Tomoki Chiba

Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice

Autophagy is a membrane-trafficking mechanism that delivers cytoplasmic constituents into the lysosome/vacuole for bulk protein degradation. This mechanism is involved in the preservation of nutrients under starvation condition as well as the normal turnover of cytoplasmic component. Aberrant autophagy has been reported in several neurodegenerative disorders, hepatitis, and myopathies. Here, we generated conditional knockout mice of Atg7, an essential gene for autophagy in yeast. Atg7 was essential for ATG conjugation systems and autophagosome formation, amino acid supply in neonates, and starvation-induced bulk degradation of proteins and organelles in mice. Furthermore, Atg7 deficiency led to multiple cellular abnormalities, such as appearance of concentric membranous structure and deformed mitochondria, and accumulation of ubiquitin-positive aggregates. Our results indicate the important role of autophagy in starvation response and the quality control of proteins and organelles in quiescent cells.

Oxidative Stress Sensor Keap1 Functions as an Adaptor for Cul3-Based E3 Ligase To Regulate Proteasomal Degradation of Nrf2

ABSTRACT Transcription factor Nrf2 is a major regulator of genes encoding phase 2 detoxifying enzymes and antioxidant stress proteins in response to electrophilic agents and oxidative stress. In the absence of such stimuli, Nrf2 is inactive owing to its cytoplasmic retention by Keap1 and rapid degradation through the proteasome system. We examined the contribution of Keap1 to the rapid turnover of Nrf2 (half-life of less than 20 min) and found that a direct association between Keap1 and Nrf2 is required for Nrf2 degradation. In a series of domain function analyses of Keap1, we found that both the BTB and intervening-region (IVR) domains are crucial for Nrf2 degradation, implying that these two domains act to recruit ubiquitin-proteasome factors. Indeed, Cullin 3 (Cul3), a subunit of the E3 ligase complex, was found to interact specifically with Keap1 in vivo. Keap1 associates with the N-terminal region of Cul3 through the IVR domain and promotes the ubiquitination of Nrf2 in cooperation with the Cul3-Roc1 complex. These results thus provide solid evidence that Keap1 functions as an adaptor of Cul3-based E3 ligase. To our knowledge, Nrf2 and Keap1 are the first reported mammalian substrate and adaptor, respectively, of the Cul3-based E3 ligase system.

CHIP is a chaperone-dependent E3 ligase that ubiquitylates unfolded protein

The ubiquitin–proteasome system catalyses the immediate destruction of misfolded or impaired proteins generated in cells, but how this proteolytic machinery recognizes abnormality of cellular proteins for selective elimination remains elusive. Here, we report that the C‐terminus of Hsc70‐interacting protein (CHIP) with a U‐box domain is an E3 ubiquitin‐ligase collaborating with molecular chaperones Hsp90 and Hsc70. Thermally denatured firefly luciferase was multiubiquitylated by CHIP in the presence of E1 and E2 (Ubc4 or UbcH5c) in vitro, only when the unfolded substrate was captured by Hsp90 or Hsc70 and Hsp40. No ubiquitylating activity was detected in CHIP lacking the U‐box region. CHIP efficiently ubiquitylated denatured luciferase trapped by the C‐terminal region of Hsp90, which contains a CHIP binding site. CHIP also showed self‐ubiquitylating activity independent of target ubiquitylation. Our results indicate that CHIP can be regarded as ‘a quality‐control E3’ that selectively ubiquitylates unfolded protein(s) by collaborating with molecular chaperones.

NEDD8 recruits E2-ubiquitin to SCF E3 ligase.

NEDD8/Rub1 is a ubiquitin (Ub)‐like post‐translational modifier that is covalently linked to cullin (Cul)‐family proteins in a manner analogous to ubiquitylation. NEDD8 is known to enhance the ubiquitylating activity of the SCF complex (composed of Skp1, Cul‐1, ROC1 and F‐box protein), but the mechanistic role is largely unknown. Using an in vitro reconstituted system, we report here that NEDD8 modification of Cul‐1 enhances recruitment of Ub‐conjugating enzyme Ubc4 (E2) to the SCF complex (E3). This recruitment requires thioester linkage of Ub to Ubc4. Our findings indicate that the NEDD8‐modifying system accelerates the formation of the E2–E3 complex, which stimulates protein polyubiquitylation.

E3 ubiquitin ligase that recognizes sugar chains

N-glycosylation of proteins in the endoplasmic reticulum (ER) has a central role in protein quality control. Here we report that N-glycan serves as a signal for degradation by the Skp1–Cullin1–Fbx2–Roc1 (SCFFbx2) ubiquitin ligase complex. The F-box protein Fbx2 (ref. 4) binds specifically to proteins attached to N-linked high-mannose oligosaccharides and subsequently contributes to ubiquitination of N-glycosylated proteins. Pre-integrin β1 is a target of Fbx2; these two proteins interact in the cytosol after inhibition of the proteasome. In addition, expression of the mutant Fbx2ΔF, which lacks the F-box domain that is essential for forming the SCF complex, appreciably blocks degradation of typical substrates of the ER-associated degradation pathway. Our results indicate that SCFFbx2 ubiquitinates N-glycosylated proteins that are translocated from the ER to the cytosol by the quality control mechanism.

Covalent modification of all members of human cullin family proteins by NEDD8.

Recently we found that NEDD8, a ubiquitin-like protein, was linked covalently to human cullin-4A (abbreviated Cul-4A) by a new ubiquitin-related pathway that is analogous to but distinct from the ligating system for SUMO1, another ubiquitin-like protein. However, it remained unknown whether the other five members of the family of human cullin/Cdc53 proteins are modified by NEDD8. Here we report that all Hs-Cul family proteins, such as Cul-1, Cul-2, Cul-3, Cul-4B, and Cul-5, in addition to Cul-4A, were modified by covalent attachment of NEDD8 in rabbit reticulocyte lysates. Moreover, by comprehensive Northern-blot analyses, we examined multiple tissue distributions of the messages for all Cul-family proteins, NEDD8, and the NEDD8-ligating system consisting of APP-BP1/hUba3, and hUbc12, which function as E1- and E2-like enzymes, respectively. The expressions of Cul-1, Cul-2, and Cul-3 resembled each other and were apparently correlated to those of NEDD8 and the NEDD8-ligating system in various human cells and tissues. However, the mRNA levels of Cul-4A, Cul-4B, and Cul-5 differed considerably from each other as well as from other Cul-family proteins. The enhanced expression of all Cul-family proteins except Cul-5 was observed in a variety of tumor cell lines.

Covalent modifier NEDD8 is essential for SCF ubiquitin-ligase in fission yeast

A ubiquitin‐like modifier, NEDD8, is covalently attached to cullin‐family proteins, but its physiological role is poorly understood. Here we report that the NEDD8‐modifying pathway is essential for cell viability and function of Pcu1 (cullin‐1 orthologue) in fission yeast. Pcu1 assembled on SCF ubiquitin‐ligase was completely modified by NEDD8. Pcu1K713R defective for NEDD8 conjugation lost the ability to complement lethality due to pcu1 deletion. Forced expression of Pcu1K713R or depletion of NEDD8 in cells resulted in impaired cell proliferation and marked stabilization of the cyclin‐dependent kinase inhibitor Rum1, which is a substrate of the SCF complex. Based on these findings, we propose that covalent modification of cullin‐1 by the NEDD8 system plays an essential role in the function of SCF in fission yeast.

The NEDD8 system is essential for cell cycle progression and morphogenetic pathway in mice

NEDD8/Rub1 is a ubiquitin (Ub)-like molecule that covalently ligates to target proteins through an enzymatic cascade analogous to ubiquitylation. This modifier is known to target all cullin (Cul) family proteins. The latter are essential components of Skp1/Cul-1/F-box protein (SCF)–like Ub ligase complexes, which play critical roles in Ub-mediated proteolysis. To determine the role of the NEDD8 system in mammals, we generated mice deficient in Uba3 gene that encodes a catalytic subunit of NEDD8-activating enzyme. Uba3 − /− mice died in utero at the periimplantation stage. Mutant embryos showed selective apoptosis of the inner cell mass but not of trophoblastic cells. However, the mutant trophoblastic cells could not enter the S phase of the endoreduplication cycle. This cell cycle arrest was accompanied with aberrant expression of cyclin E and p57Kip2. These results suggested that the NEDD8 system is essential for both mitotic and the endoreduplicative cell cycle progression. β-Catenin, a mediator of the Wnt/wingless signaling pathway, which degrades continuously in the cytoplasm through SCF Ub ligase, was also accumulated in the Uba3 −/− cytoplasm and nucleus. Thus, the NEDD8 system is essential for the regulation of protein degradation pathways involved in cell cycle progression and morphogenesis, possibly through the function of the Cul family proteins.

A novel protein-conjugating system for Ufm1, a ubiquitin-fold modifier

Several studies have addressed the importance of various ubiquitin‐like (UBL) post‐translational modifiers. These UBLs are covalently linked to most, if not all, target protein(s) through an enzymatic cascade analogous to ubiquitylation, consisting of E1 (activating), E2 (conjugating), and E3 (ligating) enzymes. In this report, we describe the identification of a novel ubiquitin‐fold modifier 1 (Ufm1) with a molecular mass of 9.1 kDa, displaying apparently similar tertiary structure, although lacking obvious sequence identity, to ubiquitin. Ufm1 is first cleaved at the C‐terminus to expose its conserved Gly residue. This Gly residue is essential for its subsequent conjugating reactions. The C‐terminally processed Ufm1 is activated by a novel E1‐like enzyme, Uba5, by forming a high‐energy thioester bond. Activated Ufm1 is then transferred to its cognate E2‐like enzyme, Ufc1, in a similar thioester linkage. Ufm1 forms several complexes in HEK293 cells and mouse tissues, revealing that it conjugates to the target proteins. Ufm1, Uba5, and Ufc1 are all conserved in metazoa and plants but not in yeast, suggesting its potential roles in various multicellular organisms.

Fbs2 is a new member of the E3 ubiquitin ligase family that recognizes sugar chains

F-box proteins are substrate recognition components of Skp1-Cullin1-F-box protein-Roc1 (SCF) E3 ubiquitin-protein ligases. We reported previously that Fbs1 (F-box protein that recognizes sugar chains; equivalent to Fbx2 or NFB42) binds specifically to proteins attached with high mannose oligosaccharides and subsequently contributes to elimination of N-glycoproteins in cytosol (Yoshida, Y., Chiba, T., Tokunaga, F., Kawasaki, H., Iwai, K., Suzuki, T., Ito, Y., Matsuoka, K., Yoshida, M., Tanaka, K., and Tai, T. (2002) Nature 418, 438–442). Here we report the identification of another F-box protein that recognizes N-glycan, Fbs2 (called Fbx6b or FBG2 previously). Although the expression of Fbs1 was restricted to the adult brain and testis, the Fbs2 transcript was widely expressed. The Fbs2 protein forms an SCFFbs2 ubiquitinligase complex that targets sugar chains in N-glycoproteins for ubiquitylation. Only glycoproteins bound to concanavalin A lectin and not to wheat germ agglutinin or Ricinus communis agglutinin interacted with Fbs2 in various tissues and cell lines. Pull-down analysis using various oligosaccharides revealed that Man3–9GlcNAc2 glycans were required for efficient Fbs2 binding, whereas modifications of mannose residues by other sugars or deletion of inner GlcNAc reduced Fbs2 binding. Fbs2 interacted with N-glycans of T-cell receptor α-subunit (TCRα), a typical substrate of the endoplasmic reticulum-associated degradation (ERAD) pathway, and the forced expression of mutant Fbs2ΔF, which lacks the F-box domain essential for forming the SCF complex, and decrease of endogenous Fbs2 by small interfering RNA led to inhibition of TCRα degradation in cells. Thus, Fbs2 is a novel member of F-box protein family that recognizes N-glycans and plays a role in ERAD.