N-Terminal Acetylation of Cellular Proteins Creates Specific Degradation Signals

The retained N-terminal methionine (Met) residue of a nascent protein is often N-terminally acetylated (Nt-acetylated). Removal of N-terminal Met by Met-aminopeptidases frequently leads to Nt-acetylation of the resulting N-terminal alanine (Ala), valine (Val), serine (Ser), threonine (Thr), and cysteine (Cys) residues. Although a majority of eukaryotic proteins (for example, more than 80% of human proteins) are cotranslationally Nt-acetylated, the function of this extensively studied modification is largely unknown. Using the yeast Saccharomyces cerevisiae, we found that the Nt-acetylated Met residue could act as a degradation signal (degron), targeted by the Doa10 ubiquitin ligase. Moreover, Doa10 also recognized the Nt-acetylated Ala, Val, Ser, Thr, and Cys residues. Several examined proteins of diverse functions contained these N-terminal degrons, termed ^(Ac)N-degrons, which are a prevalent class of degradation signals in cellular proteins

Control of Protein Quality and Stoichiometries by N-Terminal Acetylation and the N-End Rule Pathway

N^α-terminal acetylation of cellular proteins was recently discovered to create specific degradation signals termed Ac/N-degrons and targeted by the Ac/N-end rule pathway. We show that Hcn1, a subunit of the APC/C ubiquitin ligase, contains an Ac/N-degron that is repressed by Cut9, another APC/C subunit and the ligand of Hcn1. Cog1, a subunit of the Golgi-associated COG complex, is also shown to contain an Ac/N-degron. Cog2 and Cog3, direct ligands of Cog1, can repress this degron. The subunit decoy technique was used to show that the long-lived endogenous Cog1 is destabilized and destroyed via its activated (unshielded) Ac/N-degron if the total level of Cog1 increased in a cell. Hcn1 and Cog1 are the first examples of protein regulation through the physiologically relevant transitions that shield and unshield natural Ac/N-degrons. This mechanistically straightforward circuit can employ the demonstrated conditionality of Ac/N-degrons to regulate subunit stoichiometries and other aspects of protein quality control

The N-end rule pathway is mediated by a complex of the RING-type Ubr1 and HECT-type Ufd4 ubiquitin ligases

Substrates of the N-end rule pathway are recognized by the Ubr1 E3 ubiquitin ligase through their destabilizing amino-terminal residues. Our previous work showed that the Ubr1 E3 and the Ufd4 E3 together target an internal degradation signal (degron) of the Mgt1 DNA repair protein. Ufd4 is an E3 enzyme of the ubiquitin-fusion degradation (UFD) pathway that recognizes an N-terminal ubiquitin moiety. Here we show that the RING-type Ubr1 E3 and the HECT-type Ufd4 E3 interact, both physically and functionally. Although Ubr1 can recognize and polyubiquitylate an N-end rule substrate in the absence of Ufd4, the Ubr1–Ufd4 complex is more processive in that it produces a longer substrate-linked polyubiquitin chain. Conversely, Ubr1 can function as a polyubiquitylation-enhancing component of the Ubr1–Ufd4 complex in its targeting of UFD substrates. We also found that Ubr1 can recognize the N-terminal ubiquitin moiety. These and related advances unify two proteolytic systems that have been studied separately for two decades

Studies of the N-End Rule Pathway in Saccharomyces cerevisiae

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.</p

IRE1α Disruption in Triple-Negative Breast Cancer Cooperates with Antiangiogenic Therapy by Reversing ER Stress Adaptation and Remodeling the Tumor Microenvironment.

Cancer cells exploit the unfolded protein response (UPR) to mitigate endoplasmic reticulum (ER) stress caused by cellular oncogene activation and a hostile tumor microenvironment (TME). The key UPR sensor IRE1α resides in the ER and deploys a cytoplasmic kinase-endoribonuclease module to activate the transcription factor XBP1s, which facilitates ER-mediated protein folding. Studies of triple-negative breast cancer (TNBC)-a highly aggressive malignancy with a dismal posttreatment prognosis-implicate XBP1s in promoting tumor vascularization and progression. However, it remains unknown whether IRE1α adapts the ER in TNBC cells and modulates their TME, and whether IRE1α inhibition can enhance antiangiogenic therapy-previously found to be ineffective in patients with TNBC. To gauge IRE1α function, we defined an XBP1s-dependent gene signature, which revealed significant IRE1α pathway activation in multiple solid cancers, including TNBC. IRE1α knockout in TNBC cells markedly reversed substantial ultrastructural expansion of their ER upon growth in vivo. IRE1α disruption also led to significant remodeling of the cellular TME, increasing pericyte numbers while decreasing cancer-associated fibroblasts and myeloid-derived suppressor cells. Pharmacologic IRE1α kinase inhibition strongly attenuated growth of cell line-based and patient-derived TNBC xenografts in mice and synergized with anti-VEGFA treatment to cause tumor stasis or regression. Thus, TNBC cells critically rely on IRE1α to adapt their ER to in vivo stress and to adjust the TME to facilitate malignant growth. TNBC reliance on IRE1α is an important vulnerability that can be uniquely exploited in combination with antiangiogenic therapy as a promising new biologic approach to combat this lethal disease. SIGNIFICANCE: Pharmacologic IRE1α kinase inhibition reverses ultrastructural distension of the ER, normalizes the tumor vasculature, and remodels the cellular TME, attenuating TNBC growth in mice

Yeast replicative lifespan and terminal morphology in culture media of varying pH

<p>The replicative lifespan of yeast (BY4742 strain background) cultured in media of varying pH. Column L (“lifespans”; LS) through AE represent the number of daughter cells produced by each mother cell and the endCode (column AF-AY) represents the terminal morphology of the corresponding numbered mother cell. End lost represents the number of cells that did not receive a terminal cell morphology designation. U = unbudded, S = small bud (<50% size of mother), L = large bud (>=50% size of mother), C = cluster of cells.</p

Disruption of IRE1α through its kinase domain attenuates multiple myeloma

International audienceMultiple myeloma (MM) arises from malignant immunoglobulin (Ig)-secreting plasma cells and remains an incurable, often lethal disease despite therapeutic advances. The unfolded-protein response sensor IRE1α supports protein secretion by deploying a kinase-endoribonuclease module to activate the transcription factor XBP1s. MM cells may co-opt the IRE1α-XBP1s pathway; however, the validity of IRE1α as a potential MM therapeutic target is controversial. Genetic disruption of IRE1α or XBP1s, or pharmacologic IRE1α kinase inhibition, attenuated subcutaneous or orthometastatic growth of MM tumors in mice and augmented efficacy of two established frontline antimyeloma agents, bortezomib and lenalidomide. Mechanistically, IRE1α perturbation inhibited expression of key components of the endoplasmic reticulum-associated degradation machinery, as well as secretion of Ig light chains and of cytokines and chemokines known to promote MM growth. Selective IRE1α kinase inhibition reduced viability of CD138 + plasma cells while sparing CD138 − cells derived from bone marrows of newly diagnosed or posttreatment-relapsed MM patients, in both US-and European Union-based cohorts. Effective IRE1α inhibition preserved glucose-induced insulin secretion by pancreatic microislets and viability of primary hepatocytes in vitro, as well as normal tissue homeostasis in mice. These results establish a strong rationale for developing kinase-directed inhibitors of IRE1α for MM therapy. multiple myeloma | endoplasmic reticulum stress | unfolded protein response | inositol-requiring enzyme 1 | kinase inhibitors M ultiple myeloma (MM) is the second most common human hematologic cancer. It carries a lifetime risk of 0.7% and occurs mainly in older individuals. MM is caused by bone marrow infiltration by malignant, monoclonal immunoglobulin (Ig)-secreting plasma cells (1). Despite significant therapeutic advances-including proteasome inhibitors (PIs), immunomodulatory agents (IMiDs), and anti-CD38 antibodies-MM remains mainly incurable, with acquired resistance to all available agents, and a 5-y survival rate of 49% (2). Considering the growth of aging populations in many countries, there is an urgent unmet need for development of novel MM therapies. The endoplasmic reticulum (ER) ensures precise folding of newly synthesized secretory proteins. Upon elevated cellular demand for protein secretion-for example, when mature B cells differentiate into Ig-secreting plasma cells-insufficient ER capacity causes accumulation of unfolded proteins (UPs) in the ER lumen. This activates a sensing-signaling network dubbed the UP response (UPR) to orchestrate ER adaptation and reestablish homeostasis (3-6). The mammalian UPR employs three pivotal ER-resident transmembrane sensors: inositol-requiring enzyme Significance Multiple myeloma (MM) is a lethal malignancy arising from plasma cells. MM cells experience endoplasmic reticulum (ER) stress due to immunoglobulin hyperproduction. The ER-resident sensor IRE1α mitigates ER stress by expanding protein-folding and secretion capacity, while supporting proteasomal degradation of ER misfolded proteins. IRE1α elaborates these functions by deploying a cytoplasmic kinase-RNase module to activate the transcription factor XBP1s. Although IRE1α has been implicated in MM, its validity as a potential therapeutic target-particularly as a kinase-has been unclear. Using genetic and pharmacologic disruption, we demonstrate that the IRE1α-XBP1s pathway is critical for MM tumor growth. We further show that the kinase domain of IRE1α is an effective and safe potential small-molecule target for MM therapy

A Comprehensive Analysis of Replicative Lifespan in 4,698 Single-Gene Deletion Strains Uncovers Conserved Mechanisms of Aging

Many genes that affect replicative lifespan (RLS) in the budding yeast Saccharomyces cerevisiae also affect aging in other organisms such as C.&nbsp;elegans and M.&nbsp;musculus. We performed a systematic analysis of yeast RLS in a set of 4,698 viable single-gene deletion strains. Multiple functional gene clusters were identified, and full genome-to-genome comparison demonstrated a significant conservation in longevity pathways between yeast and C.&nbsp;elegans. Among the mechanisms of aging identified, deletion of tRNA exporter LOS1 robustly extended lifespan. Dietary restriction (DR) and inhibition of mechanistic Target of Rapamycin (mTOR) exclude Los1 from the nucleus in a Rad53-dependent manner. Moreover, lifespan extension from deletion of LOS1 is nonadditive with DR or mTOR inhibition, and results in Gcn4 transcription factor activation. Thus, the DNA damage response and mTOR converge on Los1-mediated nuclear tRNA export to regulate Gcn4 activity and aging