TY - CHAP
T1 - Functional Analysis of Keratin-Associated Proteins in Intestinal Epithelia Heat-Shock Protein Chaperoning and Kinase Rescue
AU - Mashukova, Anastasia
AU - Forteza, Radia
AU - Salas, Pedro J.
N1 - Funding Information:
This work was supported by a grant to P.J.S. from the National Institute of Diabetes, Digestive and Kidney Diseases (R01-DK076652) and a NIH Ruth L. Kirschstein National Research Service Award Fellowship to R.F. (F32-DK095503).
Publisher Copyright:
© 2016 Elsevier Inc. All rights reserved.
PY - 2016
Y1 - 2016
N2 - 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.
AB - 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.
KW - Bag-1
KW - Hsc70
KW - Hsp40
KW - Hsp70
KW - In vitro chaperoning assay
KW - Intermediate filament
KW - Polarity complex
KW - Subcellular fractionation
KW - aPKC
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U2 - 10.1016/bs.mie.2015.08.019
DO - 10.1016/bs.mie.2015.08.019
M3 - Chapter
C2 - 26778557
AN - SCOPUS:84954372830
T3 - Methods in Enzymology
SP - 139
EP - 154
BT - Methods in Enzymology
PB - Academic Press Inc.
ER -