Many intracellular proteins are either conditionally or constitutively short-lived, with in vivo half-lives that can be as brief as a minute or so. The regulated and processive degradation of intracellular proteins is carried out largely by the ubiquitin (Ub)-proteasome system (UPS), in conjunction with molecular chaperones, autophagy, and lysosomal proteolysis. The N-end rule pathway, the first specific pathway of UPS to be discovered, relates the in vivo half-life of a protein to the identity of its N-terminal residue. Physiological functions of the N-end rule pathway are strikingly broad and continue to be discovered. In bacteria and in eukaryotic organelles mitochondria and chloroplasts all nascent proteins bear the pretranslationally formed N-terminal formyl-methionine (fMet) residue. What is the main biological function of this metabolically costly, transient, and not strictly essential modification of N-terminal Met, and why has Met formylation not been eliminated during bacterial evolution? One possibility is that the formyl groups of N-terminal Met in Nt formylated bacterial proteins may signify a proteolytic role of Nt-formylation. My colleagues and I addressed this hypothesis experimentally, as described in Chapter 3 of this thesis.
Among the multitude of biological functions of the mammalian Arg/N-end rule pathway are its roles in the brain, including the regulation of synaptic transmission and the regulation of brain’s G-protein circuits. This regulation is mediated, in part, by the its Ate1-mediated arginylation branch of the Arg/N-end rule pathway. One role of the Ate1 arginyltransferase (R-transferase) is to mediate the conditional degradation of three G-protein down-regulators, Rgs4, Rgs5, and Rgs16. Ate1-/- mice, which lack the Ate1 R-transferase, exhibit a variety of abnormal phenotypes. Chapter 4 describes our studies of neurological abnormalities in Ate1-/- mice (and also in mice that express Ate1 conditionally, upon the addition of doxycycline), with an emphasis on the propensity of these mice to epileptic seizures.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, month = {July}, advisor = {Varshavsky, Alexander J.}, } @phdthesis{10.7907/Z91V5BZQ, author = {Wadas, Brandon Christopher}, title = {Biochemical and Genetic Studies of the N-End Rule Pathway in Yeast and Mammals}, school = {California Institute of Technology}, year = {2016}, doi = {10.7907/Z91V5BZQ}, url = {https://resolver.caltech.edu/CaltechTHESIS:06022016-162251299}, abstract = {
Regulation of the in vivo half-lives of intracellular proteins is an important cellular process. Many intracellular proteins are short-lived, owing to their regulated and processive degradation by the Ubiquitin (Ub)-Proteasome System (UPS). In eukaryotes, the N-end rule pathway is one specific pathway within the UPS. The N-end rule pathway relates the identity of the N-terminal residue of a protein, or a protein fragment, to its in vivo half-life. Substrates of the N-end rule pathway are recognized by the presence of degradation signals, termed N-degrons. Recognition components of the N-end rule pathway are E3 ubiquitin ligases that are capable of binding to N-degrons. The N-end rule pathway consists of two distinct branches: the Arg/N-end rule pathway and the Ac/N-end rule pathway.
In the present studies, we demonstrate a complementary targeting of the rat serotonin N-acetyltransferase (AANAT), an important mediator of circadian physiology, by both branches of the N-end rule pathway. The co-targeting results from incomplete N-terminal (Nt-) acetylation of a Met-Ф motif at the N-terminus of AANAT in vivo. In the same study, we demonstrate that human AANAT is substantially longer-lived than its rat counterpart, owing to differences in their N-terminal sequences. This molecular genetic investigation of the degradation of a physiological N-end rule substrate followed an analogous earlier study, in which we reported that a clinically-relevant (blood pressure-increasing) Q2L mutant of human RGS2 (termed ML-RGS2), a regulator of G proteins, could likewise be co-targeted by both branches of the N-end rule pathway. Together, AANAT and RGS2 are the first identified and characterized physiological substrates of the Ac/N-end rule pathway in mammals.
We also report on the development and use of in vitro N-terminal arginylation (Nt-arginylation) assays using CelluSpots peptide arrays, in conjunction with pulse-chase assays in rabbit reticulocyte extract, for the systematic investigation of the effects of N-terminus-proximal sequence context on the Nt-arginylation activity of the Ate1 arginyltransferase, a component of the Arg/N-end rule pathway. These experiments help to define the sequence requirements for efficient Nt-arginylation by Ate1. Finally, we demonstrate that Rec8, a subunit of the cohesin protein complex during meiosis, is a natural short-lived substrate of the mammalian Arg/N-end rule pathway.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Varshavsky, Alexander J.}, } @phdthesis{10.7907/09SW-FR88, author = {Shemorry, Anna}, title = {Studies of the N-End Rule Pathway in Saccharomyces cerevisiae}, school = {California Institute of Technology}, year = {2013}, doi = {10.7907/09SW-FR88}, url = {https://resolver.caltech.edu/CaltechTHESIS:01282013-132123935}, abstract = {
Many intracellular proteins are either conditionally or constitutively short-lived, with in vivo half-lives that can be as brief as a few minutes. The regulated and processive degradation of intracellular proteins is carried out largely by the ubiquitin (Ub)-proteasome system (UPS). In eukaryotes, the N-end rule pathway is a part of the UPS. The N-end rule relates the regulation of the in vivo half-life of a protein to the identity of its N-terminal residue. Degradation signals (degrons) that are targeted by the N-end rule pathway include a set called N-degrons. E3 Ub ligases of the N-end rule pathway are called N-recognins. They bind to primary destabilizing N-terminal residues of N-end rule substrates. The N-end rule pathway comprises two major branches, the Arg/N-end rule pathway and the Ac/N-end rule pathway.
The Arg/N-end rule branch involves the N-terminal arginylation of protein substrates and also the targeting of specific unmodified N-terminal residues by E3 N-recognins. The S. cerevisiae Arg/N-end rule pathway contains a single N-recognin, Ubr1. The Ub-fusion degradation (UFD) pathway is also a part of the UPS. This pathway recognizes a “nonremovable” N-terminal Ub moiety of a Ub fusion as a primary degron. My collaborator, Cheol-Sang Hwang, and I demonstrated that the RING-type Ubr1 E3 and the HECT-type Ufd4 E3 interact, both physically and functionally. We showed that the Ubr1-Ufd4 complex targets the S. cerevisiae Mgt1 DNA repair enzyme through a degron near its N-terminus, in addition to mediating the Arg/N-end rule pathway and a part of the UFD pathway as well. We also further characterized the physical interaction between Ubr1 and Ufd4.
I also report the discovery of the other branch of the N-end rule pathway, the Ac/N-end rule pathway, which recognizes N-terminally acetylated residues as N-degrons, termed Ac/N-degrons. We showed that Ac/N-degrons are recognized by the Doa10 E3 Ub ligase and apparently by other E3s as well. Given the prevalence of Ac/N-degrons, as nearly 90% of human proteins are Nt-acetylated, we also demonstrated the physiological role of Ac/N-degrons in protein quality, including the regulation of input stoichiometries of subunits in oligomeric proteins.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Varshavsky, Alexander J.}, } @phdthesis{10.7907/S3VT-0641, author = {Du, Fangyong}, title = {Allosteric Activation of the Ubiquitin ligase UBR 1 by Short Peptides: Molecular Mechanisms and Physiological Functions}, school = {California Institute of Technology}, year = {2002}, doi = {10.7907/S3VT-0641}, url = {https://resolver.caltech.edu/CaltechTHESIS:02232012-110128910}, abstract = {
The N-end rule relates the in vivo half life of a protein to the identity of its N-terminal residue. UBR1, the E3 of the N-end rule pathway in Sacchnromzyces cerevisiae, targets proteins that bear destabilizing N-terminal residues for Ub-dependent, processive degradation. UBR1 binds protein substrates or dipetides through two distinct sites: the type 1 site, specific for basic residues, and the type 2 site, specific for bulky hydrophobic residues. UBR1 also recognizes an internal degradation signal of the 35 kDa homeodomain protein CUP9, a transcriptional repressor of the di- and tripeptide transporter PTR2.
Here I report that the internal degradation signal of CUP9 is recognized by UBR1 through its third, distinct substrate-binding site. Occupation of the type 1 or type 2 sites of UBR1 by dipeptides allosterically stimulates the UBR1-dependent multi-ubiquitylation of CUP9 in an in vitro system, which consists of purified components of the yeast N-end rule pathway. UBR1 is the first E3 shown to be allosterically regulated by small compounds. This regulation underlies, in vivo, the accelerated UBR1-dependent degradation of CUP9 in the presence of dipeptides with destabilizing N-terminal residues. The result is a positive feedback circuit that controls the peptide import in S. cerevisiae. Specifically, the imported dipeptides bind to UBR1 and accelerate the UBR1-dependent degradation of CUP9, thereby derepressing the transcription of PTR2 and increasing the cell’s capacity to import peptides.
I also describe a new, autoinhibition-based molecular mechanism underlying the activation of UBR1 by dipeptides. UBR1 is an autoinhibited protein, in that the binding of dipeptides to the type 1 and type 2 sites of UBR1 enhances the dissociation of the C-terminal autoinhibitory domain of UBR1 from its substrate-binding N-terminal region. Moreover, this dissociation, which allows the interaction between UBRl and CUP9, is strongly increased only if both type 1 and type 2 sites of UBRl are occupied by dipeptides. An autoinhibitory mechanism discovered in the S. cerevisiae UBRl is likely to recur in metazoan homologs of UBRl, and may also be involved in controlling the activity of other Db-dependent pathways.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Varshavsky, Alexander J.}, } @phdthesis{10.7907/X5T1-TS17, author = {Turner, Glenn Cameron}, title = {Functions of the ubiquitin-proteasome system in Saccharomyces cerevisiae : cotranslational protein degradation and regulation of the UBR1 pathway}, school = {California Institute of Technology}, year = {2000}, doi = {10.7907/X5T1-TS17}, url = {https://resolver.caltech.edu/CaltechETD:etd-09222005-131809}, abstract = {
The ubiquitin-proteasome system is the major pathway for protein degradation in the cytoplasm of eukaryotic cells. This pathway serves two main functions: protein quality control - removing damaged or misfolded proteins, and concentration control - regulating levels of the protein components of biochemical switches and oscillators.
Misfolded proteins expose hydrophobic patches that act as degradation signals recognized by the ubiquitin-proteasome system. Nascent proteins being synthesized by the ribosome expose similar patches that might also serve as degradation signals. I show here that nascent polypeptides carrying a strong degradation signal of the Ub-proteasome system experience a kinetic competition between degradation and biogenesis. These results suggest that there may be a proofreading pathway for protein folding that recognizes and degrades proteins that fail to fold correctly.
Levels of regulatory proteins must be adjusted in response to many different signals, both environmental and cell-intrinsic. I show here that the activity of a specific ubiquitin-protein ligase (E3), Ubr1, is allosterically regulated. UbrI regulates dipeptide uptake in saccharomyces cerevisiae by controlling the degradation of Cup9, a homeodomain-containing repressor of the dipeptide transporter Ptr2. UbrI is allosterically activated by dipeptides bearing destabilizing residues according to the N-end rule. The import of these dipeptides stimulates Ubri, increasing Cup9 degradation, thereby de-repressing ptr2 expression. Thus, the expression of the machinery required for dipeptide uptake is coupled to the availability of dipeptides.
I also outline a novel pathway governing Ubrl activity. Free amino acids induce Ptr2 expression via a signal transduction cascade containing Ssy1, a putative transmembrane amino acid receptor, and Ptr3, a novel downstream signaling component. One of the targets of this signal transduction pathway is Ubr1. Ubrl is activated in the presence of amino acids, accelerating Cup9 degradation, thus inducing Ptr2.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Varshavsky, Alexander J.}, }