KPC1-mediated ubiquitination and proteasomal processing of NF-κB1 p105 to p50 restricts tumor growth.

NF-κB is a key transcriptional regulator involved in inflammation and cell proliferation, survival, and transformation. Several key steps in its activation are mediated by the ubiquitin (Ub) system. One uncharacterized step is limited proteasomal processing of the NF-κB1 precursor p105 to the p50 active subunit. Here, we identify KPC1 as the Ub ligase (E3) that binds to the ankyrin repeats domain of p105, ubiquitinates it, and mediates its processing both under basal conditions and following signaling. Overexpression of KPC1 inhibits tumor growth likely mediated via excessive generation of p50. Also, overabundance of p50 downregulates p65, suggesting that a p50-p50 homodimer may modulate transcription in place of the tumorigenic p50-p65. Transcript analysis reveals increased expression of genes associated with tumor-suppressive signals. Overall, KPC1 regulation of NF-κB1 processing appears to constitute an important balancing step among the stimulatory and inhibitory activities of the transcription factor in cell growth control.

The N-end rule pathway.

The N-end rule pathway is a proteolytic system in which N-terminal residues of short-lived proteins are recognized by recognition components (N-recognins) as essential components of degrons, called N-degrons. Known N-recognins in eukaryotes mediate protein ubiquitylation and selective proteolysis by the 26S proteasome. Substrates of N-recognins can be generated when normally embedded destabilizing residues are exposed at the N terminus by proteolytic cleavage. N-degrons can also be generated through modifications of posttranslationally exposed pro-N-degrons of otherwise stable proteins; such modifications include oxidation, arginylation, leucylation, phenylalanylation, and acetylation. Although there are variations in components, degrons, and hierarchical structures, the proteolytic systems based on generation and recognition of N-degrons have been observed in all eukaryotes and prokaryotes examined thus far. The N-end rule pathway regulates homeostasis of various physiological processes, in part, through interaction with small molecules. Here, we review the biochemical mechanisms, structures, physiological functions, and small-molecule-mediated regulation of the N-end rule pathway.

The N-Degron Pathway Mediates ER-phagy.

The endoplasmic reticulum (ER) is susceptible to wear-and-tear and proteotoxic stress, necessitating its turnover. Here, we show that the N-degron pathway mediates ER-phagy. This autophagic degradation initiates when the transmembrane E3 ligase TRIM13 (also known as RFP2) is ubiquitinated via the lysine 63 (K63) linkage. K63-ubiquitinated TRIM13 recruits p62 (also known as sequestosome-1), whose complex undergoes oligomerization. The oligomerization is induced when the ZZ domain of p62 is bound by the N-terminal arginine (Nt-Arg) of arginylated substrates. Upon activation by the Nt-Arg, oligomerized TRIM13-p62 complexes are separated along with the ER compartments and targeted to autophagosomes, leading to lysosomal degradation. When protein aggregates accumulate within the ER lumen, degradation-resistant autophagic cargoes are co-segregated by ER membranes for lysosomal degradation. We developed synthetic ligands to the p62 ZZ domain that enhance ER-phagy for ER protein quality control and alleviate ER stresses. Our results elucidate the biochemical mechanisms and pharmaceutical means that regulate ER homeostasis.

UBR2 targets myosin heavy chain IIb and IIx for degradation: Molecular mechanism essential for cancer-induced muscle wasting.

Cancer cachexia is a lethal metabolic syndrome featuring muscle wasting with preferential loss of fast-twitching muscle mass through an undefined mechanism. Here, we show that cancer induces muscle wasting by selectively degrading myosin heavy chain (MHC) subtypes IIb and IIx through E3 ligase UBR2-mediated ubiquitylation. Induction of MHC loss and atrophy in C2C12 myotubes and mouse tibialis anterior (TA) by murine cancer cells required UBR2 up-regulation by cancer. Genetic gain or loss of UBR2 function inversely altered MHC level and muscle mass in TA of tumor-free mice. UBR2 selectively interacted with and ubiquitylated MHC-IIb and MHC-IIx through its substrate recognition and catalytic domain, respectively, in C2C12 myotubes. Elevation of UBR2 in muscle of tumor-bearing or free mice caused loss of MHC-IIb and MHC-IIx but not MHC-I and MHC-IIa or other myofibrillar proteins, including α-actin, troponin, tropomyosin, and tropomodulin. Muscle-specific knockout of UBR2 spared KPC tumor-bearing mice from losing MHC-IIb and MHC-IIx, fast-twitching muscle mass, cross-sectional area, and contractile force. The rectus abdominis (RA) muscle of patients with cachexia-prone cancers displayed a selective reduction of MHC-IIx in correlation with higher UBR2 levels. These data suggest that UBR2 is a regulator of MHC-IIb/IIx essential for cancer-induced muscle wasting, and that therapeutic interventions can be designed by blocking UBR2 up-regulation by cancer.

UBR7 functions with UBR5 in the Notch signaling pathway and is involved in a neurodevelopmental syndrome with epilepsy, ptosis, and hypothyroidism.

The ubiquitin-proteasome system facilitates the degradation of unstable or damaged proteins. UBR1-7, which are members of hundreds of E3 ubiquitin ligases, recognize and regulate the half-life of specific proteins on the basis of their N-terminal sequences ("N-end rule"). In seven individuals with intellectual disability, epilepsy, ptosis, hypothyroidism, and genital anomalies, we uncovered bi-allelic variants in UBR7. Their phenotype differs significantly from that of Johanson-Blizzard syndrome (JBS), which is caused by bi-allelic variants in UBR1, notably by the presence of epilepsy and the absence of exocrine pancreatic insufficiency and hypoplasia of nasal alae. While the mechanistic etiology of JBS remains uncertain, mutation of both Ubr1 and Ubr2 in the mouse or of the C. elegans UBR5 ortholog results in Notch signaling defects. Consistent with a potential role in Notch signaling, C. elegans ubr-7 expression partially overlaps with that of ubr-5, including in neurons, as well as the distal tip cell that plays a crucial role in signaling to germline stem cells via the Notch signaling pathway. Analysis of ubr-5 and ubr-7 single mutants and double mutants revealed genetic interactions with the Notch receptor gene glp-1 that influenced development and embryo formation. Collectively, our findings further implicate the UBR protein family and the Notch signaling pathway in a neurodevelopmental syndrome with epilepsy, ptosis, and hypothyroidism that differs from JBS. Further studies exploring a potential role in histone regulation are warranted given clinical overlap with KAT6B disorders and the interaction of UBR7 and UBR5 with histones.

The N-terminal cysteine is a dual sensor of oxygen and oxidative stress.

Cellular homeostasis requires the sensing of and adaptation to intracellular oxygen (O2) and reactive oxygen species (ROS). The Arg/N-degron pathway targets proteins that bear destabilizing N-terminal residues for degradation by the proteasome or via autophagy. Under normoxic conditions, the N-terminal Cys (Nt-Cys) residues of specific substrates can be oxidized by dioxygenases such as plant cysteine oxidases and cysteamine (2-aminoethanethiol) dioxygenases and arginylated by ATE1 R-transferases to generate Arg-CysO2(H) (R-CO2). Proteins bearing the R-CO2 N-degron are targeted via Lys48 (K48)-linked ubiquitylation by UBR1/UBR2 N-recognins for proteasomal degradation. During acute hypoxia, such proteins are partially stabilized, owing to decreased Nt-Cys oxidation. Here, we show that if hypoxia is prolonged, the Nt-Cys of regulatory proteins can be chemically oxidized by ROS to generate Arg-CysO3(H) (R-CO3), a lysosomal N-degron. The resulting R-CO3 is bound by KCMF1, a N-recognin that induces K63-linked ubiquitylation, followed by K27-linked ubiquitylation by the noncanonical N-recognin UBR4. Autophagic targeting of Cys/N-degron substrates is mediated by the autophagic N-recognin p62/SQTSM-1/Sequestosome-1 through recognition of K27/K63-linked ubiquitin (Ub) chains. This Cys/N-degron-dependent reprogramming in the proteolytic flux is important for cellular homeostasis under both chronic hypoxia and oxidative stress. A small-compound ligand of p62 is cytoprotective under oxidative stress through its ability to accelerate proteolytic flux of K27/K63-ubiquitylated Cys/N-degron substrates. Our results suggest that the Nt-Cys of conditional Cys/N-degron substrates acts as an acceptor of O2 to maintain both O2 and ROS homeostasis and modulates half-lives of substrates through either the proteasome or lysosome by reprogramming of their Ub codes.

Phosphorylation of ß-catenin Ser60 by polo-like kinase 1 drives the completion of cytokinesis.

ß-Catenin is a multifunctional protein and participates in numerous processes required for embryonic development, cell proliferation, and homeostasis through various molecular interactions and signaling pathways. To date, however, there is no direct evidence that ß-catenin contributes to cytokinesis. Here, we identify a novel p-S60 epitope on ß-catenin generated by Plk1 kinase activity, which can be found at the actomyosin contractile ring of early telophase cells and at the midbody of late telophase cells. Depletion of ß-catenin leads to cytokinesis-defective phenotypes, which eventually result in apoptotic cell death. In addition, phosphorylation of ß-catenin Ser60 by Plk1 is essential for the recruitment of Ect2 to the midbody, activation of RhoA, and interaction between ß-catenin, Plk1, and Ect2. Time-lapse image analysis confirmed the importance of ß-catenin phospho-Ser60 in furrow ingression and the completion of cytokinesis. Taken together, we propose that phosphorylation of ß-catenin Ser60 by Plk1 in cooperation with Ect2 is essential for the completion of cytokinesis. These findings may provide fundamental knowledge for the research of cytokinesis failure-derived human diseases.

The N-end rule pathway: emerging functions and molecular principles of substrate recognition.

The N-end rule defines the protein-destabilizing activity of a given amino-terminal residue and its post-translational modification. Since its discovery 25 years ago, the pathway involved in the N-end rule has been thought to target only a limited set of specific substrates of the ubiquitin-proteasome system. Recent studies have provided insights into the components, substrates, functions and structural basis of substrate recognition. The N-end rule pathway is now emerging as a major cellular proteolytic system, in which the majority of proteins are born with or acquire specific N-terminal degradation determinants through protein-specific or global post-translational modifications.

Excess of the NF-ĸB p50 subunit generated by the ubiquitin ligase KPC1 suppresses tumors via PD-L1- and chemokines-mediated mechanisms.

Nuclear factor-ĸB (NF-ĸB) transcription factor is a family of essential regulators of the immune response and cell proliferation and transformation. A typical factor is a heterodimer made of either p50 or p52, which are limited processing products of either p105 or p100, respectively, and a member of the Rel family of proteins, typically p65. The transcriptional program of NF-ĸB is tightly regulated by the composition of the dimers. In our previous work, we demonstrated that the ubiquitin ligase KPC1 is involved in ubiquitination and proteasomal processing of p105 to generate p50. Its overexpression and the resulting high level of p50 stimulates transcription of a broad array of tumor suppressors. Here we demonstrate that additional mechanisms are involved in the p50-mediated tumor-suppressive effect. p50 down-regulates expression of a major immune checkpoint inhibitor, the programmed cell death-ligand 1 (PD-L1), both in cells and in tumors. Importantly, the suppression is abrogated by overexpression of p65. This highlights the importance of the cellular quantities of the two different subunits of NF-ĸB which determine the composition of the dimer. While the putative p50 homodimer is tumor-suppressive, the "canonical" p50p65 heterodimer is oncogenic. We found that an additional mechanism is involved in the tumor-suppressive phenomenon: p50 up-regulates expression of the proinflammatory chemokines CCL3, CCL4, and CCL5, which in turn recruit into the tumors active natural killer (NK) cells and macrophages. Overall, p50 acts as a strong tumor suppressor via multiple mechanisms, including overexpression of tumor suppressors and modulation of the tumor microenvironment by recruiting active immune cells.

Site-specific ubiquitination of pathogenic huntingtin attenuates its deleterious effects.

Huntington's disease (HD) is a progressive incurable neurodegenerative disorder characterized by motor and neuropsychiatric symptoms. It is caused by expansion of a cytosine-adenine-guanine triplet in the N-terminal domain of exon 1 in the huntingtin (HTT) gene that codes for an expanded polyglutamine stretch in the protein product which becomes aggregation prone. The mutant Htt (mHtt) aggregates are associated with components of the ubiquitin-proteasome system, suggesting that mHtt is marked for proteasomal degradation and that, for reasons still debated, are not properly degraded. We used a novel HD rat model, proteomic analysis, and long-term live neuronal imaging to characterize the effects of ubiquitination on aggregation of mHtt and subsequent cellular responses. We identified two lysine residues, 6 and 9, in the first exon of mHtt that are specifically ubiquitinated in striatal and cortical brain tissues of mHtt-transgenic animals. Expression of mHtt exon 1 lacking these ubiquitination sites in cortical neurons and cultured cells was found to slow aggregate appearance rates and reduce their size but at the same time increase the number of much smaller and less visible ones. Importantly, expression of this form of mHtt was associated with elevated death rates. Proteomic analysis indicated that cellular reactions to mHtt expression were weaker in cells expressing the lysineless protein, possibly implying a reduced capacity to cope with the proteotoxic stress. Taken together, the findings suggest a novel role for ubiquitination-attenuation of the pathogenic effect of mHtt.