Ubiquitin Chains: Degradation and Beyond

119 Pages·2015·4.602 MB·English

by  Arnab De (auth.)

Springer Theses
Recognizing Outstanding Ph.D. Research
Arnab De
Ubiquitin
Chains:
Degradation
and Beyond
Springer Theses
Recognizing Outstanding Ph.D. Research
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Arnab De
Ubiquitin Chains:
Degradation and Beyond
Doctoral Thesis accepted by
Columbia University
New York, NY
Author Supervisor
Dr. Arnab De Prof. Sankar Ghosh
Department of Microbiology and Chair, Silverstein and Hutt Family Professor
Immunology of Microbiology and Immunology
Columbia University Medical Center Department of Microbiology and
New York , NY , USA Immunology
Columbia University Medical Center
New York, NY, USA
ISSN 2190-5053 ISSN 2190-5061 (electronic)
Springer Theses
ISBN 978-3-319-14964-6 ISBN 978-3-319-14965-3 (eBook)
DOI 10.1007/978-3-319-14965-3
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Dedi cated to
The Inspiration of Sachin Tendulkar
&
My Alma Mater
( Presidency College , Calcutta , India
and Columbia University , New York , USA :
two temples of learning , in corners
of the world )
While dedication of a doctoral thesis
to places of learning is considered common
practice, many have pondered, why dedicate
a Ph.D. thesis to a sportsman? A brief word
might be apt here.
Sachin Tendulkar is the greatest batsman
the cricket world has seen in the post war
era. Some of the greatest feats of Sachin
Tendulkar (including the fi rst double century
ever in history) happened when I was
at Columbia University, away from home
studying in New York. However, this thesis
is not dedicated to the cricketing genius
of the man. While he excelled in cricket,
many others have done equally well
in their respective fi eld of enterprise.
Instead, this thesis is dedicated to Sachin
Tendulkar for the way he has inspired
the Indian youth of my generation (and me)
for the past 25 years. He did so with
his immense dedication and love for his work
(game of cricket); and his unwavering intent
to continue to be better at it, even when
he was the best in the world. The confi dence
of the Indian youth today is a refl ection
of the certainty that Sachin gradually
brought to his cricket; that is his ultimate
gift to India.
In my opinion , Tendulkar is an idea , a n idea
that will be cherished by everyone who
believes in work and letting “ karma ” talk .
Supervisor’s Foreword
K48-linked ubiquitination is well known as a signal for degradation. However, there
has been a growing body of work which suggests that K63-linked polyubiquitin
chains are non-degradative and play a role in regulating cell-signaling. The case for
regulatory ubiquitination has been to a large extent predicated upon the presumed
deubiquitinase activity of A20, long considered a key regulator of infl ammatory
responses as mice lacking A20 die from multiorgan infl ammation and cachexia.
Consistent with its role in limiting infl ammatory responses through its deubiquitin-
ase activity, A20 has been implicated in a wide variety of autoimmune diseases and
cancer. Multiple biochemical and knockdown approaches in cell culture have sug-
gested that A20 functions as a deubiquitinase by disassembling regulatory K63-
ubiquitin chains on upstream signaling molecules such as RIP1; thereby
downregulating the NF-κB-mediated infl ammatory response. This thesis examines
the evidence for regulatory ubiquitination by focusing on A20 and reports the cre-
ation and characterization of a knock-in mouse that expresses a mutated form of
A20 that selectively lacks the deubiquitinase activity. The knock-in mice surprisingly
display completely normal NF-κB activation with no accompanying infl ammatory
phenotype. Therefore, these results clearly demonstrate that the well-characterized
role of A20 in limiting infl ammatory responses is due to effects other than deubiq-
uitination of K63-ubiquitin chains. Thus, it might be important to revisit the role of
K63-linked polyubiquitination cell-signaling.
Chair, Silverstein and Hutt Family Professor Sankar Ghosh, Ph.D.
of Microbiology and Immunology
Department of Microbiology and Immunology
Columbia University Medical Center
New York , NY , USA
vii

Abstract
K 48-linked ubiquitination is well known as a signal for degradation, and the fi nding
was recognized by the Nobel Prize for Chemistry in 2004. Recent work has sug-
gested that K63-linked ubiquitination does not lead to degradation; instead, it has
been proposed to be important for cell-signaling. Indeed, there is a growing body of
in vitro evidence that suggests that non-degradative K63-linked polyubiquitin chains
play an important role in activation of transcription factor NF-κB.
W hile prompt activation of NF-κB is essential for optimal immune response, it is
equally important to terminate the response in order to avoid tissue damage and
perhaps even death resulting from organ failure. A20 is an essential inhibitor of
NF-κB-mediated infl ammation as mice lacking A20 die from multiorgan infl amma-
tion and cachexia. Multiple biochemical approaches have suggested that A20 func-
tions as a deubiquitinase by disassembling K63-linked regulatory ubiquitin chains
from upstream adapter molecules like RIP1. The importance of K63-linked polyu-
biquitin chains in abetting cell-signaling has to a large extent been predicated on the
deubiquitinase activity of A20. To determine the contribution of the deubiquitinase
role of A20 in downregulating NF-κB, we generated and characterized a knock-in
mouse lacking the deubiquitinase activity of A20. However, we fi nd that these mice
surprisingly display normal NF-κB activation and show no signs of infl ammation.
Our work clearly demonstrates that the deubiquitinase activity of A20 is dispens-
able for downregulating NF-κB and the well-characterized role of A20 in limiting
infl ammatory responses is due to effects other than deubiquitination of K63-
ubiquitin chains. Hence, it might be critical to revisit and explore the general role of
K63-linked polyubiquitination in cell-signaling.
ix