Degradation of Hepatic Stearyl CoA Δ9-Desaturase

Δ9-Desaturase is a key enzyme in the synthesis of desaturated fatty acyl-CoAs. Desaturase is an integral membrane protein induced in the endoplasmic reticulum by dietary manipulations and then rapidly degraded. The proteolytic machinery that specifically degrades desaturase and other short-lived proteins in the endoplasmic reticulum has not been identified. As the first step in identifying cellular factors involved in the degradation of desaturase, liver subcellular fractions of rats that had undergone induction of this enzyme were examined. In livers from induced animals, desaturase was present in the microsomal, nuclear (P-1), and subcellular fractions (P-2). Incubation of desaturase containing fractions at physiological pH and temperature led to the complete disappearance of the enzyme. Washing microsomes with a buffer containing high salt decreased desaturase degradation activity. N-terminal sequence analysis of desaturase freshly isolated from the P-1 fraction without incubation indicated the absence of three residues from the N terminus, but the mobility of this desaturase preparation on SDS-PAGE was identical to the microsomal desaturase, which contains a masked N terminus under similar purification procedures. Addition of concentrated cytosol or the high-salt wash fraction did not enhance the desaturase degradation in the washed microsomes. Extensive degradation of desaturase in the high-salt washed microsomes could be restored by supplementation of the membranes with the lipid and protein components essential for the reconstituted desaturase catalytic activity. Lysosomotrophic agents leupeptin and pepstatin A were ineffective in inhibiting desaturase degradation. The calpain inhibitor, N-acetyl-leucyl-leucyl-methional, or the proteosome inhibitor, Streptomyces metabolite, lactacystin, did not inhibit the degradation of desaturase in the microsomal or the P-1 and P-2 fractions. These results show that the selective degradation of desaturase is likely to be independent of the lysosomal and the proteosome systems. The reconstitution of complete degradation of desaturase in the high-salt–washed microsomes by the components essential for its catalytic activity reflects that the degradation of this enzyme may depend on a specific orientation of desaturase and intramembranous interactions between desaturase and the responsible protease.

Degradation of a Short-lived Glycoprotein from the Lumen of the Endoplasmic Reticulum: The Role of N-linked Glycans and the Unfolded Protein Response

We are studying endoplasmic reticulum–associated degradation (ERAD) with the use of a truncated variant of the type I ER transmembrane glycoprotein ribophorin I (RI). The mutant protein, RI332, containing only the N-terminal 332 amino acids of the luminal domain of RI, has been shown to interact with calnexin and to be a substrate for the ubiquitin-proteasome pathway. When RI332 was expressed in HeLa cells, it was degraded with biphasic kinetics; an initial, slow phase of ∼45 min was followed by a second phase of threefold accelerated degradation. On the other hand, the kinetics of degradation of a form of RI332 in which the single used N-glycosylation consensus site had been removed (RI332-Thr) was monophasic and rapid, implying a role of the N-linked glycan in the first proteolytic phase. RI332 degradation was enhanced when the binding of glycoproteins to calnexin was prevented. Moreover, the truncated glycoprotein interacted with calnexin preferentially during the first proteolytic phase, which strongly suggests that binding of RI332 to the lectin-like protein may result in the slow, initial phase of degradation. Additionally, mannose trimming appears to be required for efficient proteolysis of RI332. After treatment of cells with the inhibitor of N-glycosylation, tunicamycin, destruction of the truncated RI variants was severely inhibited; likewise, in cells preincubated with the calcium ionophore A23187, both RI332 and RI332-Thr were stabilized, despite the presence or absence of the N-linked glycan. On the other hand, both drugs are known to trigger the unfolded protein response (UPR), resulting in the induction of BiP and other ER-resident proteins. Indeed, only in drug-treated cells could an interaction between BiP and RI332 and RI332-Thr be detected. Induction of BiP was also evident after overexpression of murine Ire1, an ER transmembrane kinase known to play a central role in the UPR pathway; at the same time, stabilization of RI332 was observed. Together, these results suggest that binding of the substrate proteins to UPR-induced chaperones affects their half lives.

Exceptionally potent inhibitors of fatty acid amide hydrolase: The enzyme responsible for degradation of endogenous oleamide and anandamide

The development of exceptionally potent inhibitors of fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of oleamide (an endogenous sleep-inducing lipid), and anandamide (an endogenous ligand for cannabinoid receptors) is detailed. The inhibitors may serve as useful tools to clarify the role of endogenous oleamide and anandamide and may prove to be useful therapeutic agents for the treatment of sleep disorders or pain. The combination of several features—an optimal C12–C8 chain length, π-unsaturation introduction at the corresponding arachidonoyl Δ8,9/Δ11,12 and oleoyl Δ9,10 location, and an α-keto N4 oxazolopyridine with incorporation of a second weakly basic nitrogen provided FAAH inhibitors with Kis that drop below 200 pM and are 102–103 times more potent than the corresponding trifluoromethyl ketones.

Oncogenic mutation in the Kit receptor tyrosine kinase alters substrate specificity and induces degradation of the protein tyrosine phosphatase SHP-1

Activating mutations in the Kit receptor tyrosine kinase have been identified in both rodent and human mast cell leukemia. One activating Kit mutation substitutes a valine for aspartic acid at codon 816 (D816V) and is frequently observed in human mastocytosis. Mutation at the equivalent position in the murine c-kit gene, involving a substitution of tyrosine for aspartic acid (D814Y), has been described in the mouse mastocytoma cell line P815. We have investigated the mechanism of oncogenic activation by this mutation. Expression of this mutant Kit receptor tyrosine kinase in a mast cell line led to the selective tyrosine phosphorylation of a 130-kDa protein and the degradation, through the ubiquitin-dependent proteolytic pathway, of a 65-kDa phosphoprotein. The 65-kDa protein was identified as the src homology domain 2 (SH2)-containing protein tyrosine phosphatase SHP-1, a negative regulator of signaling by Kit and other hematopoietic receptors, and the protein product of the murine motheaten locus. This mutation also altered the sites of receptor autophosphorylation and peptide substrate selectivity. Thus, this mutation activates the oncogenic potential of Kit by a novel mechanism involving an alteration in Kit substrate recognition and the degradation of SHP-1, an attenuator of the Kit signaling pathway.

Proteasome-dependent degradation of the human estrogen receptor

In eukaryotic cells, the ubiquitin–proteasome pathway is the major mechanism for the targeted degradation of proteins with short half-lives. The covalent attachment of ubiquitin to lysine residues of targeted proteins is a signal for the recognition and rapid degradation by the proteasome, a large multi-subunit protease. In this report, we demonstrate that the human estrogen receptor (ER) protein is rapidly degraded in mammalian cells in an estradiol-dependent manner. The treatment of mammalian cells with the proteasome inhibitor MG132 inhibits activity of the proteasome and blocks ER degradation, suggesting that ER protein is turned over through the ubiquitin–proteasome pathway. In addition, we show that in vitro ER degradation depends on ubiquitin-activating E1 enzyme (UBA) and ubiquitin-conjugating E2 enzymes (UBCs), and the proteasome inhibitors MG132 and lactacystin block ER protein degradation in vitro. Furthermore, the UBA/UBCs and proteasome inhibitors promote the accumulation of higher molecular weight forms of ER. The UBA and UBCs, which promote ER degradation in vitro, have no significant effect on human progesterone receptor and human thyroid hormone receptor β proteins.

GTP bound to chloroplast thylakoid membranes is required for light-induced, multienzyme degradation of the photosystem II D1 protein

Even though light is the driving force in photosynthesis, it also can be harmful to plants. The water-splitting photosystem II is the main target for this light stress, leading to inactivation of photosynthetic electron transport and photooxidative damage to its reaction center. The plant survives through an intricate repair mechanism involving proteolytic degradation and replacement of the photodamaged reaction center D1 protein. Based on experiments with isolated chloroplast thylakoid membranes and photosystem II core complexes, we report several aspects concerning the rapid turnover of the D1 protein. (i) The primary cleavage step is a GTP-dependent process, leading to accumulation of a 23-kDa N-terminal fragment. (ii) Proteolysis of the D1 protein is inhibited below basal levels by nonhydrolyzable GTP analogues and apyrase treatment, indicating the existence of endogenous GTP tightly bound to the thylakoid membrane. This possibility was corroborated by binding studies. (iii) The proteolysis of the 23-kDa primary degradation fragment (but not of the D1 protein) is an ATP- and zinc-dependent process. (iv) D1 protein degradation is a multienzyme event involving a strategic (primary) protease and a cleaning-up (secondary) protease. (v) The chloroplast FtsH protease is likely to be involved in the secondary degradation steps. Apart from its significance for understanding the repair of photoinhibition, the discovery of tightly bound GTP should have general implications for other regulatory reactions and signal transduction pathways associated with the photosynthetic membrane.

Elongin BC complex prevents degradation of von Hippel-Lindau tumor suppressor gene products

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene causes the familial cancer syndrome, VHL disease, characterized by a predisposition to renal cell carcinoma and other tumor types. Loss of VHL gene function also is found in a majority of sporadic renal carcinomas. A preponderance of the tumor-disposing inherited missense mutations detected in VHL disease are within the elongin-binding domain of VHL. This region mediates the formation of a multiprotein VHL complex containing elongin B, elongin C, cul-2, and Rbx1. This VHL complex is thought to function as an E3 ubiquitin ligase. Here, we report that VHL proteins harboring mutations which disrupt elongin binding are unstable and rapidly degraded by the proteasome. In contrast, wild-type VHL proteins are directly stabilized by associating with both elongins B and C. In addition, elongins B and C are stabilized through their interactions with each other and VHL. Thus, the entire VHL/elongin complex is resistant to proteasomal degradation. Because the elongin-binding domain of VHL is frequently mutated in cancers, these results suggest that loss of elongin binding causes tumorigenesis by compromising VHL protein stability and/or potential VHL ubiquitination functions.

Parkin functions as an E2-dependent ubiquitin– protein ligase and promotes the degradation of the synaptic vesicle-associated protein, CDCrel-1

Parkinson's disease is a common neurodegenerative disorder in which familial-linked genes have provided novel insights into the pathogenesis of this disorder. Mutations in Parkin, a ring-finger-containing protein of unknown function, are implicated in the pathogenesis of autosomal recessive familial Parkinson's disease. Here, we show that Parkin binds to the E2 ubiquitin-conjugating human enzyme 8 (UbcH8) through its C-terminal ring-finger. Parkin has ubiquitin–protein ligase activity in the presence of UbcH8. Parkin also ubiquitinates itself and promotes its own degradation. We also identify and show that the synaptic vesicle-associated protein, CDCrel-1, interacts with Parkin through its ring-finger domains. Furthermore, Parkin ubiquitinates and promotes the degradation of CDCrel-1. Familial-linked mutations disrupt the ubiquitin–protein ligase function of Parkin and impair Parkin and CDCrel-1 degradation. These results suggest that Parkin functions as an E3 ubiquitin–protein ligase through its ring domains and that it may control protein levels via ubiquitination. The loss of Parkin's ubiquitin–protein ligase function in familial-linked mutations suggests that this may be the cause of familial autosomal recessive Parkinson's disease.

Structural features of the kringle domain determine the intracellular degradation of under-γ-carboxylated prothrombin: Studies of chimeric rat/human prothrombin

Vitamin K antagonists such as warfarin inhibit the vitamin K-dependent γ-glutamyl carboxylation during protein processing and block the secretion of under-γ-carboxylated prothrombin (FII) in the rat but not in the human or bovine. Under-γ-carboxylated prothrombin is also secreted from warfarin-treated human (HepG2) cell cultures but is degraded in the endoplasmic reticulum in warfarin-treated rat (H-35) cell cultures. This differential response to warfarin has been shown to be determined by the structural difference in the proteins rather than by the origin of the cell line. When recombinant rat prothrombin (rFII) and human prothrombin (hFII) were expressed in a transformed human kidney cell line (HEK293), secretion of rFII but not hFII was drastically decreased in response to warfarin. To determine the structural signal required for this differential response, chimeric cDNAs with the propeptide/Gla domains, kringle domain, and serine protease domain exchanged between rFII and hFII were generated (FIIRHH and FIIHRR, FIIRRH and FIIHHR, FIIRHR and FIIHRH) and expressed in both warfarin-treated HEK293 cells and HepG2 cells. The presence of the hFII kringle domain changed the stability of rFII to that of hFII, and the rFII kringle domain changed the stability of hFII to that of rFII. The kringle domain therefore is critical in determining the metabolic fate of under-γ-carboxylated prothrombin precursors during processing. Prothrombin contains two kringle structures, and expression of additional rFII/hFII chimeras (FIIHrhH and FIIHhrH, FIIRrhR, and FIIRhrR) was used to determine that the first of the two kringles plays a more important role in the recognition process.

Hsp70 Molecular Chaperone Facilitates Endoplasmic Reticulum-associated Protein Degradation of Cystic Fibrosis Transmembrane Conductance Regulator in Yeast

Membrane and secretory proteins fold in the endoplasmic reticulum (ER), and misfolded proteins may be retained and targeted for ER-associated protein degradation (ERAD). To elucidate the mechanism by which an integral membrane protein in the ER is degraded, we studied the fate of the cystic fibrosis transmembrane conductance regulator (CFTR) in the yeast Saccharomyces cerevisiae. Our data indicate that CFTR resides in the ER and is stabilized in strains defective for proteasome activity or deleted for the ubiquitin-conjugating enzymes Ubc6p and Ubc7p, thus demonstrating that CFTR is a bona fide ERAD substrate in yeast. We also found that heat shock protein 70 (Hsp70), although not required for the degradation of soluble lumenal ERAD substrates, is required to facilitate CFTR turnover. Conversely, calnexin and binding protein (BiP), which are required for the proteolysis of ER lumenal proteins in both yeast and mammals, are dispensable for the degradation of CFTR, suggesting unique mechanisms for the disposal of at least some soluble and integral membrane ERAD substrates in yeast.

Ligand-dependent Degradation of Smad3 by a Ubiquitin Ligase Complex of ROC1 and Associated Proteins

Smads are signal mediators for the members of the transforming growth factor-β (TGF-β) superfamily. Upon phosphorylation by the TGF-β receptors, Smad3 translocates into the nucleus, recruits transcriptional coactivators and corepressors, and regulates transcription of target genes. Here, we show that Smad3 activated by TGF-β is degraded by the ubiquitin–proteasome pathway. Smad3 interacts with a RING finger protein, ROC1, through its C-terminal MH2 domain in a ligand-dependent manner. An E3 ubiquitin ligase complex ROC1-SCFFbw1a consisting of ROC1, Skp1, Cullin1, and Fbw1a (also termed βTrCP1) induces ubiquitination of Smad3. Recruitment of a transcriptional coactivator, p300, to nuclear Smad3 facilitates the interaction with the E3 ligase complex and triggers the degradation process of Smad3. Smad3 bound to ROC1-SCFFbw1a is then exported from the nucleus to the cytoplasm for proteasomal degradation. TGF-β/Smad3 signaling is thus irreversibly terminated by the ubiquitin–proteasome pathway.

Direct Evidence for Rapid Degradation of Bacillus thuringiensis Toxin mRNA as a Cause of Poor Expression in Plants1

It is well established that the expression of Bacillus thuringiensis (B.t.) toxin genes in higher plants is severely limited at the mRNA level, but the cause remains controversial. Elucidating whether mRNA accumulation is limited transcriptionally or posttranscriptionally could contribute to effective gene design as well as provide insights about endogenous plant gene-expression mechanisms. To resolve this controversy, we compared the expression of an A/U-rich wild-type cryIA(c) gene and a G/C-rich synthetic cryIA(c) B.t.-toxin gene under the control of identical 5′ and 3′ flanking sequences. Transcriptional activities of the genes were equal as determined by nuclear run-on transcription assays. In contrast, mRNA half-life measurements demonstrated directly that the wild-type transcript was markedly less stable than that encoded by the synthetic gene. Sequences that limit mRNA accumulation were located at more than one site within the coding region, and some appeared to be recognized in Arabidopsis but not in tobacco (Nicotiana tabacum). These results support previous observations that some A/U-rich sequences can contribute to mRNA instability in plants. Our studies further indicate that some of these sequences may be differentially recognized in tobacco cells and Arabidopsis.

Ubiquitin-protein ligase activity of X-linked inhibitor of apoptosis protein promotes proteasomal degradation of caspase-3 and enhances its anti-apoptotic effect in Fas-induced cell death

The inhibitor of apoptosis (IAP) family of anti-apoptotic proteins regulate programmed cell death and/or apoptosis. One such protein, X-linked IAP (XIAP), inhibits the activity of the cell death proteases, caspase-3, -7, and -9. In this study, using constitutively active mutants of caspase-3, we found that XIAP promotes the degradation of active-form caspase-3, but not procaspase-3, in living cells. The XIAP mutants, which cannot interact with caspase-3, had little or no activity of promoting the degradation of caspase-3. RING finger mutants of XIAP also could not promote the degradation of caspase-3. A proteasome inhibitor suppressed the degradation of caspase-3 by XIAP, suggesting the involvement of a ubiquitin-proteasome pathway in the degradation. An in vitro ubiquitination assay revealed that XIAP acts as a ubiquitin-protein ligase for caspase-3. Caspase-3 was ubiquitinated in the presence of XIAP in living cells. Both the association of XIAP with caspase-3 and the RING finger domain of XIAP were essential for ubiquitination. Finally, the RING finger mutants of XIAP were less effective than wild-type XIAP at preventing apoptosis induced by overexpression of either active-form caspase-3 or Fas. These results demonstrate that the ubiquitin-protein ligase activity of XIAP promotes the degradation of caspase-3, which enhances its anti-apoptotic effect.

Degradation of CYC1 mRNA in the yeast Saccharomyces cerevisiae does not require translation.

Several studies have indicated that degradation of certain mRNAs is tightly coupled to their translation, whereas, in contrast, other observations suggested that translation can be inhibited without changing the stability of the mRNA. We have addressed this question with the use of altered CYC1 alleles, which encode iso-1-cytochrome c in the yeast Saccharomyces cerevisiae. The cyc1-1249 mRNA, which lacks all in-frame and out-of-frame AUG triplets, was as stable as the normal mRNA. This finding established that translation is not required for the degradation of CYC1 mRNAs. Furthermore, poly(G)18 tracks were introduced within the CYC1 mRNA translated regions to block exonuclease degradation. The recovery of 3' fragments revealed that the translatable and the AUG-deficient mRNAs are both degraded 5'-->3'. Also, the increased stability of CYC1 mRNAs in xrn1-delta strains lacking Xrn1p, the major 5'-->3' exonuclease, established that the normal and AUG-deficient mRNAs are degraded by the same pathway. In addition, deadenylylation, which activates the action of Xrn1p, occurred at equivalent rates in both normal and AUG-deficient mRNAs. We conclude that translation is not required for the normal degradation of CYC1 mRNAs, and that translatable and untranslated mRNAs are degraded by the same pathway.

The Mos/mitogen-activated protein kinase (MAPK) pathway regulates the size and degradation of the first polar body in maturing mouse oocytes.

Mos is an upstream activator of mitogen-activated protein kinase (MAPK) and, in mouse oocytes, is responsible for metaphase II arrest. This activity has been likened to its function in Xenopus oocytes as a component of cytostatic factor. Thus, Mos-deficient female mice (MOS-/-) are less fertile and oocytes derived from these animals fail to arrest at metaphase II and undergo parthenogenetic activation [Colledge, W. H., Carlton, M. B. L., Udy, C. B. & Evans, M. J. (1994) Nature (London) 370, 65-68 and Hashimoto, N., Watanabe, N., Furuta. Y., Tamemoto, B., Sagata, N., Yokoyama, M., Okazaki, K., Nagayoshi, M., Takeda, N., Ikawa, Y. & Aizawa, S. (1994) Nature (London) 370, 68-71]. Here we show that maturing MOS-/- oocytes fail to activate MAPK throughout meiosis, while p34cdc2 kinase activity is normal until late in metaphase II when it decreases prematurely. Phenotypically, the first meiotic division of MOS-/- oocytes frequently resembles mitotic cleavage or produces an abnormally large polar body. In these oocytes, the spindle shape is altered and the spindle fails to translocate to the cortex, leading to the establishment of an altered cleavage plane. Moreover, the first polar body persists instead of degrading and sometimes undergoes an additional cleavage, thereby providing conditions for parthenogenesis. These studies identify meiotic spindle formation and programmed degradation of the first polar body as new and important roles for the Mos/MAPK pathway.