Selective Ubiquitin-dependent and -independent Proteolysis; Regulation of Polyamine Biosynthesis
We are interested in the mechanisms and principles of selective
proteolysis in eukaryotic cells. As a model system in our studies
we employ the yeast Saccharomyces cerevisiae, the first
eukaryote for which the sequence of the entire genome has become
available. High metabolic instability is characteristic for abnormal
and defective proteins, but also for many regulatory proteins,
prime examples being cyclins and Cdk inhibitors. Substrate proteins
that are destined for degradation by a complex protease, termed
the proteasome, are marked by conjugation to a chain of ubiquitin,
a small protein present in all eukaryotic cells. We are interested
in the nature of the degradation signals and their recognition
that result in ubiquitylation and subsequent degradation by the
proteasome.
Some
substrates, however, are recognised by the proteasome without prior
ubiquitylation. On such substrate ist ornithine decarboxylase (ODC),
the rate-limiting enzyme in the synthesis of polyamines. We have
identified a yeast ODC antizyme, which disrupts the ODC dimer and
thereby targets it to the proteasome. We are dissecting the mechanism
of ubiquitin-independent targeting of ODC. The expression of ODC
antizyme involves a +1 ribosomal frameshifting, which is induced by high
levels of polyamines. These conditions, in addition, lead to a
stablization of ODC antizyme, which is degraded by the proteasome in a
ubiquitin-dependent manner (Palanimurugan et al., 2004; Gödderz et al. 2011; Kurian et al., 2011; Beenukumar et al. 2015; Palanimurugan et al., 2018; Halwas et al. 2022).
Heat-Inducible degron and the generation of conditional mutants:
A method was developed that allows for a rapid design-based generation of temperature sensitive mutants in Saccharomyces cerevisiae. The method employs a temperature-inducible degron (td),
which, when linked to the N terminus of a protein to be studied,
targets it for rapid degradation via the ubiquitin-dependent N-end rule
pathway. The degron, however, is only active at elevted
("restrictive") temperatures whereas it is inactive at lower
("permissive") temperatures.
References and information on the method
Low-temperature-inducible(lt) degron and the generation of conditional mutants:
In collaboration with the group of Nico Dissmeyer, we developed a variant of the heat-inducible degron that allows a switch from a more stable state to an unstable state at lower temperatures (e.g. 17 degree C to 25 degree C). This allows to generate yeast mutants which allow shutoff of a function at normal growth temperature (30 degree C), or to generate lt mutants in plants (Faden et al. 2005).
Assembly and maturation of a complex protease
The 26S proteasome is a complex 2-Megadalton protease composed
of at least 32 different subunits. It is formed by two subcomplexes,
a 19S ATPase particle responsible for the recognition of ubiquitylated
proteins, and a proteolytic core particle termed the 20S proteasome.
We have identified a maturation factor that is specifically required
for efficient assembly and maturation of the 20S proteasomes.
This maturation factor, Ump1, is assembled into half-proteasome
precursor complexes containing 7 alpha and 6 beta-subunits. Some
of the beta-subunits are in a precursor form bearing propeptides.
Incorporation of the beta-7 drives the dimerization of two such
precursor complexes yielding the 20S proteasome core particle. During
this process, Ump1 becomes encased. After Ump1-assisted activation of
the
proteolytic sites through processing of beta-subunits, Ump1 is
rapidly degraded making it the first substrate of newly formed
proteasomes (Ramos et al, 1998).
Several beta-subunits are distinguished from their bacterial
counterparts by striking C-terminal extensions. We investigated
the role of such an extension on subunit beta-2 in Saccharomyces
cerevisiae. A truncated version of beta-2 lacking this extension
still assembles into proteasomal complexes in the presence of
full length beta-2. A yeast mutant expressing only the truncated
version, however, is inviable, indicating the beta-2 C-terminal
extension is essential for the formation of functional 26S proteasomes.
The C-terminal extension of beta-7, which extends from one half
to the other, appears to act as a clamp that stabilizes the interaction
of two halfproteasome precursor complexes during assembly. Halfproteasome
precursor complexes accumulate to unusually high levels in mutants
lacking this extension. These mutants are moreover characterized
by a complete loss of the post-acidic activity. H-bonds between
residues in the beta-7 tail and beta-1 are apparently stabilizing
an active conformation of the postacidic side that resides in
the beta-1 subunit. Incorporation of the beta-7 subunit is a rate-limiting step for precursor complex dimerization (Ramos et al., 2004; Marques et al, 2007; Ramos and Dohmen, 2008; Marques et al. 2009; Kock et al. 2015). Ump1 interacts with parts of beta-7, in particular its propeptide, to promote the incorporation of this subunit (Zimmermann et al. 2022). Human mutations in the UMP1 (alias POMP) gene lead to a disease characterized by autoinflammation and immune dysregulation (PRAID) (Meinhardt et al. 2021).
Defekts in the proteasome or overloading with substrates leads to an induction of the expression of proteasome subunit genes by the transcription factor Rpn4. This transcription factor is therefore a central component in a regulatory feedback control of proteasome regulation (London et al. 2007; (Dohmen et al., 2007).
Protein modification by conjugation of the
small ubiquitin-related modifier SUMO
SUMO, a protein with sequence
similarity to ubiquitin is conjugated to substrate proteins in
an enzymatic cascade similar to that of ubiquitin conjugation.
It involves the activity of specific activating and conjugating
enzymes. We have iscovered another enzyme of this system,
a specific SUMO deconjugating enzyme, Ulp2, which is located in the
cell nucleus and required for proper cell cycle progression and
response to DNA damage. We are currently studying the balance
in the system that is provided by the conjugating and deconjugating
enzymes, and will try to learn more about the system's function
through characterization of the substrates modified by SUMO conjugation.
In the yeast Saccharomyces cerevisiae SUMO conjugation
is required for transport of proteins with so-called classical
nuclear localization signals (NLS) into the cell nucleus (Dohmen et al., 1995; Johnson et al., 1997; Schwienhorst et al. 2000; Stade et al. 2002; Eckhoff and Dohmen 2015, 2016; Pabst et al. 2019).
