PhD
1983 Free University Berlin and the Max Planck
Institute of Molecular Genetics, Berlin,
Germany
Postdoctoral
work
at
Massachusetts Institute of Technology (MIT),
Cambridge, USA
Group
Leader
at Friedrich Miescher Laboratory of the Max
Planck Society, Tuebingen, Germany
Professor
at
Center for Molecular Biology (ZMBH),
Ruprecht-Karls University, Heidelberg, Germany
Director
at MPI of Biochemistry and Member
of the Max Planck Society since
1998
At
the Max Planck Institute of Biochemistry since
1998
Louis-Jeantet
Prize for Medicine (2011)
Honorary Professorship of Fudan University,
Shanghai (2006)
Max-Planck Research Award (2003)
Otto Bayer Award (1996)
Gottfried Wilhelm Leibniz Prize (1993)
Otto Klung Prize for Chemistry (1992)
Elected member of EMBO, German Academy of
Sciences Leopoldina, Academia Europaea
Protein
Degradation
and Targeting
DNA Transactions and Chromatin Biology
RESEARCH
INTERESTS
Our
laboratory has a long-standing interest in protein
modification by the ubiquitin system. Covalent modification of
proteins by attachment of the small protein ubiquitin
("ubiquitylation") mediates a variety of different cellular
functions. The best-known function of ubiquitylation is to
label proteins for degradation by the 26S proteasome. Other, non-proteolytic functions
of ubiquitylation are regulatory roles in e.g. protein
sorting, gene expression and DNA repair. In addition to
ubiquitin, eukaryotes also express ubiquitin-like proteins and
some of these can form conjugates with cellular proteins
similar to ubiquitin.
Our research focuses primarily on
functional aspects of the ubiquitin pathway. We identify from
yeast and mammalian cells the enzymatic components of this
system, clone the corresponding genes, and study their
functions in vitro and in vivo. We are increasingly interested in the
non-proteolytic functions of ubiquitylation (in particular in
DNA transactions), the role of the ubiquitin-selective Cdc48
chaperone-like enzyme, and the function of the ubiquitin-like
proteins SUMO and Hub1.
A
novel mechanism for alternative splicing Role of the ubiquitin-like protein Hub1 in
splice-site usage and alternative splicing.
Mishra et al., Nature474, 173-178,
2011[PDF]
Timing
replication and DNA damage tolerance
The RAD6 DNA damage
tolerance pathway operates uncoupled from the
replication fork and is functional beyond S-phase. Karras and Jentsch, Cell 141,
255-267, 2010 [PDF]
Meiotic checkpoint control by Red1
Synaptonemal complex formation and meiotic
checkpoint signaling are linked to the lateral element
protein Red1.
Eichinger and Jentsch,PNAS107,
11370-11375, 2010[PDF]
Unexpected
behavior of broken chromosomes
Chromosome-wide Rad51 spreading and
SUMO-H2A.Z-dependent chromosome fixation in
response to a persistent DNA double-strand
break. Kalocsayet al., Mol. Cell33, 335-343,
2009 [PDF]
A
fate switch in the ubiquitin pathway Prolyl isomerase Pin1
acts as a switch to control the degree of
substrate ubiquitylation. Siepe and Jentsch, Nat. Cell Biol
11,
967-972, 2009 [PDF]
A
molecular Swiss army knife mediates abscission
Final stages of
cytokinesis and midbody ring formation are controlled
by BRUCE. & Midbody ring disposal by
autophagy is a post-abscission event of
cytokinesis. Pohl and
Jentsch, Cell132,
832-845, 2008 [PDF]. Pohl and
Jentsch, Nat.
Cell Biol11,
65-70, 2009 [PDF]
Modification
of proteins by covalent conjugation to the small protein
ubiquitin ("ubiquitylation") mediates several distinct
cellular functions. A central function of ubiquitylation is to
earmark proteins for degradation by the 26S proteasome, a
large protease of the cytosol and the nucleus of eukaryotic
cells. Selective protein degradation by the
ubiquitin-proteasome system (UPS) plays an important role in
cellular regulation. Moreover, it is used to eliminate
aberrant proteins generated under normal and in particular
under stress conditions. Ubiquitin is joined to substrate
proteins via an isopeptide linkage between its carboxyl-(C)
terminus and theepsilon-amino group of an internal lysine
residue of the target protein.
Structure of ubiquitin.
It has a tightly packed globular structure with a
five-strand beta sheet wrapped around one alpha
helix. Ubiquitin becomes conjugated to other
proteins via its C-terminus (red).
A
space-filling representation of ubiquitin is shown
as page background.
(Data from Vijay-Kumar, S., Bugg, C.E., and Cook,
W.J. (1987) J. Mol. Biol.194,
531-544.)
Ubiquitylation
involves E1, E2, and E3 enzymes. Multiple rounds of
ubiquitylation reactions result in the formation of
polyubiquitin chains. Within these chains ubiquitin
can be linked via distinct lysine residues of
ubiquitin.
Red symbol,
ubiquitin; K, lysine residue of a target
protein.
Ubiquitin
conjugation
requires
typically
three
classes
of
enzymes.
E1
(ubiquitin-activating
enzyme)
hydrolyses
ATP
and
forms
a
thioester-linked
complex
between
itself
and
ubiquitin.
E2
(ubiquitin-conjugating
enzyme)
receives
ubiquitin
from
E1
and
forms
a
similar
thioester-linked
intermediate
with
ubiquitin.
E3
(ubiquitin
ligase)
finally
binds
both
the
E2
and
a
substrate
and
catalyzes
the
transfer
of
ubiquitin
to
the
substrate.
Ubiquitin
itself
is
often
a
substrate
for
further
ubiquitylation,
which
results
in
the
formation
of
so-called
polyubiquitin
chains.
Ubiquitin
has
seven
lysine
residues,
and
depending
on
the
lysine
residue
used
for
ubiquitin-ubiquitin
chain
formation,
the
polyubiquitin
chain
can
signal
different
functions.
Proteins
modified
by
lysine-48
(K48)
or
lysine-29
(K29)
linked
chains
are
usually
degraded
by
the proteasome. In contrast, modification by K63-linked chains
or by a single ubiquitin moiety (monoubiquitylation) seem to
trigger other functions, e.g. protein sorting, gene
expression, and DNA repair (see below).
Ubiquitin in chains.
Modification of proteins by polyubiquitin chains
linked via lysine (K) 48 or K29 appear to label
proteins for proteasomal degradation. In contrast,
monoubiquitylation or modification by K63-linked
multi-ubiquitin chains do not generally promote
protasomal degradation, but rather it seems to alter
the function of the substrate or to mediate
protein-protein interactions (important for protein
targeting, endocytosis and DNA repair).
In the past years we have characterized the family of E2
enzymes from the budding yeast Saccharomyces
cerevisiae. Yeast possesses 11 genes (UBC genes)
for ubiquitin-conjugating enzymes. Our genetic data have
indicated that E2s mediate a surprising variety of functions,
including DNA repair, cell cycle progression, transcriptional
regulation, and heat and heavy metal tolerance. Among
the known substrates of the ubiquitin/proteasome system are
cellular regulators (e.g. cyclins, CDK inhibitors,
transcription factors, signal transducers) and aberrant
proteins. Yeast
cells also possess a large variety of E3 enzymes (more than 40 in yeast).
One class of E3s is distinguished by the presence of a
so-called HECT domain, which catalyzes the transfer of
ubiquitin to cellular substrates. A second class
of E3s possess RING finger domains, which function as
recruiting domains for E2 enzymes. Some RING finger E3s are
monomeric enzymes, others (e.g. APC or SCF) are large
oligomeric protein complexes.
REGULATION
OF DNA DAMAGE TOLERANCE AND REPAIR BY UBIQUITIN AND
SUMO: THE PCNA SWITCH
Accurate
maintenance
of the genetic information of a cell is one of the most
important aspects of life. However, DNA is a rather unstable
molecule that is constantly subject to damage both by
environmental influences and by spontaneous decay due to its
inherent reactivity. It is therefore not surprising that cells
support a complex enzymatic machinery for the maintenance and
repair of their genomes. In yeast and also in higher
eukaryotes DNA damage repair is executed by several largely
independent groups of enzymes that correspond to
mechanistically different repair systems. Our research is
focused on the pathway of postreplication repair (DNA damage
tolerance, DDT), which is activated upon damaged DNA
templates. DNA lesions in the template strand that have
remained unrepaired during S phase cause a stalling of the
replication and lead to gaps in the newly synthesized strand.
These gaps are filled by the postreplication repair system in
order to allow mitosis to proceed. The filling of daughter
strand gaps can occur via an error-prone mechanism by a damage-tolerant
mutagenic polymerase or alternatively in an error-free fashion, most
likely using information from the undamaged sister chromatid.
The
RAD6 pathway of DNA damage tolerance (DDT)
Until the late
1980ies, ubiquitin was commonly believed to function exclusively
in protein degradation pathways. However, this view was
challenged by the finding that the DNA repair gene RAD6 encodes a
ubiquitin-conjugating enzyme (Jentsch et
al., 1987). In fact, this report established for the
first time a link between the ubiquitin system and DNA repair.
Now, two
decades later, the relationship between ubiquitin, its cousin
SUMO, and DNA repair pathways is the topic of a hot and
exciting field. Breast cancer, Fanconi anemia,
Xeroderma pigmentosum are among those numerous
human diseases now known to be linked to defective ubiquitin
modifications.
Rad6 is an
enzyme that can monoubiquitylate histones such as histone H2B (Jentsch et
al., 1987). In addition, it is central to the RAD6 DDT pathway.
Subsequently, two other components of the ubiquitin system were
identified as members of this pathway: Mms2, a Ubc homolog that
is lacking the active-site cysteine conserved in genuine Ubcs,
and Ubc13, which associates with Mms2 to form a catalytically
active heterodimer. Pickart and colleagues have demonstrated
that this complex assembles polyubiquitin chains that are linked
in a non-standard way, via K63.
We showed
that Rad6 and the heteromeric Ubc13-Mms2 complex physically
interact and functionally cooperate in DNA repair (see Figure; Ulrich
and Jentsch, 2000). We also discovered that two
chromatin-associated RING finger proteins, Rad18 and Rad5, play
a central role in mediating physical contacts between the
members of the RAD6
pathway. Rad5 recruits the Ubc13-Mms2 complex to DNA by means of
its RING finger domain. Moreover, Rad5 association with Rad18
brings Ubc13-Mms2 into contact with the Rad6/Rad18 complex.
Interaction between the two RING finger proteins thus promotes
the formation of a heteromeric complex in which the two distinct
ubiquitin-conjugating activities of Rad6 and Ubc13-Mms2 can be
closely coordinated. Surprisingly, Ubc13 and Mms2 are largely
cytosolic proteins, but DNA damage triggers their redistribution
to the nucleus.
Left: Model for alternative repair
complexes within the RAD6 pathway of DNA repair.
A schematic representation of the DNA repair proteins shows
the distribution of RING finger-Ubc complexes that would
result from a competition between Rad18 and Rad5 homo- and
heterodimerization. Homodimerization of Rad18 would lead to
assemblies comprising Rad6 as the sole Ubc, whereas
heterodimerization with Rad5 would promote the recruitment
of Ubc13-Mms2 for the assembly of lysine (K) 63-linked
polyubiquitin chains. Right: Ubc13 and Mms2 redistribute to the
nucleus upon treatment with DNA damaging agents (MMS; far
right pictures). The proteins are visualized by
immunofluorescence (red), the nucleus is stained for DNA
with DAPI (blue).
See Ulrich
and Jentsch (2000) for details.
Ubiquitin and SUMO as decision
makers: The PCNA switch
Continuing
on this project, we discovered that PCNA (proliferating cell
nuclear antigen) is
the cruciual substrate of the RAD6 pathway of DNA
repair. PCNA is positioned at the crossroads of multiple
replication-linked pathways (Hoege
et al., 2002). It is involved in leading and
lagging strand DNA synthesis, cell cycle arrest,
replication-linked DNA silencing, mismatch repair, nucleotide-
and base-excision repair, and postreplicative error-free and
error-prone DNA repair. PCNA directly associates with various
DNA polymerases and functions as a sliding clamp, thereby
stimulating accurate and processive DNA synthesis. In
addition, PCNA appears to function as a platform for accessory
factors. Previous studies have shown that PCNA is controlled
by at least two distinct mechanisms. One important principle
appears to be regulated, ATP-dependent loading/unloading of
the trimeric PCNA ring on the DNA template by an ATP-dependent
clamp loader (replication factor C, RFC). A second mechanism
may involve PCNA inhibition through interaction with
repressing molecules, the most prominent being the cell-cycle
checkpoint protein p21.
We
showed that PCNA is exquisitely modulated by covalent
modification with ubiquitin or the "small ubiquitin-like
modifier" SUMO (termed
Smt3 in yeast). Yeast PCNA (Pol30) is a target for
SUMOylation, monoubiquitylation, and K63-linked
polyubiquitylation. Intriguingly, these modifications are
differentially regulated and mediate different functions. We
identified two target sites for modification: K164, a
conserved modification site, which is SUMOylated and
ubiquitylated, and K127, a yeast-specific site, which appears
to be exclusively SUMOylated. Both residues are positioned
distally from the encircled DNA on the outside rim of the
trimeric PCNA ring. K164 is located in one of the protruding
tips of PCNA, whereas K127 lies within a large loop that
connects the two domains of a PCNA monomer. This connecting
loop is known to mediate interaction with polymerases,
suggesting that SUMOylation at this site might interfere with
polymerase binding.
Model
of modified PCNA - The PCNA switch. Shown in
yellow is the ring-shaped PCNA trimer, which
encircles the DNA strand. Ubiquitin, shown in red,
can be conjugated to lysine 164 (K164) of PCNA.
This modification is required for postreplicative
DNA repair. Monoubiquitylation (as shown) appears
to mediate error-prone DNA repair. In contrast,
K-63-linked polyubiquitylation (i.e. conjugation
of additional ubiquitin moieties to pre-conjugated
ubiquitin via lysine 63 of ubiquitin) switches on
error-free DNA repair. SUMOylation of K164 of PCNA
recruits the helicase Srs2 to prevent
recombination at the replication fork.
PCNA
structure
by
Krishna et al., 1994; Ubiquitin
structure by Vijay-Kumar et al., 1987. Model by M.
Groll.
SUMOylation of PCNA occurs during S-phase even in the absence
of endogenous DNA damage and modifies predominantly K164 and,
to a lesser extent, also K127 of yeast PCNA. Our data indicate
that SUMOylation of PCNA inhibits a DNA repair pathway and suggests a role in
conjunction with normal DNA replication.
Ubiquitylation
of
PCNA
is
clearly
linked
to
DNA
damage
and
strictly
depends
on
the
RAD6 pathway of DNA repair. It specifically targets the
conserved K164 residue of yeast and human PCNA. Moreover, we
found that PCNA ubiquitylation is elementary for the RAD6-dependent
DNA repair. Previous data have indicated a complex
orchestration of RAD6-dependent functions and revealed
distinct pathways for error-free and error-prone modes of
repair. These pathways may be subdivided into distinct
error-free branches and an error-prone mode that uses the
translesion polymerases Rev1-Pol-zeta (encoded by REV3 and REV7)
and Pol-eta (encoded by RAD30).
Our findings provide a new conceptual framework for the
function of the RAD6 pathway as it suggests that the
stalled replication machinery may be switched to the different
modes of repair through distinct PCNA modifications.
We
discovered that polyubiquitylation of PCNA at K164 is pivotal
for the error-free branch of RAD6-dependent DNA
repair. We also showed that two ubiquitin-conjugating enzymes,
Rad6 and Ubc13/Mms2, and two putative ubiquitin ligases, the
RING-finger proteins Rad18 and Rad5, are involved in PCNA
ubiquitylation.
The PCNA switch.
(Structure of yeast PCNA is derived from Krishna et al., 1994) (top left). K127 and K164 modification
sites are indicated.
DNA damage
induces monoubiquitylation of PCNA at K164, which is
catalyzed by ubiquitin-activating enzyme Uba1 (not
shown), Rad6, and Rad18. DNA damage induces nuclear
import of Ubc13 and Mms2, which are recruited to
chromatin by Rad5. The complete enzyme assembly
catalyzes K63-linked multiubiquitylation of PCNA. In
the absence of DNA damaging agents, PCNA is
SUMOylated during S-phase involving SUMO-activating
enzyme Uba2/Aos1 (not shown), SUMO-conjugating
enzyme Ubc9, and SUMO ligase Siz1 (not shown).
Enzymes required for the modification reactions are
labeled in red. Ubiquitin- and SUMO-conjugating
enzymes are shown in blue; ubiquitin ligases in
green.
Triggered
by DNA damage, Rad6 appears to initially monoubiquitylate PCNA
at residue . This reaction additionally requires Rad18, which
recruits Rad6 to DNA-bound PCNA. As mentioned above, DNA
damage also induces nuclear import of Ubc13 and Mms2, which
results in an association of the heterodimeric
ubiquitin-conjugating enzyme with chromatin-bound Rad5.
Moreover, through Rad5-Rad18 interaction, Ubc13/Mms2 is
brought into contact with Rad6. In a second enzymatic
reaction, this assembly of two ubiquitin-conjugating enzymes
and two ubiquitin ligases (together with ubiquitin-activating
enzyme) seems to catalyse the conjugation of additional
ubiquitin molecules onto the ubiquitin moiety of
monoubiquitylated PCNA, thereby forming K63-linked
polyubiquitin chains. These chains, in contrast to K48-linked
chains, do not promote proteasomal degradation.
Our data indicate that K63-linked polyubiquitylation activates
PCNA for being engaged in error-free repair. Postreplicative
error-free repair is thought to involve a transient template
switch to the undamaged sister chromatid. An attractive model
is that the ubiquitin chains on PCNA may stimulate interaction
of the stalled replication machinery with proteins associated
with the undamaged sister duplex. Interestingly, recent
studies have indicated that K63-linked polyubiquitylation
involving Ubc13/Mms2 can also activate certain protein
kinases. Moreover K63-linked polyubiquitylation has been shown
to play a role in ribosome activity and endocytosis. It will
be important to identify the parallels between these roles in
order to understand the mechanism through which K63-linked
polyubiquitin chains function.
Conservation.
PCNA is a highly conserved protein with residues
sharing 35% identity between yeast and human
orthologues. Notably, the modification site K164 of
the S. cerevisiae protein is conserved and
found at identcal positions in PCNA from yeasts,
plants and higher eukaryotes, including humans. Also
human PCNA was found to be ubiquitylated at K164 on
DNA damage.
Monoubiquitylation
of
PCNA
is
also
relevant
for
DNA
repair.
Because
error-free
repair
requires
K63-linked
polyubiquitination,
monoubiquitylation
of
PCNA
is
expected
to
mediate
another,
possibly
error-prone
mode
of
repair.
It
is
now
believed
that
monoubiquitylated
PCNA
binds
polymerases
involved
in
translesion
synthesis.
PCNA
ubiquitylation
is
conserved
from
yeast
to
mammals
and
occurs
at
the
same
K164
residue
(see
Figure
above).
Monoubiquitylation
of
PCNA
seems
to
be
prevalent
in
human
HeLa
cells,
yet
polyubiquitylated
forms
can
also
be
detected
at
lower
levels.
Importantly,
mammals
have
homologs
of
Ubc9,
Siz1,
Rad6,
Rad18,
Ubc13,
Mms2,
and
possibly
Rad5
as
well.
Given
this
conservation,
it
seems
likely
that
the
mechanisms
and
functions
are
highly
conserved,
and,
indeed,
genetic
studies
have
confirmed
that
human Rad18 and Mms2 are highly relevant for DNA repair.
SUMO-modified
PCNA
recruits Srs2 to prevent recombination during S-phase
We discoverd an intriguing
affiliation of the SUMOylation system with the
ubiquitin-conjugating machinery. We demonstrated that
SUMOylation and ubiquitylation of PCNA are physically and
functionally linked. We observed that Ubc9 not only interacts
with PCNA but also with the ubiquitin ligases Rad18 and Rad5.
Importantly, ubiquitylation and SUMOylation targets the same
K164 residue of PCNA, demonstrating that the two modifications
are mutually exclusive. SUMO-conjugation enzyme Ubc9 not only
interacts with PCNA but also with the ubiquitin ligases Rad18
and Rad5. The most plausible model is that the
ubiquitylation/SUMOylation enzymes are part of a regulatory
switchboard, which directs PCNA for alternative functions.
Several previous studies have provided evidence for a link
between SUMOylation and ubiquitylation. In cases where these
two processes target the same lysine residue, studies suggest
that SUMO might function as an antagonist for K48-linked
polyubiquitylation, thereby inhibiting proteasomal degradation
of the substrate.
Our data indicate that SUMOylation of PCNA
at K164 does not merely block ubiquitylation at this site, but
that it directs the substrate for other functions. We noticed
previously (Hoege
et al., 2002)
that in Saccharomyces
cerevisiae SUMOylation of PCNA at K127 and K164 are
detrimental for DNA damage tolerance in the absence of PCNA
ubiquitination. It has been suggested by others that
(analogous to the model for mono-ubiquitinated PCNA)
SUMOylation promotes error-prone synthesis through recruitment
of a translesion polymerase. However, we demonstrated recently that
SUMOylated PCNA functionally cooperates with Srs2, a helicase
that blocks recombinational repair by disrupting Rad51
nucleoprotein filaments (Pfander
et al., 2005).
Moreover, we detected that
Srs2 also interacts physically preferentially with the
SUMOylated form of PCNA, owing to a specific binding site in
its C-terminal tail (see Figure). Our data are consistant with a model
in which SUMOylated PCNA recruits Srs2 in S-phase in order to
prevent unwanted recombination events of replicating
chromosomes.
Diagram
of Srs2. Binding sites for PCNA-SUMO and
Rad51 and the helicase domain are indicated. The
SUMO-PCNA-binding site is located at Srs2's
extreme C-terminus.
Together, our findings
emphasize the importance of PCNA modifications for
decision-making at the replication fork. While PCNA
ubiquitination mediates post-replicative lesion bypasses,
modification with SUMO - which occurs even in the absence of
exogenous DNA damage - seems to be a guarding mechanism that
prevents unwanted recombination during replication.
SUMOylation and ubiquitination might represent autonomous
triggers, which operate independently from each other.
However, since components of the two modification pathways
interact, they might build a switchboard in which the
PCNA-SUMO-Srs2 check might facilitate channeling into the RAD6-dependent bypass.
PCNA controls establishment of
sister chromatid cohesion during S phase - control by PCNA
SUMOylation
Accurate segregation of the genetic
material during cell division requires that sister chromatids
are kept together by cohesion proteins until anaphase, when
the chromatids become separated and distributed to the two
daughter cells. Studies in yeast revealed that chromatid
cohesion is essential for viability and is triggered by the
conserved protein Eco1 (Ctf7). Cohesion must be established
already in S-phase in order to tie up sister chromatids
instantly after replication, but how this crucial timing is
achieved remains enigmatic. Recently, we discovered that in
yeast and humans Eco1 is directly physically coupled to the
replication protein PCNA. Binding to PCNA is crucial as yeast
Eco1 mutants deficient in Eco1-PCNA interaction are defective
in cohesion and inviable. Remarkably, binding of Eco1 to
PCNA is prevented by a two-step process. First, the
SUMO-conjugation enzyme Ubc9 compets with Eco1 for PCNA
binding; second, this inhibition is fixed by covalent
modification of PCNA by SUMO.
The RAD6 DNA damage tolerance
pathway operates uncoupled from the replication fork
and is functional beyond S-phase
Although after its
discovery in the 1960s, DNA damage tolerence (DDT) was
initially coined “post-replicative DNA repair”, the
prevailing view today is that DDT acts directly at the
replication fork in S-phase. In fact, PCNA ubiquitylation
is believed to be physically coupled to the stalled fork,
and PCNA modifications were reported to promote
replication fork progression in frog egg extracts, yeast
and humans. Moreover, since the helicase activity of yeast
Rad5 appears to catalyze fork regression in vitro, it was
also suggested that Rad5 promotes template switching
directly at the replication fork. These and several other
studies led to the commonly accepted model that TLS
promotes “bypass replication” across the lesion at the
replication fork, and that the error-free
template-switching mode – either by sister chromatid
junctions or fork regression leading to a DNA structure
called “chicken foot” – acts near the replication fork,
and promotes replication restart similar to bacterial DDT.
The currently widely
accepted model is at first glance appealing as it may
superficially envision a swift rescuing mode for stalled
forks. However, recent work has shown that a fraction of
TLS can occur in the rear of the fork. This issue still
remains unsettled, as it was thus far not directly tested
when and in which phase of the cell cycle the RAD6 DDT
pathway has to operate. This question is not only central
from a mechanistic point of view, but also of singular
general importance, as DDT is highly crucial for cell
survival upon DNA damage, genome stability and hence tumor
biology.
We recently put the
hitherto accepted model directly to test by expressing key
components of both the error-prone and the error-free
pathway specifically in the G2/M phase of the cell cycle.
Expression in G2/M was ensured by the use of G2/M-specific
promoters and by making the proteins highly unstable in
G1. Surprisingly, when we restricted the expression of TLS
polymerases necessary for the error-prone pathway, or the
Rad5 and Sgs1 proteins, which are both essential for the
error-free branch, to the G2/M phase of the cell cycle,
they fully supported DDT virtually identical to wild-type
(WT) cells. Importantly, we also found that replication of
damaged DNA continues even in the absence of the PCNA
polyubiquitylation enzyme Rad5. These findings strongly
suggest that both branches of DDT in eukaryotes operate
post chromosomal replication (even outside S-phase) on
ss-gaps left behind the replication fork. We propose that
this uncoupling allows rapid replication completion and
protects genomic integrity. Additionally, this mechanism
may also make more time for TLS and template switching and
may thus be crucial for evolution and mutagenesis.
Time does matter
Left:
Layman's View:
The RAD6 DNA damage
tolerance pathway acts uncpoupled from the moving replication
fork (trains) even outside S-phase in G2/M of the cell cycle.
Depicted here is the error-free branch, which utilizes the
information of the undamaged sister duplex for repair. PCNA
(center stage) receives instructions from workers acting at the
undamaged sister duplex. This infomation is used by (busier)
workers for the repair of the damaged tract. (Drawing
by
G.I. Karras)
Right: Expert's View:
Bulky DNA adducts on the template strand (black) block DNA
synthesis by the replicative polymerases (a). DDT is activated
in an identical manner by lesions blocking the lagging-strand
(b, left) or the leading-strand
polymerases (b, right).
Leading strand synthesis (yellow) stalling promotes re-priming
downstream of the lesion, thereby leaving a ssDNA stretch
(ss-gap) that contains the lesion behind. Similarly, ss-gaps are
formed by lagging strand stalling (incomplete Okazaki
fragments). The ss-gaps participate in D-loop formation (c),
which are resolved by three different mechanisms. Modification
of PCNA (blue ring) by SUMO (crimson) triggers recruitment of
the helicase Srs2 (brown triangle), which removes the
recombinase Rad51 (gray) from chromatin. The first mechanism
(left row) involves polyubiquitylated (orange) PCNA and sister
chromatid junctions (SCJs), which are dissolved by the
Sgs1-Top3-Rmi1 complex (green triangles). The second mechanism
(middle row) is activated by the DNA damage checkpoint (DDC) and
involves PCNA monoubiquitylation, which triggers the recruitment
of specific TLS polymerases for induced TLS. The third mechanism
(right row) is activated in the absence of PCNA SUMOylation.
This pathway involves Rad51-dependent recombination and the
resolution of double Holliday junctions (RDH) involving the
Sgs1-Top3-Rmi1 complex (green) or specific nucleases. (Drawing
G.I. Karras)
MECHANISMS
OF HOMOLOGOUS RECOMBINATION AND REGULATION BY UBIQUITIN
AND SUMO
Control of Rad52
recombination activity by double-strand break-induced
SUMO modification
Homologous recombination
is essential for genetic exchange, meiosis, and
error-free repair of double-strand breaks. Central to
this process is Rad52, a conserved, homo-oligomeric
ring-shaped protein, which mediates the exchange of
the early recombination factor RPA by Rad51, and
promotes strand annealing. W discovered that Rad52 of
S. cerevisiae is modified by the ubiquitin-like protein
SUMO chiefly at two sites that flank the conserved
Rad52 domain. SUMOylation is induced upon DNA damage
and triggered by MRX-governed double-strand breaks.
Although SUMOylation-defective Rad52 is largely
recombination proficient, mutant analysis revealed
that the SUMO modification sustains Rad52 activity and
concomitantly shelters the protein against accelerated
proteasomal degradation. Importantly, our data suggest
that SUMOylation becomes particularly relevant for
those Rad52 molecules that are engaged in
recombination.
SUMOylation of Rad52. Rad52
becomes SUMOylated at two sites upon DSB
formation. This requires the MRX complex and DNA
resecetion. SUMOylated species accumulate if DSB
repair is blocked downstream of Rad52 in the
pathway (e.g. in dmc1 mutants).
In collaboration with M. Lisby and his co-workers we found
that recombinational repair of a DSB in rDNA involves the
transient relocalization of the lesion and the associated
recombination machinery at an extranucleolar site.
Remarkably, SUMOylation of Rad52 is required for this
process. Mutants defective in Rad52 SUMOylation form foci
within the nucleolus and cause rDNA hyperrecombination and
the excision of extrachromosomal rDNA circles.
See movie by M. Lisby
showing that repair sites (green) locate outside the
nucleolus (red) if Rad52 is modified by SUMO.
Chromosome-wide Rad51
spreading and SUMO-H2A.Z-dependent chromosome
fixation in response to a persistent double-strand
break
DNA double strand breaks
(DSBs) are acutely hazardous for cells as they can cause
genome instability. DSB repair involves the sequential
recruitment of repair factors to the DSBs, followed by
Rad51-mediated homology probing, DNA synthesis, and ligation.
Although previous studies have established a detailed
choreography of events during HR-directed DSB repair, little
is known how cells react if no homology is found and DSBs
persist. We recently addressed this question by using a
defined genetic setup in yeast that generates a single DSB,
which cannot be repaired. When we followed the cellular events
over time, we discovered that cells react by a unique,
multifaceted and chromosome-wide response. The most striking
events are the continuous distribution of the Rad51
recombinase on the broken chromosome indicative of ongoing
homology search (see figure below, left panel), and the late
fixation of the DSB to the nuclear envelope (right panel).
Importantly, the latter process depends on SUMOylated H2A.Z,
which thus functions as a specific chromatin mark for
chromosome relocation.
Behavior of broken chromosomes
Left:Rad51 spreading
along the double-strand break (DSB)-harboring chromosome. (A) Rad51-directed
ChIP-on-chip analysis (genome-wide), 2 hours after the
formation of a single DSB (HO cut site) on chromosome III.
Shown are all chromosomes. Note that Rad51 spreads only on
chromosome III, which harbors the DSB. (B) As above (only
chromosome III is shown) but ChIPs were done at the
indicated time points after DSB-induction. Chromosome III is
shown enlarged with the position of the persisting DSB at
200.85 kb (red line). The asterisks label homology present
on the tiling array, which is, however, deleted in the
strain used. In both panels, filled circles denote the
position of centromeres. Note that Rad51 spreads
bidirectionally from the break and covers the entire
chromosome after some hours.
Right: Recruitment of the DSB to the nuclear
periphery. (Top)
Scheme of chromosome III, which harbors the HO-cut site
(DSB) labeled by an LacO array. GFP-tagged LacI will bind
this array, thereby labeling the position of the DSB in
vivo. (Bottom) Using
this technique we could show that this DNA segment (green)
localizes in the nucleoplasm before the DSB is formed.
However, five hours after DSB formation, the DSB relocalizes
to the nuclear periphery (red).
PROTEASOME-MEDIATED PROTEIN
PROCESSING: THE OLE PATHWAY
The OLE pathway
A few years ago, we discovered
a novel regulatory pathway in yeast, termed the OLE pathway, which is intriguing
with respect to cell physiology and its unique mode of action
(Hoppe
et al., 2000). We found that two related
transcription activators, Spt23 and Mga2, are kept in a
dormant state in the cytosol by anchoring to membranes of the
ER/nuclear envelope via their C-terminal tails. Both proteins
are endoproteolytically processed in a novel
ubiquitin/proteasome-dependent reaction (see below), thereby
enabling the liberated transcription factors to migrate into
the nucleus (see Figure).
Dormant and active transcription factor.
Immunofluorescence and deconvolution imaging of
cells expressing an epitope-tagged version of Spt23.
The Spt23 precursor (left) localizes around the
nucleus. The precursor is cleaved at the membrane by
a ubiquitin/proteasome- dependent reaction, which
liberates the active transcription factor. This
enables the transcription factor to move into the
nucleus (right) and to drive OLE1
transcription. OLE1 encodes delta-9 fatty acid desaturase, an
ER-bound enzyme that catalyzes the formation of
unsaturated fatty acids. High levels of unsaturated
fatty acids in turn repress Spt23 processing
(negative feed-back control).
Importantly, OLE1, which
encodes the ER-bound enzyme delta-9 fatty
acid desaturase, is among the known Spt23/Mga2 targets. Ole1
is a key enzyme in lipid and membrane synthesis pathways and
catalyzes the desaturation of C16
and C18
fatty acids, forming palmitoleic and oleic acid (right
picture). The correct ratio of saturated to unsaturated fatty
acids is crucially important for maintaining the optimal
fluidity of the membrane. Low levels of unsaturated fatty
acids lead to a severe impairment of cellular membrane
systems, most notably of the nuclear envelope and of
mitochondria.
Particularly
intriguing
is
our
observation
that
Spt23
processing
is
completely
blocked
by
adding
unsaturated
fatty
acids
to
the
growth
medium.
Importantly,
we
found
that
not
oleic
acid
(18:1),
but
the
-
by
two
carbons
shorter
-
palmitoleic
acid
(16:1)
and
polyunsaturated
fatty
acids,
linoleic
(18:2)
or
linolenic
acid
(18:3),
are
specifically
potent
in
preventing
Spt23
processing.
Our
findings
suggests
that
the
trigger
that
regulates
Spt23
processing
may
not
be
the
chemical
nature
of
the
fatty
acid
but
rather
the
fluidity
and/or
the
thickness
of
the
membrane.
We
propose
that
the
precursor
of
Spt23
(or
a
protein
that
interacts
with
Spt23)
functions
as
the
sensor
of
membrane
composition
because
it
is
exposed
to
the
membrane
system
which
is
altered
by the activity of Ole1. A change in membrane fluidity or
composition could induce a conformational change into the
Spt23 molecule or a binding partner, which thereby could
trigger an association of Spt23 with the processing machinery.
This type of control would be reminiscent of the pathways that
regulate the processing of the membrane-bound precursor of
SREBP by sterols.
The
previously discovered processing reactions of membrane
proteins (e.g. SREBP, ATF6, IRE1, Notch, APP) are thought to
be mediated by regulated intramembrane proteolysis or "RIP",
involving site-specific, membrane-localized proteases. Spt23
and Mga2, however, are processed by a strikingly different
mechanism that we term "regulated
ubiquitin/proteasome-dependent processing" or "RUP".
We found that
Spt23 directly interacts with Rsp5, a hect E3 ubiquitin
ligase, which catalyzes the ubiquitylation of Spt23. Rsp5
interacts with its substrate through the most C-terminal of
Rsp5's three so-called WW (protein interaction) domains.
Importantly, Spt23 precursor processing was found to depend on
Rsp5 and the proteasome. Proteasome action commonly results in
the complete degradation of the substrate protein to small
peptides. An exception to this rule is the processing of p105,
the precursor of the p50 NF-kB transcription factor. Processing of
p105 by proteasomes proceeds by a mechanism in which the
C-terminal half of the molecule is rapidly degraded whereas
the N-terminal portion (p50) is left intact. Intriguingly,
Spt23 and Mga2 are structurally related to NF-kB p105.
Spt23 and Mga2 have immunoglobulin-like putative DNA binding
(IPT) domains similar to NF-kB
and possess C-terminally positioned ankyrin repeats
analogously to p105 (see Figure below).
Diagram
of Spt23 and Mga2, yeast homologs of mammalian NF-kB. The
proteins possess distinct activation domains (blue), a
region of homology to NF-kB (red), two ankyrin repeats (dark
gray), and a single transmembrane span (black). The
approximate location of the cleavage site that results in
p90 production is indicated by an arrow head.
We
discovered that Spt23/Mga2 precursors are endoproteolytically
processed by proteasomes (see Figure below and Rape
and Jentsch, 2002 and Piwko
and Jentsch, 2006). Our model derives from the fact that
the molecules are anchored to membranes via their C-terminal
tails and that the N-terminal domains are spared from
degradation. We propose that the polypeptide chains of
Spt23/Mga2 enter the openings of the proteasomes as loops in
order to contact the active sites that are located within the
central cavity of the protease.
Taking a bite - a
polypeptide loop as a possible substrate for the
proteasome.
Left. The model shows a vertically sliced
and opened 20S proteasome, as determined by X-ray
cristallography studies (Groll et al., Nature386, 463-471, 1997). A hairpin loop of
a substrate composed of an unstructured polypeptide
chain is modelled into the structure (yellow). A
folded NF-kB (p50) domain of a
hypothetical substrate is shown for comparision. The
dimensions of the gate are ~13Å and the distance
from the outside of the 20S proteasome to the active
sites (red dots)
are ~70Å (equivalent to about 20 amino acids of an
extended polypeptide chain.
Right. Model
for proteasome-mediated degradation and processing.
The proteasome harbors within its 20S particle a
central catalytic cavity and two
antechambers.Complete substrate degradation (first row) may
commence by the insertion of an internal polypeptide
loop through the openings of the proteasome into the
proteolytic chamber. Degradation initiates by a cut
within the loop and continues bi-directionally (red
arrows) towards both the N- and C-terminal ends of
the polypeptide (middle column). If there are no
steric constraints (first
row), degradation continues in both
directions (redarrows)
until the polypeptide is completely degraded to
small peptides, which exit the proteasome by
diffusion. Protein processing (rows 2-4) occurs
if the polypeptide contains tightly folded
dimerization domains or other tightly folded
domains. These domains cannot enter the central
cavity and therefore cause incomplete degradation,
i.e. degradation proceeds only towards the end that
is not hindered by barriers. The processing product
contains the tightly folded (or dimerization)
domain, and additionally segments distal from these
domains and a short polypeptide stretch that
reflects the distance from the proteasome openings
to the active sites. Degradation of substrates that
contain tightly folded domains both N- and
C-terminally of the inserted loop yield two
processing products (last row). Only the authentic N- and
C-termini are labeled.
Indeed,
the
known
crystal
structure
of
yeast
proteasomes
suggests
that
the
openings
are
sufficiently
wide
to
accommodate
two
juxtaposed
polypeptide
chains.
The
distance
from
the
active
sites
to
the
outside
of
the
20S
proteasome
equals
approximately
an
extended
polypeptide
chain
of
20
amino
acids,
indicating
that
at
least
40
amino
acids
of
the
substrate
has
to
enter
the
proteasome
barrel.
An
important
element
in
p105
processing
is
a
glycine-rich
repeat
within
the
center
of
the
molecule.
Spt23
and
Mga2
do
not
possess
this
element,
but
both
proteins
have
several
stretches
enriched
in
asparagine
residues.
It
seems
attractive
to
speculate
that
these
low
complexity
domains
might
promote
loop
formation.
We
were
consistently
unable
to
recover
a
C-terminal
cleavage
product of Spt23 and hence we assume that, analogous to the
situation for p105, the C-terminal tail gets completely
degraded. Degradation of Spt23's tail, in contrast, would
require membrane extraction, and this could be achieved in a
manner that resembles proteasome-mediated ER-associated
degradation. Interestingly, previous data have indicated that
also NF-kB p50 is generated by a similar
endoproteolytic reaction, suggesting that this mode of action
might be common for proteasomes. In fact, proteasomal
degradation sometimes leads to an accumulation of a stable
protein fragment (Seufert
and Jentsch, 1992), possibly suggesting that proteasomal
degradation is frequently initiated by an internal cleavage.
Model for Spt23 Processing and
Mobilization. Spt23 (green) at the ER might
dimerize via the IPT domains before Rsp5-mediated
monoubiquitylation (red ball). Processing of Spt23
by the proteasome (gray barrel) leads to p90, which is
associated with an uncleaved p120 partner molecule.
Processing might be assisted by Cdc48Ufd1/Npl4 (yellow
crown). The processed p90 has retained its ubiquitin
modification, and Cdc48Ufd1/Npl4 removes p90 from its p120 partner. Subsequently,
p90 can enter the nucleus to drive OLE1
transcription.
Cdc48Ufd1/Npl4,
a ubiquitin-selective chaperone (segregase)
We
discovered that Spt23 processing results in p90 bound to an
unprocessed Spt23 p120 partner molecule at the membrane (see
Figure above). Interestingly, p90 has retained its ubiquitin
modification after processing and we discovered that a
chaperone-like enzyme, designated Cdc48Ufd1/Npl4,
separates p90 from p120 by an ATP-dependent mechanism, thereby
mobilizing p90 for nuclear targeting (Rape
et al., 2001). Intriguingly, the chaperone
displays selectivity towards ubiquitylated proteins,
suggesting that this enzyme has the potential to selectively
segregate ubiquitylated proteins from other proteins of
oligomeric protein complexes (see Figure). We suggest the term
"segregase" for this and related activities.
Notably,
Cdc48Ufd1/Npl4 is also required for ER-associated
protein degradation (ERAD) (see below),
indicating that the segregase is a basic component of the
ubiquitin system (Braun
et al., 2002).
Cdc48 associates with distinct
'substrate-processing cofactors' that control the degree of
ubiquitination of bound substrates. We recently proposed that Cdc48
functions similar to a "gearbox" in a car. In this model,
the 'substrate-processing cofactors' shift the
polyubiquitination reaction either into 'forward',
'neutral', or 'reverse'.
Speculative model of a gearbox function of Cdc48.
Mono- or oligo-ubiquitinated substrates (brown; ubiquitin,
red) are recruited to the Cdc48 (p97) gearbox (gray). In the
position ‘forward’ (F) the E4 enzyme Ufd2 polyubiquitinates
the substrate, thereby promoting proteasomal degradation. In
‘neutral’ (N), the WD-40 protein Ufd3 competes with Ufd2 for
Cdc48 binding, thereby preventing further ubiquitination of
the substrate by Ufd2. In the position ‘reverse’ (R), the
deubiquitination enzyme Otu1 removes the ubiquitin
modification of the substrate. Substrates released from
Cdc48 through the ‘N’ and ‘R’ positions of the gearbox are
either mono- (oligo-)ubiquitinated or unmodified and thus
metabolically stable.
SUBSTRATE DELIVERY TO THE PROTEASOME: THE ESCORT PATHWAY
Polyubiquitylation factor E4
Proteins
modified by polyubiquitin chains are the preferred substrates
of the proteasome. Previous work has suggested that E1, E2,
and E3 enzymes are both required and sufficient for the
formation of polyubiquitylated substrates. However, we
discovered that efficient polyubiquitylation of a model
substrate (ubiquitin fusions; UFD substrates) requires an
additional conjugation factor, which we termed E4 (Koegl
et al., 1999). This protein, previously known as
Ufd2 in yeast, binds to the ubiquitin moieties of preformed
conjugates and catalyzes ubiquitin chain assembly in
conjunction with E1, E2, and E3 (see Figure). Intriguingly,
Ufd2 defines a new protein family that contain a conserved
domain, which we coined the U-box. These domains are related
to RING fingers and are involved in polyubiquitylation
reactions. In yeast, Ufd2/E4 activity is linked to cell
survival under stress conditions and mediates the degradation
of Spt23 p90 (see above). Ufd2 is largely
in nuclear protein and its nuclear localization depends on the
activity of the Cdc48Ufd1/Npl4 complex (Richly
et al., 2005).
E4 activity.
Left: Degradation of a model substrate (ubiquitin
fusions) by the "Ubiquitin Fusion Degradation" (UFD)
pathway. E1, E2, and E3 enzymes catalyze the conjugation of
only a few ubiquitin moieties to the substrate (Initiation).
In the presence of E4 (Ufd2) these enzymes catalyze
polyubiquitin chain assembly. Right: The
picture demonstrates the influence of increasing
concentrations of E4 on polyubiquitin chain formation (gel
electrophoresis).
See Koegl
et al. (1999) for details.
An escort pathway to the proteasome
Protein
degradation in eukaryotes usually requires polyubiquitylation
and subsequent delivery of the tagged substrates to the
proteasome. Recent studies suggest the involvement of the AAA
ATPase Cdc48, its co-factors, and other ubiquitin-binding
factors in protein degradation, but how these proteins work
together was unknown until recently. We found that
these factors cooperate sequentially through protein-protein
interactions and thereby escort ubiquitin-protein conjugates
to the proteasome. Central to this pathway is the chaperone
Cdc48/p97, which coordinates substrate recruitment,
E4-catalyzed polyubiquitin chain assembly, and proteasomal
targeting. In yeast
this escort pathway guides a transcription factor from its
activation in the cytosol to its final degradation and
also mediates proteolysis at the endoplasmic reticulum by
the ERAD pathway.
On the eve of destruction.
A protein substrate (brown; possibly bound to a partner
protein) is modified by one or two ubiquitin moieties
(red) using E1, E2, and E3 enzymes. The oligoubiquitylated
substrate is then recognized by the Cdc48Ufd1/Npl4
complex (dark gray) and liberated from its potential
partner protein. Cdc48 recruits E4 (Ufd2; green), which
extends the ubiquitin chain by a few extra ubiquitin
moieties (size-restricted chains). Subsequently, Ufd2
recruits Rad23 (or Dsk2; blue), which binds the
ubiquitin-protein conjugate and delivers it to the
proteasome for degradation. See Richly
et al. for
details.
We also found that Cdc48
prevents the formation of excessive polyubiquitin chain
sizes ("size restriction") that are surplus to requirements
for degradation (see Figure below). Intriguingly, the
size-restricted polyubiquitin chains formed by E4 in the
presence of Cdc48 have the optimal length for binding to
Rad23. Rad23 finally escorts the ubiquitin-conjugate to the
proteasome.
Size restriction.
Ubiquitylation of specific substrates (UFD proteins)
by E1, E2, and E3 leads to the conjugation of only
one or two ubiuqitin moieties (first lane from
left). In the presence of E4, however,
polyubiquitylation is processive and leads to the
formation of very long multiubiquitin chains (second
lane). Increasing concentrations of Cdc48 to the
ubiquityletion reactions restricts the chain length
to 4-6 ubiquitin moieties in a chain. Intriguingly,
this chain length is optimal for binding to Rad23
(see figure above). See Richly
et al.
for details.
The
traditional view of an escort pathway to hell is best illustrated by Giotto in the Capella
degli Scrovegni (Arena chapel), Padova,
Italy.
Proteins,
which are translocated into the ER, and fail to fold or
assemble properly, are subject to a proteolytic quality
control pathway, termed "ER-associated degradation" or ERAD.
Our studies provided the first evidence that ERAD is mediated
by the cytosolic ubiquitin/proteasome pathway (Sommer
and Jentsch, 1993). We identified an integral membrane
ubiquitin-conjugating enzyme (Ubc6) and demonstrated that it
mediates degradation of ER membrane proteins. Subsequent
studies have shown that also substrates of the lumen of the ER
are degraded by the ubiquitin/proteasome pathway. These
substrates are first transported back into the cytosol by a
mechanism that involves the Sec61 translocon. Proteolysis of
ER-membrane proteins appears to be mediated by a similar
pathway , but how membrane extraction, ubiquitination, and
proteasomal degradation are functionally connected has
remained an enigma.
By
using a designed model substrate we discovered that the
proteasome itself participates in the extraction of an
ER-membrane protein destined for cytosolic destruction (Mayer
et al., 1998). In yeast mutants expressing
functionally attenuated proteasomes, we found that degradation
of a short-lived doubly membrane-spanning protein proceeds
rapidly through the amino-terminal cytosolic domain of the
substrate, but slows down considerably when continued
degradation of the molecule requires membrane extraction. From
these data we conclude that proteasomes engaged in ERAD can
directly process transmembrane proteins through a mechanism in
which the dislocation of the substrate and its proteolysis are
coupled (see Figure). Intriguingly, ERAD also requires the
activity of the ubiquitin-selcelive segregase Cdc48Ufd1/Npl4 (Braun
et al., 2002). We propose that retrograde
transport of short-lived substrates might be driven through
the action of the AAA-ATPase Cdc48Ufd1/Npl4
in conjunction with the proteasome.
Model for the role of the
proteasome in ERAD. Degradation of integral
membrane proteins (green) of the ER is mediated by the
ubiquitin/proteasome pathway (ERAD). Ubc6 and Ubc7 are
membrane associated. Membrane extraction is coupled to
degradation by proteasomes (gray cylinders). It has been
proposed by others that the Sec61 complex facilitates the
reaction, probably by providing a hydrophilic environment.(not
drawn to scale). See Mayer
et al., (1998) for details.
REGULATION OF APOPTOSIS AND CYTOKINESIS
BY BRUCE, A GIANT E2/E3 UBIQUITIN-LIGASE
BRUCE, a giant
ubiquitin-conjugating enzyme related to IAP apoptosis
inhibitors
By
homology probing we identified a novel murine E2 enzyme and
cloned the corresponding ~16kb cDNA by cDNA walking (Hauser
et al., 1998). The cDNA sequence predicts a
giant protein of 530 kDa, which we termed BRUCE (BIR
Repeat-containing Ubiquitin-Conjugating Enzyme). BRUCE is one
of the largest proteins known to date, but, unlike most other
giant proteins, nonrepetitive in structure. BRUCE possesses a
C-terminal ubiquitin-conjugating enzyme (UBC) domain and an
N-terminal BIR repeat. BIR repeats have been been found in
apoptosis inhibitors of the IAP-related protein family and are
thought to inhibit caspase activity through binding.
BRUCE's BIR domain is ~40% identical in sequence to those of
other IAP relatives and bears the hallmarks of this domain,
including a characteristic cysteine/histidine motif that is
thought to function in zinc binding.
Structure of BRUCE.
Top: Scheme of the protein structure of
BRUCE. The 530 kDa protein has only two recognizable
protein domains, an N-terminally positioned BIR repeat
and a UBC (ubiquitin-conjugating enzyme) domain close
to its C-terminus. See Hauser
et al.(1998) for details. Bottom: Neon art by Bruce
Nauman, 1968; "My name as though it were written on
the surface of the moon".
BRUCE is a peripheral membrane
protein and localizes to the Golgi compartment and the
vesicular system.
BRUCE localizes mainly to the trans-Golgi
network (bright green) and the vesicular
systemin interphase.
Primary rat neurons.
Dual role of BRUCE as an anti-apoptotic IAP and a chimeric
E2/E3 ubiquitin ligase
Apoptotic cell
death and survival is controlled by pro- and anti-apoptotic
proteins. Because these proteins act on each other, cell fate is
dictated by the relative activity of pro- versus anti-apoptotic
proteins. Recently, we reported that BRUCE protects cells
against apoptosis and functions as a novel inhibitor of
apoptosis (IAP). By using wild-type and mutant forms we show
that BRUCE inhibits caspase activity and apoptosis depending on
the integrity of the BIR domain. Upon apoptosis induction, BRUCE
is antagonized by three mechanisms: first, through binding to
Smac, second, by proteolysis catalyzed by the mitochondrial
derived protease HtrA2, and third, by caspase-mediated cleavage.
In addition to its IAP activity BRUCE has the distinctive
property to function as a chimeric E2/E3 ubiquitin ligase with
Smac being a substrate. This suggests that, owing to its two
activities and its localization, BRUCE may function as
specialized regulator of cell death pathways.
BRUCE is required for normal placenta development and mouse
survival
To study the function of BRUCE in vivo we have
recently generated BRUCE knock out mice by gene
targeting. Complete inactivation of the BRUCE gene resulted in
perinatal lethality and growth retardation discernable after
embryonic day 14. The growth defect is linked to an impaired
placental development and may be caused by insufficient oxygen
and nutrient transfer across the placenta. Chorioallantoic
placentation initiated normally but the mutant placenta showed
an impaired maturation of the labyrinth layer and a
significant reduction of the spongiotrophoblast. No evidence
for an elevated apoptosis rate was detectable in embryonic and
extra-embryonic tissues and in knockout fibroblasts. Thus,
although BRUCE is
broadly expressed in embryonic, extra-embryonic and adult
mouse tissues, this bi-functional protein might plays a unique
role in normal trophoblast differentiation and embryonic
survival.
Final stages
of cytokinesis and midbody ring formation are controlled by
BRUCE
Cytokinesis involves the formation of a cleavage furrow,
followed by abscission, the cutting of the midbody channel,
the final bridge between dividing cells. Recent findings
suggest that the midbody ring is central for abscission. We
found that BRUCE in addition to its antiapoptotic function
acts as a major regulator of abscission. During cytokinesis,
BRUCE moves from the vesicular system to the midbody ring (MR;
see Figure) and serves as a platform for the membrane delivery
machinery and mitotic regulators.
The midbody ring. The
midbody ring (Flemming body) localized to the center
of the intercellular tube that connects two
prospective daughter cells during cell division. Left:
BRUCE (green) is part of the ring, which seems to
encircle microtubules (red). Right:Midbody ring
visualized with a C-terminal fregment of BRUCE
harboring a midbody ring-targeting domain (MRD). Watchmovie to see how mobile the
MR is during abscission.
See Pohl
and Jentsch (2008) for details.
Depletion of BRUCE in cell cultures
causes defective abscission (watch movie) and
cytokinesis-associated apoptosis, accompanied by a block of
vesicular targeting and defective formation of the midbody and
the MR. Notably, ubiquitin relocalizes from midbody
microtubules to the MR during cytokinesis (see Figure below
and watchmovie) and depletion of BRUCE
disrupts this process. We propose that BRUCE coordinates
multiple steps required for abscission, and that
ubiquitylation may be a crucial trigger.
BRUCE and ubiquitin
during cytokineses. A small portion of
BRUCE (green) localizes to the midbody ring during
cytokinesis. Ubiquitin (red) initially concentrates
flanking the midbody ring, but co-localizes finally
with BRUCE on the midbody ring. DAPI stains DNA.
Circular images represent blow-ups of the midbody
region.
Watch movie showing the behavior
of GFP-tagged ubiquitin during cytokinesis
The
MR materializes in telophase, localizes to the intercellular
bridge during cytokinesis, and moves asymmetrically into one
cell after abscission (see Figure below and watchmovie).
Because there is just one MR, but two daughter cells,
presumably all mammalian cell divisions are asymmetric. Daughter cells rarely
accumulate MRs of previous divisions, but how these large
structures finally disappear was an open question.
Asymmetry of cell
division. The single, active midbody ring
(green) is discarded into one daughter cell after
cytokinesis. Vesicle flow comes mainly from one
prospective daughter cell. Old midbody rings (red)
can accumulate, lose their ubiquitin modification,
and are finally eliminated by autophagy.
Midbody ring
disposal by autophagy is a post-abscission event of
cytokineis
We found that MRs are in fact discarded by autophagy.
This involves their sequestration into autophagosomes and
delivery to lysosomes for degradation. Notably, autophagy
factors, like the ubiquitin-adaptor p62 and the
ubiquitin-related protein Atg8, associate with the MR already
during abscission, suggesting that autophagy is coupled to
cytokinesis. Moreover, MRs accumulate in cells of patients
with lysosomal storage disorders, indicating that defective
midbody ring disposal is characteristic for these diseases.
Thus autophagy plays a broader role than previously assumed,
and we popose that cell renovation by clearing from
superfluous large macromolecular assemblies like MRs is an
important autophagic function.
Midbody
rings are eliminated by autophagy. Midbody
rings (green) accumulate upon inhibition of
autophagy or lysosomal degradation.
Ubiquitin
and
ubiquitin-like
modifiers
(UBLs)
form
covalent
complexes
with
other
proteins
by
isopeptide
formation
between
their
carboxyl
(C)-termini
and
-amino
groups
of
lysine
residues
of
acceptor
proteins.
A
hallmark
of
UBLs
is
a
protruding
C-terminal
tail
with
a
terminal
glycine
residue,
which
is
required
for
ATP-dependent
conjugation.
Recently, it has been reported that the
highly conserved protein Hub1 (homologous to ubiquitin 1)
functions as a UBL following C-terminal processing (Dittmar et
al., Science295, 2442-2446, 2002).
Hub1 exhibits sequence similarity with ubiquitin, but it lacks
a C-terminal tail bearing a glycine residue (see figure
below). We showed that Hub1 can indeed form SDS-resistant
complexes with cellular proteins, but provide evidence that
these adducts are not formed through covalent C-terminal
conjugation of Hub1 to substrates (Lüders
et al., 2003). The adducts are still formed when
the C-terminus of Hub1 was altered by epitope tagging,
amino-acid exchange or deletion, or when cells were depleted
of ATP. Moreover, we
found no evidence for C-terminal processing of Hub1. Thus, our data indicate that Hub1 is a
ubiquitin-domain protein (UDP), rather than a UBL, and that
the observed Hub1-protein adducts arise probably through noncovalent protein-protein
interactions. An attractive speculation is that Hub1 may form
tight associations (some of which being SDS resistant) with
proteins or protein complexes in order to regulate their
function, localization, or stability. Indeed, gel filtration
experiments indicate that Hub1-protein adducts may be part of
larger protein complexes.
Ubiquitin and its
cousins. Structural ribbon (top) and
molecular surface representations (bottom) of Hub1 (S.
cerevisiae; Ramelot et al., 2003),
ubiquitin (Homo sapiens; Vijay-Kumar et al.,
1987), Rub1/NEDD8 (H. sapiens; Whitby et
al., 1998) and SUMO-1 (H. sapiens; Bayer
et al., 1998).
Pichler*, A.,
Knipscheer*, P., Oberhofer, E., van Dijk, W.J., Körner, R.,
Olsen, J.V., Jentsch, S., Melchior, F., and Sixma, T.K.
(2005). SUMO
modification of the ubiquitin conjugating enzyme E2-25K. Nat. Struct.
Mol. Biol.12,
264-269. (Published
online: 20 February 2005) [PDF]
Scheffner, M.,
Smith, S., and Jentsch, S. (1998).
The
ubiquitin-conjugation system.
In: Ubiquitin
and the Biology of the Cell. J. M. Peters, J. R. Harris , and D. Finley, eds.,
Plenum Press, New
York, pp 65-98.
Jentsch, S. and
Bachmair, A. (1992).
Principles of
protein turnover - Possible manipulations.
In: Protein
Engineering, a Practical Approach. A. R. Rees, R.
Wetzel, and M. J. E. Sternberg, eds.,
IRL Press, Oxford
UK, pp 221-228.
Finley, D.,
Özkaynak, E., Jentsch, S., McGrath, J.P., Pazin, M., Snapka,
R.M.,
Bartel, B., and
Varshavsky, A. (1988).
Molecular genetics
of the ubiquitin-system.
In: Ubiquitin.
M. Rechsteiner, ed., Plenum Press, New York, pp 39-75.
2011
Elected member
of Academia Europaea
1998
Elected member of the German Academy of Sciences, Leopoldina
1995
Elected member of the
European Molecular Biology Organization, EMBO
2007
Swerling Lecture, Dana
Farber Cancer Center, Harvard, Boston, USA
2001
Appointment to
Honorary Professor of Biology at Ludwig Maximilians
University, Munich, Germany
2011 Klaus
Tschira Prize for Understandable Science to Frank
Striebel
2011 Otto-Hahn
Medal to Georgios Karras
2010 MPIB Junior
Research Award to Georgios Karras
2008 MPIB Junior Research Award to Christian Pohl
2007
MPIB Junior Research Award to Meik Sacher
2007
MPIB Junior Research Award to Lucian Moldovan
2006
Otto-Hahn
Group
Award to Boris Pfander
2006
Otto-Hahn
Medal to Boris Pfander
2006
MPIB Junior Research Award to Boris Pfander
2005
MPIB Junior Research Award to Till Bartke
2002
Otto-Hahn
Medal to Michael Rape
2002
MPIB
Junior Research Award to Carsten Hoege
2001
MPIB
Junior
Research Award to Michael Rape
2000
MPIB
Junior
Research Award to Thorsten Hoppe
LINKS
Max Planck Society Home
Page
Max Planck Institute of
Biochemistry
Center
for Integrated Protein Science Munich
Rubicon, EU Network of Excellence
for Ubiquitin Research
UbiRegulators, Marie
Curie Research Training Network
International Max Planck Research
School
European Research Institute for
Integrated Cellular Pathology
Ludwig-Maximilians
Universitaet Muenchen
Genzentrum der LMU
Technische Universitaet
Muenchen
Fudan University Shanghai,
China
EMBO, European Molecular Biology Organization
Deutsche Forschungsgemeinschaft
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RESEARCH
INTERESTS 
Regulation
of
the splicesome by the ubiquitin-like protein Hub1
SnapShot:The SUMO pathway. Creton and Jentsch,
Cell 143, 2010
REGULATION
OF DNA DAMAGE TOLERANCE AND REPAIR BY UBIQUITIN AND
SUMO: THE PCNA SWITCH
