[Anticancer therapy for symptom relief? : A systematic review of clinical trials in oncology]. / Symptomlinderung durch Tumortherapie? : Eine systematische Übersicht über onkologische Therapiestudien.

BACKGROUND: When patients suffer from incurable cancer, drug-based, systemic anticancer therapy is usually used with the aim of longer survival, while minimizing toxicity and ensuring a tolerable quality of life. It is unclear to what extent and with what degree of success systemic tumor therapy can be used to specifically improve pre-existing pain and an already compromised quality of life (QoL). METHODS: Therefore, a systematic review of oncological therapy studies (PubMed) was conducted. Only studies that identified the pain-relieving and QoL-enhancing effects of systemic anticancer therapy as the primary endpoint were selected and evaluated descriptively. RESULTS: Of the 2229 abstracts identified using a piloted search string, only 13 studies met the inclusion criteria. Of these, 10 studies showed an improvement in QoL-parameters through the use of systemic tumor therapies. DISCUSSION: Only a few studies focused primarily on the improvement of parameters related to quality of life-although this is the primary therapeutic goal for many patients suffering from incurable and advanced cancer. The study results encourage regular inclusion of symptom- and QoL-related data in clinical studies and to more explicitly address the potential of systemic anticancer therapy in relieving pain and other symptoms, thereby supporting the goals of palliative care.

PARP-1 deficiency blocks IL-5 expression through calpain-dependent degradation of STAT-6 in a murine asthma model.

BACKGROUND: We recently showed that poly(ADP-ribose)polymerase-1 (PARP-1) may play a role in allergen (ovalbumin)-induced airway eosinophilia, potentially through a specific effect on IL-5 production. We also reported that while IL-5 replenishment promotes reversal of eosinophilia in lungs of PARP-1(-/-) mice, IL-4 or Immunoglobulin E replenishment do not, suggesting a potentially significant regulatory relationship between PARP-1 and IL-5. OBJECTIVE: To explore the mechanism by which PARP-1 regulates IL-5 production and to determine how PARP-1 inhibition blocks allergen-induced eosinophilia. METHODS: This study was conducted using a murine model of allergic airway inflammation and primary splenocytes. RESULTS: PARP-1 knockout-associated reduction in IL-5 upon allergen exposure occurs at the mRNA level. Such an effect appears to take place after IL-4 receptor activation as PARP-1 inhibition exerted no effect on JAK1/JAK3 activation. Signal transducer and activator of transcription-6 (STAT-6) protein was severely downregulated in spleens of PARP-1(-/-) mice without any effect on mRNA levels, suggesting an effect on protein integrity rather than gene transcription. Interestingly, the degradation of STAT-6 in PARP-1(-/-) mice required allergen stimulation. Additionally, PARP-1 enzymatic activity appears to be required for STAT-6 integrity. The downregulation of STAT-6 coincided with mRNA and protein reduction of GATA-binding protein-3 and occupancy of its binding site on the IL-5 gene promoter. IL-4 was sufficient to induce STAT-6 downregulation in both PARP-1(-/-) mice and isolated splenocytes. Such degradation may be mediated by calpain, but not by proteasomes. CONCLUSION: These results demonstrate a novel function of PARP-1 in regulating IL-5 expression during allergen-induced inflammation and explain the underlying mechanism by which PARP-1 inhibition results in IL-5 reduction.

Hdm2 is a ubiquitin ligase of Ku70-Akt promotes cell survival by inhibiting Hdm2-dependent Ku70 destabilization.

Earlier, we have reported that 70 kDa subunit of Ku protein heterodimer (Ku70) binds and inhibits Bax activity in the cytosol and that ubiquitin (Ub)-dependent proteolysis of cytosolic Ku70 facilitates Bax-mediated apoptosis. We found that Hdm2 (human homolog of murine double minute) has an ability to ubiquitinate Ku70 and that Hdm2 overexpression in cultured cells causes a decrease in Ku70 expression levels. An interaction between Ku70 and Hdm2 was shown by means of immunoprecipitation, whereas none could be shown between 80 kDa subunit of Ku protein heterodimer and Hdm2. Vascular endothelial growth factor (VEGF) is known to inhibit endothelial cell (EC) apoptosis through an Akt-mediated survival kinase signal; however, the mechanism underlying this inhibition of apoptosis has not been fully elucidated. We found that VEGF inhibited cytosolic Ku70 degradation induced by apoptotic stress. It is known that Akt-dependent phosphorylation of Hdm2 causes nuclear translocation of Hdm2 followed by Hdm2-mediated inactivation of p53. We found that VEGF stimulated nuclear translocation of Hdm2 in EC and efficiently inhibited Ku70 degradation. We also found that constitutively active Akt, but not kinase-dead Akt, inhibited Ku70 degradation in the cytosol. Furthermore, Ku70 knockdown diminished antiapoptotic activity of Akt. Taken together, we propose that Hdm2 is a Ku70 Ub ligase and that Akt inhibits Bax-mediated apoptosis, at least in part, by maintaining Ku70 levels through the promotion of Hdm2 nuclear translocation.

Pregnancy and interferon-tau upregulate gene expression of members of the 1-8 family in the bovine uterus.

The 1-8 family (1-8U, 1-8D, Leu-13/9-27) of interferon (IFN)-inducible genes encodes proteins that are components of multimeric complexes involved with transduction of antiproliferative and homotypic adhesion signals. Human 1-8 family members are highly similar and are regulated by type 1 and type 2 IFNs. Because the bovine uterus is bathed in conceptus-derived IFN tau during early pregnancy, it was hypothesized that members of the 1-8 family were upregulated in the bovine uterus during early pregnancy. Oligonucleotide primers were designed based on human and rat 1-8U and Leu-13 cDNAs and used in reverse transcription polymerase chain reactions to amplify bovine cDNAs from endometrial RNA. The bovine 1-8U cDNA was sequenced, found to be 84% identical to the human 1-8U, and used to screen a bovine endometrial cDNA library to isolate the full-length 1-8U and Leu-13 cDNAs. The inferred amino acid sequences of bovine 1-8U and Leu-13 were 72% and 73% identical to their respective human counterparts. Bovine 1-8U and Leu-13 retain an amino acid motif that is conserved in other 1-8 family members and in some ubiquitin-conjugating enzymes (E2s). This motif is critical for function of E2s in covalently linking ubiquitin to targeted proteins. Northern blotting revealed that bovine endometrial 1-8U and Leu-13 mRNAs were upregulated on Day 15 of pregnancy (P < 0.0001) and continued to accumulate through Day 18 of pregnancy (P < 0.05) when compared with endometrium from nonpregnant cows. The bovine 1-8U and Leu-13 mRNAs were also upregulated (P < 0.05) by IFN tau (25 nM) within 3 h, continued to accumulate through 12 h, and reached a plateau at 12-24 h in cultured bovine endometrial cells. In situ hybridization revealed that mRNAs encoding 1-8 family members were heavily localized to glandular epithelium but also were present to a lesser extent in the luminal epithelium and stroma. The temporal upregulation of 1-8U and Leu-13 mRNAs by pregnancy and IFN tau and tissue distribution of these mRNAs paralleled closely that of the ubiquitin homolog, IFN-stimulated gene product 17. These IFN-induced proteins probably work together to prepare the endometrium for adhesion of the developing conceptus.

N-end rule specificity within the ubiquitin/proteasome pathway is not an affinity effect.

The N-end rule relates the amino terminus to the rate of degradation through the ubiquitin/26 S proteasome pathway. Proteins bearing basic (type 1) or large hydrophobic (type 2) amino termini are assumed to be targeted through this pathway by their higher affinity for binding to the responsible E3 ligase compared with proteins bearing other residues (type 3). Paradoxically, a significant fraction of eukaryotic protein degradation occurs through the N-end rule pathway, although the majority of cellular proteins are type 3 substrates. We have exploited specific interactions between ubiquitin carrier proteins (E2/Ubc) and their cognate E3 ligases to purify for the first time the mammalian N-end rule ligase E3alpha/Ubr1 to near homogeneity. In vitro studies show that E3alpha forms lysine 48-linked polyubiquitin degradation signals on type 1-3 substrates and is absolutely dependent on Ubc2/Rad6 orthologs. Biochemically defined kinetic studies show that the basis of N-end rule specificity is a k(cat) rather than the K(m) effect originally proposed, since all three substrate classes show similar binding affinities (K(m) approximately 5 microm) but V(max) values that are 100- and 50-fold greater for type 1 and 2 versus type 3 model substrates, respectively. In addition, the N-end rule dipeptides lysylalanine and phenylalanylalanine are general noncompetitive inhibitors for E3alpha-catalyzed ubiquitination of type 1-3 substrates rather than type-specific competitive inhibitors as predicted. These observations are consistent with a model in which the N-end rule effect reflects substrate binding-induced transitions in E3alpha to a catalytically competent conformer, the equilibrium for which depends on the identity of the amino terminus or the presence of basic or hydrophobic surface features. The model reconciles conflicts between specific predictions and empirical observations relating N-end rule targeting in addition to explicating the efficacy of selected dipeptides as potent in vivo inhibitors of this pathway.

Kinetic analysis of the conjugation of ubiquitin to picornavirus 3C proteases catalyzed by the mammalian ubiquitin-protein ligase E3alpha.

The 3C proteases of the encephalomyocarditis virus and the hepatitis A virus are both type III substrates for the mammalian ubiquitin-protein ligase E3alpha. The conjugation of ubiquitin to these proteins requires internal ten-amino acid-long protein destruction signal sequences. To evaluate how these destruction signals modulate interactions that must occur between E3alpha and the 3C proteases, we have kinetically analyzed the formation of ubiquitin-3C protease conjugates in a reconstituted system of purified E1, HsUbc2b/E2(14Kb), and human E3alpha. Our measurements show that the encephalomyocarditis virus 3C protease is ubiquitinated in this system with K(m) = 42 +/- 11 microm and V(max) = 0.051 +/- 0.01 pmol/min whereas the parameters for the ubiquitination of the hepatitis A virus 3C protease are K(m) = 20 +/- 5 microm and V(max) = 0.018 +/- 0.003 pmol/min. Mutations in the destruction signal sequences resulted in changes in the rate at which E3alpha conjugates ubiquitin to the altered 3C protease proteins. The K(m) and V(max) values for these reactions change proportionally in the same direction. These results suggest differences in rates of conjugation of ubiquitin to 3C proteases are primarily a k(cat) effect. Replacing specific encephalomyocarditis virus 3C protease lysine residues with arginine residues was found to increase, rather than decrease, the rate of ubiquitin conjugation, and the K(m) and V(max) values for these reactions are both higher than for the wild type protein. The ability of E3alpha to catalyze the conjugation of ubiquitin to both 3C proteases was found to be inhibited by lysylalanine and phenylalanylalanine, demonstrating that the same sites on E3alpha that bind destabilizing N-terminal amino acids in type I and II substrates also interact with the 3C proteases.

Variations in site and levels of expression of chondrocyte nucleotide pyrophosphohydrolase with aging.

The aim of this study was to identify changes in cartilage intermediate layer protein/nucleotide pyrophosphohydrolase (CILP/NTPPH) expression in articular cartilage during aging. Adult (3-4 years old) and young (7-10 days old) porcine articular hyaline cartilage and fibrocartilage were studied by Northern blot analysis, in situ hybridization, and immunohistochemistry using a complementary DNA (cDNA) probe encoding porcine CILP/NTPPH and antibody to a synthetic peptide corresponding to a CILP/NTPPH sequence. Northern blot analysis of chondrocytes showed lower expression of CILP/NTPPH messenger RNA (mRNA) in young cartilage than in adult cartilage. In adult cartilage, extracellular matrix from the surface to the middeep zone was immunoreactive for CILP/NTPPH, especially in the pericellular matrix surrounding the middeep zone chondrocytes. In young cartilage, chondrocytes were moderately immunoreactive for CILP/NTPPH throughout all zones except the calcified zone. The matrix of young cartilage was negative except in the superficial zone. In young cartilage, CILP/NTPPH mRNA expression was undetectable. In adult cartilage, chondrocytes showed strong mRNA expression for CILP/NTPPH throughout middeep zones. Protein and mRNA signals were not detectable below the tidemark. CILP/NTPPH secretion into matrix around chondrocytes increases with aging. In this extracellular site it may generate inorganic pyrophosphate and contribute to age-related calcium pyrophosphate dihydrate crystal deposition disease.

Novel mechanism of surface catalysis of protein adduct formation. NMR studies of the acetylation of ubiquitin.

Reactivity of surface lysyl residues of proteins with a broad range of chemical agents has been proposed to be dependent on the catalytic microenvironment of the residue. We have investigated the acetylation of wild type ubiquitin and of the UbH68N mutant to evaluate the potential contribution of His-68 to the reactivity of Lys-6, which is about 4 A distant. These studies were performed using [1-(13)C]acetyl salicylate or [1,1'-(13)C(2)]acetic anhydride, and the acetylated products were detected by two-dimensional heteronuclear multiple quantum coherence spectroscopy. The results demonstrate that His-68 makes a positive contribution to the rate of acetylation of Lys-6 by labeled aspirin. Additionally, a pair of transient resonances is observed after treatment of wild type ubiquitin with the labeled acetic anhydride but not upon treatment of the H68N mutant. These resonances are assigned to the acetylated His-68 residue. The loss of intensity of the acetylhistidine resonances is accompanied by an increase in intensity of the acetyl-Lys-6 peak, supporting the existence of a transacetylation process between the acetylhistidine 68 and lysine 6 residues located on the protein surface. Hence, this may be the first direct demonstration of a catalytic intermediate forming on the protein surface.

Ubiquitination and degradation of the zebrafish paired-like homeobox protein VSX-1.

Vsx-1 is a paired-like : CVC homeobox protein dynamically expressed during zebrafish development. Previous results indicate that Vsx-1 influences bipolar cell differentiation and maintenance of these cells in the adult retina. To understand the developmental regulation of this transcription factor, we investigated ubiquitination as a possible posttranslational mechanism. In vitro, Vsx-1 was conjugated with multiple ubiquitin moieties. Proteasome inhibitors and added ubiquitin increased the accumulation of Vsx-1-ubiquitin(n) complexes and stabilized unmodified Vsx-1. Also, in transiently transfected COS-7 cells, Vsx-1 is ubiquitinated, and pulse-chase experiments show that Vsx-1 proteolysis occurs. Vsx-1 proteins with C-terminal deletions retained the capacity for initial modification by ubiquitin but lost the capacity for efficient chain elongation. These results show that Vsx-1 is a substrate of the ubiquitin/proteasome pathway and suggest that C-terminal sequences of Vsx-1 are critical for ubiquitin chain elongation. In addition, our findings suggest that ubiquitin-dependent proteolysis regulates Vsx-1 during zebrafish retinal development.

Identification of a novel SCAN box-related protein that interacts with MZF1B. The leucine-rich SCAN box mediates hetero- and homoprotein associations.

The SCAN box or leucine-rich (LeR) domain is a conserved motif found within a subfamily of C(2)H(2) zinc finger proteins. The function of a SCAN box is unknown, but it is predicted to form alpha-helices that may be involved in protein-protein interactions. Myeloid zinc finger gene-1B (MZF1B) is an alternatively spliced human cDNA isoform of the zinc finger transcription factor, MZF1. MZF1 and MZF1B contain 13 C(2)H(2) zinc finger motifs, but only MZF1B contains an amino-terminal SCAN box. A bone marrow cDNA library was screened for proteins interacting with the MZF1B SCAN box domain and RAZ1 (SCAN-related protein associated with MZF1B) was identified. RAZ1 is a novel cDNA that encodes a SCAN-related domain and arginine-rich region but no zinc finger motifs. Co-immunoprecipitation assays demonstrate that the SCAN box domain of MZF1B is necessary for association with RAZ1. By yeast two-hybrid analysis, the carboxyl terminus of RAZ1 is sufficient for interaction with the MZF1B SCAN box. Furthermore, MZF1B and RAZ1 each self-associate in vitro via a SCAN box-dependent mechanism. These data provide evidence that the SCAN box is a protein interaction domain that mediates both hetero- and homoprotein associations.

Development of substance abuse problems among drug-involved offenders. Evidence for the telescoping effect.

PURPOSE: The present study was designed to evaluate gender differences in the development of substance abuse disorders among drug-involved offenders and to determine whether women in this population exhibit a telescoping effect (i.e., acceleration in the progression from substance use to substance abuse), which has been observed in other setting. METHOD: Participants consisted of 160 polysubstance-abusing individuals (118 men, 42 women) who were admitted to two Florida drug court programs. Data were obtained from the Addiction Severity Index, intake interviews, and archival court records. RESULTS: Female and male offenders differed significantly in the developmental trajectory of their addiction. Women offenders initiated alcohol and marijuana use significantly later in life than their male cohorts but began using cocaine earlier in the course of their addiction. Women also reported more problems related to cocaine use and significantly more prior treatment episodes. Women were found to have a shorter latency from first use of cocaine to cocaine abuse. Findings are consistent with those of previous studies examining gender differences among individuals referred for substance abuse treatment. Future directions for research and implications for treatment planning are discussed.

The binding site for UCH-L3 on ubiquitin: mutagenesis and NMR studies on the complex between ubiquitin and UCH-L3.

The ubiquitin fold is a versatile and widely used targeting signal that is added post-translationally to a variety of proteins. Covalent attachment of one or more ubiquitin domains results in localization of the target protein to the proteasome, the nucleus, the cytoskeleton or the endocytotic machinery. Recognition of the ubiquitin domain by a variety of enzymes and receptors is vital to the targeting function of ubiquitin. Several parallel pathways exist and these must be able to distinguish among ubiquitin, several different types of polymeric ubiquitin, and the various ubiquitin-like domains. Here we report the first molecular description of the binding site on ubiquitin for ubiquitin C-terminal hydrolase L3 (UCH-L3). The site on ubiquitin was experimentally determined using solution NMR, and site-directed mutagenesis. The site on UCH-L3 was modeled based on X-ray crystallography, multiple sequence alignments, and computer-aided docking. Basic residues located on ubiquitin (K6, K11, R72, and R74) are postulated to contact acidic residues on UCH-L3 (E10, E14, D33, E219). These putative interactions are testable and fully explain the selectivity of ubiquitin domain binding to this enzyme.

Precursor processing of pro-ISG15/UCRP, an interferon-beta-induced ubiquitin-like protein.

Induction of the 17-kDa ubiquitin-like protein ISG15/UCRP and its subsequent conjugation to cellular targets is the earliest response to type I interferons. The polypeptide is synthesized as a precursor containing a carboxyl-terminal extension whose correct processing is required for subsequent ligation of the exposed mature carboxyl terminus. Recombinant pro-ISG15 is processed in extracts of human lung fibroblasts by a constitutive 100-kDa enzyme whose activity is unaffected by type I interferon stimulation. The processing enzyme has been purified to apparent homogeneity by a combination of ion exchange and hydrophobic chromatography and found to be stimulated 12-fold by micromolar concentrations of ubiquitin. Analysis of the products of pro-ISG15 processing enzyme demonstrates specific cleavage exclusively at the Gly(157)-Gly(158) peptide bond to generate a mature ISG15 carboxyl terminus. Irreversible inhibition of pro-ISG15 processing activity by thiol-specific alkylating agents and a pH rate dependence conforming to titration of a single group of pK(a) 8.1 indicate the 100-kDa enzyme is a thiol protease. Partial sequencing of a trypsin-derived peptide indicates the enzyme is either the human ortholog of yeast Ubp1 or a Ubp1-related protein. As yeast do not contain ISG15, these results suggest that a ubiquitin-specific enzyme was recruited for pro-ISG15/UCRP processing by adaptive divergence.

An NMR analysis of the reaction of ubiquitin with [acetyl-1-13C]aspirin.

The acetylation of ubiquitin by [acetyl-1-13C]aspirin has been studied using 2D NMR methods. Studies performed in a 50:50 H2O:D2O medium show doubling of the acetyl carbonyl resonances, indicating that all of the stable adducts formed involved amide linkages. Assignment of the heteronuclear multiple quantum coherence (HMQC) resonances was accomplished based on comparison of resonance intensities with the results of an Edman degradation analysis, pH titration studies of acetylated ubiquitin, and analysis of two ubiquitin mutants, K33R and K63R. The presence of a single tyrosine residue in close proximity to lysine-48 suggested another assignment strategy. Nitration of tyrosine-59 resulted in a small, pH-dependent shift of the resonance assigned to lysine-48, with a pK of 7.0, close to that expected for the nitrotyrosyl hydroxyl group. An additional adduct resonance with very low intensity also was observed and tentatively assigned to the acetylated N-terminal methionine residue. The relative rates of acetylation of the various lysine residues were obtained from time-dependent HMQC studies. Since no sample preparation artifacts were introduced, the levels of modification of the various residues could be determined with relatively high accuracy. Based on the time-dependent intensity data, the relative rate constants for modification of K6, K48, K63, K11, K33, and M1 were 1.0, 0.59, 0.43, 0.26, 0.23, and 0.03, respectively. These results were in much better agreement with amino accessibility predictions based on the crystal structure of the ubiquitin monomer than with predictions based on the ubiquitin structure in the crystallized dimeric and tetrameric forms. This approach provides a useful basis for understanding how local environmental factors can influence protein adduct formation, as well as for comparing the extent and specificity of various acetylation reagents.

Neurite outgrowth in PC12 cells. Distinguishing the roles of ubiquitylation and ubiquitin-dependent proteolysis.

Nerve growth factor (NGF)-induced neurite outgrowth from rat PC12 cells was coincident with elevated (>/=2-fold) levels of endogenous ubiquitin (Ub) protein conjugates, elevated rates of formation of 125I-labeled Ub approximately E1 (Ub-activating enzyme) thiol esters and 125I-labeled Ub approximately E2 (Ub carrier protein) thiol esters in vitro, and enhanced capacity to synthesize 125I-labeled Ub-protein conjugates de novo. Activities of at least four E2s were increased in NGF-treated cells, including E2(14K), a component of the N-end rule pathway. Ubiquitylation of 125 I-labeled beta-lactoglobulin was up to 4-fold greater in supernatants from NGF-treated cells versus untreated cells and was selectively inhibited by the dipeptide Leu-Ala, an inhibitor of Ub isopeptide ligase (E3). However, Ub-dependent proteolysis of 125I-labeled beta-lactoglobulin was not increased in supernatants from NGF-treated cells, suggesting that neurite outgrowth is promoted by enhanced rates of synthesis (rather than degradation) of Ub-protein conjugates. Consistent with this observation, neurite outgrowth was induced by proteasome inhibitors (lactacystin and clasto-lactacystin beta-lactone) and was associated with elevated levels of ubiquitylated protein and stabilization of the Ub-dependent substrate, p53. Lactacystin-induced neurite outgrowth was blocked by the dipeptide Leu-Ala (2 mM) but not by His-Ala. These data 1) demonstrate that the enhanced pool of ubiquitylated protein observed during neuritogenesis in PC12 cells reflects coordinated up-regulation of Ub-conjugating activity, 2) suggest that Ub-dependent proteolysis is a negative regulator of neurite outgrowth in vitro, and 3) support a role for E2(14K)/E3-mediated protein ubiquitylation in PC12 cell neurite outgrowth.

Modeling of alcohol use mediates the effect of family history of alcoholism on adolescent alcohol expectancies.

The relative impact of biological family history of alcoholism and exposure to abusive parental drinking on alcohol effect expectancies of adolescent offspring were investigated in the present study. Exposure to familial models of alcohol abuse and biological family history were both predictive of positive alcohol effect expectancies of adolescent offspring. Degree of exposure to an alcohol-abusing family member mediated the relationship between biological family history of alcoholism and adolescent alcohol outcome expectancies. These results support prior findings of expectancy differences between youths with and without a family background of alcoholism and provide evidence supporting the significance of family modeling influences in the development of adolescents' alcohol expectancies.

Serum nucleotide pyrophosphohydrolase activity; elevated levels in osteoarthritis, calcium pyrophosphate crystal deposition disease, scleroderma, and fibromyalgia.

OBJECTIVE: Quantification of serum nucleotide pyrophosphohydrolase (NTPPHase) activity in healthy subjects and in patients with various rheumatic diseases or with quad/hemiplegia, hemodialysis, or renal transplant. METHODS: Colorimetric assay of enzyme activity in serum. RESULTS: Serum NTPPHase activity in 85 healthy subjects was independent of age or sex and was highly reproducible in each individual. The biologic and methodologic coefficients of variation were nearly identical. Elevated enzyme levels were found in sera from patients with osteoarthritis/spondylosis, calcium pyrophosphate dihydrate (CPPD) crystal deposition, scleroderma, fibromyalgia, or hemodialysis. Renal transplant patients receiving cyclosporine had the highest enzyme activity of any group, whereas transplant patients not taking this drug had normal levels. Histograms of values in all groups showed a normal distribution. CONCLUSION: Serum NTPPHase activity levels were significantly elevated in patients with degenerative arthritis whether or not CPPD crystals were present, in patients with either scleroderma or fibromyalgia, and in patients receiving hemodialysis therapy or taking cyclosporine.

Defects in the ubiquitin pathway induce caspase-independent apoptosis blocked by Bcl-2.

Apoptosis requires the activation of caspases (formerly interleukin 1beta-converting enzyme-like proteases), in particular those related to the caspase-3/7/6 subfamily. Recent data, however, revealed that, although caspase-specific inhibitors delay apoptosis, they are often incapable of preventing it. To obtain evidence for caspase-independent steps of apoptosis, we artificially created a high amount of short-lived or aberrant proteins by blocking the ubiquitin degradation pathway. A temperature-sensitive defect in the ubiquitin-activating enzyme E1 induced apoptosis independent of the activation of caspase-3 and -6 and the cleavage of their respective substrates poly(ADP-ribose) polymerase and lamin A. In addition, neither the caspase 3/7-specific inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone nor the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone were capable of blocking this type of cell death. By contrast, Bcl-2 overexpression effectively protected cells from apoptosis induced by a defect in the E1 enzyme at the nonpermissive temperature. Bcl-2 acted downstream of the accumulation of short-lived or aberrant proteins because it did not prevent the overexpression of the short-lived proteins p53, p27(kip1), and cyclins D1 and B1 under conditions of decreased ubiquitination. These results suggest the existence of short-lived proteins that may serve the role of caspase-independent effectors of apoptosis and attractive targets of the death-protective action of Bcl-2.

Pathways of ubiquitin conjugation.

The covalent attachment of the polypeptide ubiquitin to proteins marks them for degradation by the ubiquitin/26S proteasome-dependent degradation pathway. This pathway functions in regulating many fundamental processes required for cell viability. Phylogenetic analysis of ubiquitin sequences reveals greater variability among lower eukaryotes and defines essential residues, many of which are conserved among the three ubiquitin-like proteins known to undergo parallel ligation pathways. The hierarchical design of the ubiquitin conjugation mechanism provides great flexibility for the divergent evolution of new functions mediated by this posttranslational modification. Within this hierarchy, a single ubiquitin-activating enzyme provides charged intermediates to multiple targeting pathways defined by cognate ubiquitin carrier protein (E2)/ligase (E3) pairs. Sequence analysis of E2 isozymes shows that the E2 superfamily is composed of distinct function-specific families. The apparent lack of E2/E3 specificity suggested in the literature results from the presence of multiple isozymes within many E2 families and erroneous family assignments based on incomplete data sets. Other apparent inconsistencies are explained by interfamily sequence relationships among some E2 isoforms.