Please use this identifier to cite or link to this item: doi:10.22028/D291-30213
Notice: temporarily not accessible for legal reasons
Title: Optoregulation of collagen biosynthesis and remodeling in collagen associated diseases
Author(s): Khan, Essak
Language: English
Year of Publication: 2019
DDC notations: 540 Chemistry
Publikation type: Doctoral Thesis
Abstract: Tissue regeneration and remodeling after damage requires enhanced collagen deposition at the site of damage. In collagen disorders like keratoconus and brittle bone disease this ability is lost due to collagen misfolding, poor crosslinking and deposition. For this purpose, tools that allow to control and regulate collagen biosynthesis and folding are required. Ideally, such tools should be collagen-specific and allow remote control, which available strategies fail to fulfill.In this context, a collagen-specific molecular chaperone, Hsp47, was chosen as it has multiple roles in collagen biosynthesis. Recombinant Hsp47 can be delivered in the endoplasmic reticulum of mammalian cells via KDEL receptor mediated endocytosis. Exogenous delivery of Hsp47 stimulates fibrillar collagen I, III and V in cells. A photoactivatable derivative of Hsp47 (H47Y<ONBY)was developed containing an un‐natural light‐responsive tyrosine (o‐nitro benzyl tyrosine (ONBY)),which renders Hsp47 inactive toward collagen binding. On-demand, localized and in situ activation of this tool, stimulating collagen production in disease-state cells, was tested in vitro.Also, this tool can be easily delivered precisely in cells of damage corneal tissue from keratoconus patients. Site-selective exposure after H47Y<ONBY treatment,allowing localized remodeling of the extracellular collagen matrix, was demonstrated ex vivo. This tool has potential to trigger collagen deposition in collagen deficient disorders.Tissue regeneration and remodeling after damage requires enhanced collagen deposition at the site of damage. In collagen disorders like keratoconus and brittle bone disease this ability is lost due to collagen misfolding, poor crosslinking and deposition. For this purpose, tools that allow to control and regulate collagen biosynthesis and folding are required. Ideally, such tools should be collagen-specific and allow remote control, which available strategies fail to fulfill.In this context, a collagen-specific molecular chaperone, Hsp47, was chosen as it has multiple roles in collagen biosynthesis. Recombinant Hsp47 can be delivered in the endoplasmic reticulum of mammalian cells via KDEL receptor mediated endocytosis. Exogenous delivery of Hsp47 stimulates fibrillar collagen I, III and V in cells. A photoactivatable derivative of Hsp47 (H47Y<ONBY)was developed containing an un‐natural light‐responsive tyrosine (o‐nitro benzyl tyrosine (ONBY)),which renders Hsp47 inactive toward collagen binding. On-demand, localized and in situ activation of this tool, stimulating collagen production in disease-state cells, was tested in vitro.Also, this tool can be easily delivered precisely in cells of damage corneal tissue from keratoconus patients. Site-selective exposure after H47Y<ONBY treatment,allowing localized remodeling of the extracellular collagen matrix, was demonstrated ex vivo. This tool has potential to trigger collagen deposition in collagen deficient disorders.
Eine lokal erhöhte Kollagenablagerung ist für die Geweberegeneration und den Gewebeaufbau nach einer Schädigung notwendig. Bei Kollagenstörungen wie Keratokonus und Brittle Bone Disease geht diese Fähigkeit durch Fehlfaltung und schlechte Vernetzung verloren. Mittel und Wege werden benötigt, um die Kollagenbiosynthese und -faltung zu regulieren. Im Idealfall sind solche Werkzeuge kollagenspezifisch und von außen steuerbar, was derzeitige Strategien nicht erfüllen. In dieser Arbeit wurde das kollagenspezifische Chaperon Hsp47 ausgewählt, da es mehrere Rollen bei der Kollagenbiosynthese hat. Rekombinantes Hsp47 kann im endoplasmatischen Retikulum von Säugetierzellen über KDEL-Rezeptor-vermittelte Endozytose abgegeben werden. Die exogene Gabe von Hsp47 stimuliert in Zellen die Bildung von fibrillärem Kollagen I, III und V. Ein neues Hsp47 (H47Y <ONBY) wurde entwickelt mit nicht-natürlichem o-Nitro-Benzyltyrosin (ONBY) an der Tyr383- Position, die inaktiv ist für die Bindung und Bildung von Kollagen. In vitro wurde in Hsp47- defizienten Zellen bewiesen, dass die kontrollierte lokale Photoaktivierung dieser Variante die Ablagerung von Kollagen stimuliert. Außerdem wurde das neue HSP47 ex vivo in Zellen von Hornhautgewebe von Keratokonus-Patienten angewendet. Hier wurde nachgewiesen, dass durch lokale Belichtung nach H47Y <ONBY-Anwendung Kollagenfasern wiederherstellt werden. Das neue HSP47 hat das Potential, gezielte Kollagenablagerungen bei Störungen mit Kollagenmangel auszulösen.
Link to this record: urn:nbn:de:bsz:291--ds-302136
hdl:20.500.11880/28733
http://dx.doi.org/10.22028/D291-30213
Advisor: del Campo, Aránzazu
Date of oral examination: 25-Nov-2019
Date of registration: 19-Feb-2020
Faculty: NT - Naturwissenschaftlich- Technische Fakultät
Department: NT - Chemie
Professorship: NT - Prof. Dr. Aránzazu del Campo
Collections:SciDok - Der Wissenschaftsserver der Universität des Saarlandes

Files for this record:
File Description SizeFormat 
final thesis kh.pdfPhD thesis Essak Khan10,15 MBAdobe PDFView/Open


Items in SciDok are protected by copyright, with all rights reserved, unless otherwise indicated.