In Methods in Enzymology (pp. 139-154). (Methods in Enzymology; Vol. 569). Academic Press Inc.. https://doi.org/10.1016/bs.mie.2015.08.019