Structural basis for the specificity of ubiquitin C-terminal hydrolases.

The release of ubiquitin from attachment to other proteins and adducts is critical for ubiquitin biosynthesis, proteasomal degradation and other cellular processes. De-ubiquitination is accomplished in part by members of the UCH (ubiquitin C-terminal hydrolase) family of enzymes. We have determined the 2.25 A resolution crystal structure of the yeast UCH, Yuh1, in a complex with the inhibitor ubiquitin aldehyde (Ubal). The structure mimics the tetrahedral intermediate in the reaction pathway and explains the very high enzyme specificity. Comparison with a related, unliganded UCH structure indicates that ubiquitin binding is coupled to rearrangements which block the active-site cleft in the absence of authentic substrate. Remarkably, a 21-residue loop that becomes ordered upon binding Ubal lies directly over the active site. Efficiently processed substrates apparently pass through this loop, and constraints on the loop conformation probably function to control UCH specificity.     

Mechanistic studies of ubiquitin C-terminal hydrolase L1

Ubiquitin C-terminal hydrolases (UCHs) cleave Ub-X bonds (Ub is ubiquitin and X an alcohol, an amine, or a protein) through a thioester intermediate that is produced by nucleophilic attack of the Cys residue of a Cys-SH/His-Im catalytic diad. We are studying the mechanism of UCH-L1, a UCH that is implicated in Parkinson's disease, and now wish to report our initial findings. (i) Pre-steady-state kinetic studies for UCH-L1-catalyzed hydrolysis of Ub-AMC (AMC, 7-amido-4-methylcoumarin) indicate that k(cat) is rate-limited by acyl-enzyme formation. Thus, K(m) = K(s), the dissociation constant for the Michaelis complex, and k(cat) = k(2), the rate constant for acyl-enzyme formation. (ii) For K(assoc) (=K(s)(-)(1)), DeltaC(p) = -0.8 kcal mol(-)(1) deg(-)(1) and is consistent with coupling between substrate association and a conformational change of the enzyme. For k(2), DeltaS(++) = 0 and suggests that in the E-S, substrate and active site residues are precisely aligned for reaction. (iii) Solvent isotope effects are (D)K(assoc) = 0.5 and (D)k(2) = 0.9, suggesting that the substrate binds to a form of free enzyme in which the active site Cys exists as the thiol. In the resultant Michaelis complex, the diad has tautomerized to ion pair Cys-S(-)/His-ImH(+). Subsequent attack of thiolate produces the acyl-enzyme species. In contrast, isotope effects for association of UCH-L1 with transition-state analogue ubiquitin aldehyde suggest that an alternative mechanistic pathway can sometimes be available to UCH-L1 involving general base-catalyzed attack of Cys-SH by His-Im.     

Development of a high-throughput screen targeting caspase-8-mediated cleavage of the amyloid precursor protein

Caspases, effectors of apoptosis, are key mediators of neuronal death in several neurodegenerative diseases. Caspase-8 and caspase-6 have been implicated in the pathogenesis of amyotrophic lateral sclerosis, multiple sclerosis, Parkinson’s disease, and Alzheimer’s disease (AD). ß-Amyloid precursor protein (APP) is cleaved at Asp664 in its intracellular domain by caspase-8. We and other laboratories recently showed that obliteration of the caspase cleavage site on APP alleviates functional AD-like deficits in a mouse model. Therefore, caspase cleavage of APP constitutes a potential novel target for therapeutic intervention. To identify chemical inhibitors of caspase-8 cleavage, we screened a subset of the chemical library at the Harvard NeuroDiscovery Center’s Laboratory for Drug Discovery in Neurodegeneration. We show that caspase-8, but not caspase-1, -3, or -9, cleaves a biotinylated peptide derived from APP at Asp664, and we report the development of a sensitive high-throughput assay for caspase-8 cleavage of APP and the use of that assay for the identification of specific small molecule “hit” compounds that potently inhibit Asp664 cleavage of APP. Furthermore, we demonstrate that one of these compounds (LDN-0021835) inhibits the cleavage of APP at Asp664 in cell-based assays.     

Length of the active-site crossover loop defines the substrate specificity of ubiquitin C-terminal hydrolases for ubiquitin chains

UCHs [Ub (ubiquitin) C-terminal hydrolases] are a family of deubiquitinating enzymes that are often thought to only remove small C-terminal peptide tails from Ub adducts. Among the four UCHs identified to date, neither UCH-L3 nor UCH-L1 can catalyse the hydrolysis of isopeptide Ub chains, but UCH-L5 can when it is present in the PA700 complex of the proteasome. In the present paper, we report that the UCH domain of UCH-L5, different from UCH-L1 and UCH-L3, by itself can process the K48-diUb (Lys48-linked di-ubiquitin) substrate by cleaving the isopeptide bond between two Ub units. The catalytic specificity of the four UCHs is dependent on the length of the active-site crossover loop. The UCH domain with a long crossover loop (usually >14 residues), such as that of UCH-L5 or BAP1 [BRCA1 (breast cancer early-onset 1)-associated protein 1], is able to cleave both small and large Ub derivatives, whereas the one with a short loop can only process small Ub derivatives. We also found that elongation of the crossover loop enables UCH-L1 to have isopeptidase activity for K48-diUb in a length-dependent manner. Thus the loop length of UCHs defines their substrate specificity for diUb chains, suggesting that the chain flexibility of the crossover loop plays an important role in determining its catalytic activity and substrate specificity for cleaving isopeptide Ub chains.     

泛素羧基末端水解酶-1(UCHL1)的分子进化分析

泛素羧基末端水解酶-1(ubiquitin C-terminal hydrolase L1,UCHL1)是一种去泛素化酶,特异性表达于脑与生殖腺,具有去泛素化活性、稳定细胞内泛素单体的功能。为确定UCHL1在脊椎动物中是否普遍存在,从而选择身体构造更简单的模式动物研究其生物学功能,通过生物信息学手段分析脊椎动物门的11种生物中基因UCHL1的分子进化情况,分析显示UCHL1基因组长度在进化过程中变化较大,但外显子个数变化较小,蛋白氨基酸残基数目也基本维持在223 aa左右。分析结果表明,mRNA有不同的剪接方式,但氨基酸序列在进化上是高度保守的,UCHL1蛋白活性位点同源性高达90%,该结果证明了蛋白UCHL1的功能对于物种正常生存是必须的。     

C-terminal amidation of neuropeptides. Gly-Lys-Arg extension an efficient precursor of C-terminal amide

Biosynthesis of the C-terminal carboxamide group of peptide hormones was studied using comparatively pGlu-His-Pro-Gly and Glu-His-Pro-Gly-Lys-Arg as putative precursors of the tripeptide, thyroliberin (TRH). Rat hypothalamus granules were found to contain an amide group forming activity which converts both peptide substrates into TRH. Comparison of the rate of conversion of the two substrates indicated that the C-terminal dibasic extension favored a 10-fold increase in the production of amidated peptide. It is suggested that this type of structure may be present in the putative biosynthetic precursor of TRH and that it may provide a better substrate for the enzyme(s) involved in C-terminal amidation.     

Essential role for the C-terminal noncatalytic region of SHIP in FcgammaRIIB1-mediated inhibitory signaling

The inositol phosphatase SHIP binds to the FcgammaRIIB1 receptor and plays a critical role in FcgammaRIIB1-mediated inhibition of B-cell proliferation and immunoglobulin synthesis. The molecular details of SHIP function are not fully understood. While point mutations of the signature motifs in the inositol phosphatase domain abolish SHIP's ability to inhibit calcium flux in B cells, little is known about the function of the evolutionarily conserved, putative noncatalytic regions of SHIP in vivo. In this study, through a systematic mutagenesis approach, we identified the inositol phosphatase domain of SHIP between amino acids 400 and 866. Through reconstitution of a SHIP-deficient B-cell line with wild-type and mutant forms of SHIP, we demonstrate that the catalytic domain alone is not sufficient to mediate FcgammaRIIB1/SHIP-dependent inhibition of B-cell receptor signaling. Expression of a truncation mutant of SHIP that has intact phosphatase activity but lacks the last 190 amino acids showed that the noncatalytic region in the C terminus is essential for inhibitory signaling. Mutation of two tyrosines within this C-terminal region, previously identified as important in binding to Shc, showed a reduced inhibition of calcium flux. However, studies with an Shc-deficient B-cell line indicated that Shc-SHIP complex formation is not required and that other proteins that bind these tyrosines may be important in FcgammaRIIB1/SHIP-mediated calcium inhibition. Interestingly, membrane targeting of SHIP lacking the C terminus is able to restore this inhibition, suggesting a role for the C terminus in localization or stabilization of SHIP interaction at the membrane. Taken together, these data suggest that the noncatalytic carboxyl-terminal 190 amino acids of SHIP play a critical role in SHIP function in B cells and may play a similar role in several other receptor systems where SHIP functions as a negative regulator.     

The Leber congenital amaurosis protein AIPL1 modulates the nuclear translocation of NUB1 and suppresses inclusion formation by NUB1 fragments

Mutations in the aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) cause the blinding disease Leber congenital amaurosis (LCA). The similarity of AIPL1 to AIP has led to suggestions that AIPL1 could function in a similar manner to AIP in facilitating protein translocation and as a component of chaperone complexes. AIPL1 interacts with the cell cycle regulator NEDD8 ultimate buster protein 1 (NUB1). As AIPL1 is predominantly cytoplasmic and NUB1 is predominantly nuclear, we tested the hypothesis that AIPL1 could modulate the nuclear translocation of NUB1. Co-transfection of AIPL1 with GFP-NUB1 resulted in a shift of GFP-NUB1 subcellular distribution toward the cytoplasm. Interestingly, AIPL1 was able to act in a chaperone-like fashion to efficiently suppress inclusion formation by NUB1 fragments. Co-transfection of AIPL1 with GFP-NUB1-N and GFP-NUB1-C resulted in an AIPL1-dependent suppression of GFP-NUB1-N perinuclear inclusions and GFP-NUB1-C intranuclear inclusions leading to the redistribution of these fragments in the cytoplasm. This chaperone-like function of AIPL1 was specific for NUB1, since AIPL1 was unable to suppress the inclusion formation by unrelated aggregation-prone proteins and AIP had no effect on NUB1 localization or inclusion formation. We examined the effect of a range of pathogenic and engineered mutations on the ability of AIPL1 to modulate NUB1 localization or inclusion formation. With the exception of W278X, which formed non-functional SDS-insoluble inclusions, all of the pathogenic mutations studied were soluble and could modulate NUB1 with varying efficiency compared with the wild-type protein. The effect of AIPL1 on NUB1 required the C-terminal region of AIPL1, as engineered C-terminal truncation mutations had no effect on NUB1. These data show that AIPL1 can modulate protein translocation and act in a chaperone-like manner and suggest that AIPL1 is an important modulator of NUB1 cellular function.     

The ubiquitin binding domain ZnF UBP recognizes the C-terminal diglycine motif of unanchored ubiquitin

Ubiquitin binding proteins regulate the stability, function, and/or localization of ubiquitinated proteins. Here we report the crystal structures of the zinc-finger ubiquitin binding domain (ZnF UBP) from the deubiquitinating enzyme isopeptidase T (IsoT, or USP5) alone and in complex with ubiquitin. Unlike other ubiquitin binding domains, this domain contains a deep binding pocket where the C-terminal diglycine motif of ubiquitin is inserted, thus explaining the specificity of IsoT for an unmodified C terminus on the proximal subunit of polyubiquitin. Mutations in the domain demonstrate that it is required for optimal catalytic activation of IsoT. This domain is present in several other protein families, and the ZnF UBP domain from an E3 ligase also requires the C terminus of ubiquitin for binding. These data suggest that binding the ubiquitin C terminus may be necessary for the function of other proteins.     

Structural insights into the ubiquitin recognition by OPTN (optineurin) and its regulation by TBK1-mediated phosphorylation

OPTN (optineurin), a ubiquitin-binding scaffold protein, functions as an important macroautophagy/autophagy receptor in selective autophagy processes. Mutations in OPTN have been linked with human neurodegenerative diseases including ALS and glaucoma. However, the mechanistic basis underlying the recognition of ubiquitin by OPTN and its regulation by TBK1-mediated phosphorylation are still elusive. Here, we demonstrate that the UBAN domain of OPTN preferentially recognizes linear ubiquitin chain and forms an asymmetric 2:1 stoichiometry complex with the linear diubiquitin. In addition, our results provide new mechanistic insights into how phosphorylation of UBAN would regulate the ubiquitin-binding ability of OPTN and how disease-associated mutations in the OPTN UBAN domain disrupt its interaction with ubiquitin. Finally, we show that defects in ubiquitin-binding may affect the recruitment of OPTN to linear ubiquitin-decorated mutant Huntington protein aggregates. Taken together, our findings clarify the interaction mode between UBAN and linear ubiquitin chain in general, and expand our knowledge of the molecular mechanism of ubiquitin-decorated substrates recognition by OPTN as well as the pathogenesis of neurodegenerative diseases caused by OPTN mutations.     

The region-specific functions of two ubiquitin C-terminal hydrolase isozymes along the epididymis.

We previously showed that gad mice, which are deficient for ubiquitin C-terminal hydrolase L1 (UCH-L1), have a significantly increased number of defective spermatozoa, suggesting that UCH-L1 functions in sperm quality control during epididymal maturation. The epididymis is the site of spermatozoa maturation, transport and storage. Region-specific functions along the epididymis are essential for establishing the environment required for sperm maturation. We analyzed the region-specific expression of UCH-L1 and UCH-L3 along the epididymis, and also assessed the levels of ubiquitin, which has specificity for UCH-L1. In wild-type mice, western blot analysis demonstrated a high level of UCH-L1 expression in the caput epididymis, consistent with ubiquitin expression, whereas UCH-L3 expression was high in the cauda epididymis. We also investigated the function of UCH-L1 and UCH-L3 in epididymal apoptosis induced by efferent duct ligation. The caput epididymides of gad mice were resistant to apoptotic stress induced by efferent duct ligation, whereas Uchl3 knockout mice showed a marked increase in apoptotic cells following ligation. In conclusion, the response of gad and Uchl3 knockout mice to androgen withdrawal suggests a reciprocal function of the two UCH enzymes in the caput epididymis.     

Functional heterogeneity of C-terminal peroxisome targeting signal 1 in PEX5-defective patients.

To investigate mechanisms related to functions of the peroxisome targeting signal (PTS) 1 receptor, Pex5p, we analyzed peroxisome matrix protein import in fibroblasts from three patients with peroxisome biogenesis disorders, all with different mutations in the PEX5 gene. The patients 2-01 (Zellweger syndrome) and 2-05 (neonatal adrenoleukodystrophy) have the reported mutations, R390X and N489K, and patient 2-03 (infantile Refsum disease) has a newly identified mutation, S563W. Fibroblasts from 2-03 (S563W) were detected in both PTS1 and PTS2 imports despite the PEX5 defect, findings in contrast with fibroblasts from 2-05 (N489K) severely defective in PTS1 import and those from 2-01 (R390X) severely defective in both PTS1 and PTS2. The PTS1 receptor in 2-03 is functional for only the C-terminal -SKL sequence (acyl-CoA oxidase) and had little or no function for C-terminal -AKL (D-bifunctional protein and sterol carrier protein 2) and -KANL (catalase) sequences, respectively. After transfection of these mutated PEX5 cDNA into the PEX5-defective CHO mutant, transformants of ZP102 revealed that each mutation was responsible for each dysfunction of the PTS1 import. It seems apparent that -AKL and -KANL are poorer variants of PTS1 and are likely to be more susceptible to effects of mutation of its receptor, Pex5p.     

SMG1 regulates adipogenesis via targeting of staufen1-mediated mRNA decay

Suppressor of morphogenesis in genitalia 1 (SMG1), a member of the phosphatidylinositol 3-kinase-related kinase family, is involved in nonsense-mediated mRNA decay (NMD). SMG1 phosphorylates Upf1, a key NMD factor. Subsequently, hyperphosphorylated Upf1 associates with SMG5-7 or proline-rich nuclear receptor coregulatory protein (PNRC2) to elicit rapid mRNA degradation. Upf1 is also known to be involved in staufen 1 (Stau1)-mediated mRNA decay (SMD), which is closely related to NMD. However, the biological and molecular roles of SMG1 in SMD remain unknown. Here, we provide evidence that SMG1 is involved in SMD. The immunoprecipitation results show that SMG1 is complexed with Stau1, Upf1, and Dcp1a. Downregulation of SMG1 or overexpression of a kinase-inactive mutant of SMG1 inhibits SMD efficiency. In addition, downregulation of SMG1 inhibits rapid degradation elicited by artificially tethered Stau1 or Upf1 downstream of the normal termination codon. Furthermore, Stau1 and Upf1 colocalize in processing bodies in an SMG1-dependent manner. We also find that the level of SMG1 increases during adipogenesis. Accordingly, downregulation of SMG1 causes the reduction in the level of Upf1 phosphorylation and delays adipogenesis, suggesting the functional involvement of SMG1 in adipogenesis via SMD.     

The C-terminal domain of plant catalases. Implications for a glyoxysomal targeting sequence.

A castor bean (Ricinus communis cv. Hale) cDNA encoding catalase was cloned and sequenced. The cDNA encoding the carboxy-terminal domain of catalase was compared to the corresponding sequences of six other plant catalases. The deduced amino acid sequences were compared according to the chemical attributes of each amino acid within each carboxy-terminal domain. A tripeptide sequence having the chemical attributes of the peroxisomal targeting sequence [Gould, S.J., Keller, G.-A., Hosken, N., Wilkinson, J. & Subramani, S. (1989) J. Cell Biol. 108, 1657-1664] was common to all the glyoxysomal/peroxisomal plant catalases. This sequence motif was located six amino acids from the carboxy terminus of each of the plant catalases. An identical motif was also found within the carboxy-terminal domain of three mammalian catalases previously sequenced. We hypothesize that these motifs are at least part of the targeting mechanism for catalase entry into plant glyoxysomes/peroxisomes.     

The ubiquitin ligase gp78 promotes sarcoma metastasis by targeting KAI1 for degradation

Metastasis is the primary cause of mortality from cancer, but the mechanisms leading to metastasis are poorly understood. In particular, relatively little is known about metastasis in cancers of mesenchymal origins, which are known as sarcomas. Approximately ten proteins have been characterized as 'metastasis suppressors', but how these proteins function and are regulated is, in general, not well understood. Gp78 (also known as AMFR or RNF45) is a RING finger E3 ubiquitin ligase that is integral to the endoplasmic reticulum (ER) and involved in ER-associated degradation (ERAD) of diverse substrates. Here we report that expression of gp78 has a causal role in the metastasis of an aggressive human sarcoma and that this prometastatic activity requires the E3 activity of gp78. Further, gp78 associates with and targets the transmembrane metastasis suppressor, KAI1 (also known as CD82), for degradation. Suppression of gp78 increases KAI1 abundance and reduces the metastatic potential of tumor cells, an effect that is largely blocked by concomitant suppression of KAI1. An inverse relationship between these proteins was confirmed in a human sarcoma tissue microarray. Whereas most previous efforts have focused on genetic mechanisms for the loss of metastasis suppressor genes, our results provide new evidence for post-translational downregulation of a metastasis suppressor by its ubiquitin ligase, resulting in abrogation of its metastasis-suppressing effects.