title: Functional Analysis of Keratin-Associated Proteins in Intestinal Epithelia: Heat-Shock Protein Chaperoning and Kinase Rescue.
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.
authors.affiliation: Department of Physiology, Nova Southeastern University, Ft. Lauderdale, Florida, USA; Department of Cell Biology, University of Miami Miller School of Medicine, Miami, Florida, USA.
authors.forename: Anastasia
authors.initials: A
authors.lastname: Mashukova
authors.affiliation: Department of Cell Biology, University of Miami Miller School of Medicine, Miami, Florida, USA.
authors.forename: Radia
authors.initials: R
authors.lastname: Forteza
authors.affiliation: Department of Cell Biology, University of Miami Miller School of Medicine, Miami, Florida, USA. Electronic address: PSalas@med.miami.edu.
authors.forename: Pedro J
authors.initials: PJ
authors.lastname: Salas
mesh_terms: D000818:Animals; D018938:Caco-2 Cells; D002458:Cell Fractionation; D006360:Heat-Shock Proteins / Q000737:chemistry*; D006801:Humans; D007382:Intermediate Filaments / Q000201:enzymology; D007413:Intestinal Mucosa / Q000166:cytology; D053564:Keratins, Hair-Specific / Q000737:chemistry*; D051379:Mice; D010766:Phosphorylation; D017510:Protein Folding; D011493:Protein Kinase C / Q000737:chemistry*; D011499:Protein Processing, Post-Translational
chemical_list: D006360:Heat-Shock Proteins; D053564:Keratins, Hair-Specific; C115015:PKC-3 protein; D011493:Protein Kinase C
keywords: Bag-1; Hsc70; Hsp40; Hsp70; In vitro chaperoning assay; Intermediate filament; Polarity complex; Subcellular fractionation; aPKC