The activation of the decapping enzyme DCP2 by DCP1 occurs on the EDC4 scaffold and involves a conserved loop in DCP1

The removal of the 5′-cap structure by the decapping enzyme DCP2 and its coactivator DCP1 shuts down translation and exposes the mRNA to 5′-to-3′ exonucleolytic degradation by XRN1. Although yeast DCP1 and DCP2 directly interact, an additional factor, EDC4, promotes DCP1–DCP2 association in metazoan. Here, we elucidate how the human proteins interact to assemble an active decapping complex and how decapped mRNAs are handed over to XRN1. We show that EDC4 serves as a scaffold for complex assembly, providing binding sites for DCP1, DCP2 and XRN1. DCP2 and XRN1 bind simultaneously to the EDC4 C-terminal domain through short linear motifs (SLiMs). Additionally, DCP1 and DCP2 form direct but weak interactions that are facilitated by EDC4. Mutational and functional studies indicate that the docking of DCP1 and DCP2 on the EDC4 scaffold is a critical step for mRNA decapping in vivo. They also revealed a crucial role for a conserved asparagine–arginine containing loop (the NR-loop) in the DCP1 EVH1 domain in DCP2 activation. Our data indicate that DCP2 activation by DCP1 occurs preferentially on the EDC4 scaffold, which may serve to couple DCP2 activation by DCP1 with 5′-to-3′ mRNA degradation by XRN1 in human cells.     

Degradation of ornithine decarboxylase in reticulocyte lysate is ATP-dependent but ubiquitin-independent

Reticulocyte lysate contains all the components of the ubiquitin-dependent proteolytic system. Several proteins are degraded in reticulocyte lysate in a ubiquitin-dependent manner. However, none of the proteins studied has a short intracellular half-life. We have investigated the degradation of ornithine decarboxylase (ODC), one of the most labile proteins in mammalian cells. ODC is efficiently degraded in reticulocyte lysate depleted of the ubiquitin activating enzyme, E1, in fraction II of reticulocyte lysate completely lacking ubiquitin, and in fraction II depleted of the entire complex of enzymes responsible for the ligation of ubiquitin to target proteins. The degradation of ODC is ATP dependent. Therefore, our results demonstrate that in addition to the ubiquitin-dependent proteolytic pathway, reticulocyte lysate contains at least one additional ATP-dependent proteolytic pathway. In vitro synthesized ODC served as a substrate in the present degradation study. Its successful utilization establishes a general strategy for investigating the degradation of short-lived proteins (for which a corresponding cDNA is available), that constitute a very small fraction of cellular proteins and for which purification is difficult or impossible. In contrast to ODC synthesized in vitro, that isolated from cells was not degraded by the reticulocyte lysate degradation system, suggesting that post-translational modifications may be involved in regulating ODC degradation.     

Characterization of sequences involved in mediating degradation of ornithine decarboxylase in cells and in reticulocyte lysate

Mouse ornithine decarboxylase is a 461-amino-acid protein that is extremely labile. A set of contiguous in-frame deletions were introduced into its C-terminal hydrophilic region. The resulting mutant proteins were expressed in cos monkey cells using an expression vector based on simian virus 40 (SV40) or by in vitro translation in reticulocyte lysate. The degradation of wild-type and mutant proteins was determined in transfected cos cells and in a degradation system based on reticulocyte lysate. Deletion mutants lacking segments of the C-terminus (amino acids 423-461, 423-435, 436-449 and 449-461) were converted into stable proteins in both experimental systems. The mutant lacking amino acids 295-309 was significantly stabilized in transfected cos cells, but was rapidly degraded in reticulocyte-lysate-based degradation mix. Our results suggest that the carboxyl-terminal region encompassing amino acids 423-461 and perhaps also amino acids 295-309 may constitute a signal recognized by the proteolytic machinery that degrades ornithine decarboxylase.     

A novel founder mutation in the RNASEL gene, 471delAAAG,is associated with prostate cancer in Ashkenazi Jews

HPC1/RNASEL was recently identified as a candidate gene for hereditary prostate cancer. We identified a novel founder frameshift mutation in RNASEL, 471delAAAG, in Ashkenazi Jews. The mutation frequency in the Ashkenazi population, estimated on the basis of the frequency in 150 healthy young women, was 4% (95% confidence interval [CI] 1.9%-8.4%). Among Ashkenazi Jews, the mutation frequency was higher in patients with prostate cancer (PRCA) than in elderly male control individuals (6.9% vs. 2.4%; odds ratio = 3.0; 95% CI 0.6-15.3; P=.17). 471delAAAG was not detected in the 134 non-Ashkenazi patients with PRCA and control individuals tested. The median age at PRCA diagnosis did not differ significantly between the Ashkenazi carriers and noncarriers included in our study. However, carriers received diagnoses at a significantly earlier age, compared with patients with PRCA who were registered in the Israeli National Cancer Registry (65 vs. 74.4 years, respectively; P<.001). When we examined two brothers with PRCA, we found a heterozygous 471delAAAG mutation in one and a homozygous mutation in the other. Loss of heterozygosity was demonstrated in the tumor of the heterozygous sib. Taken together, these data suggest that the 471delAAAG null mutation is associated with PRCA in Ashkenazi men. However, additional studies are required to determine whether this mutation confers increased risk for PRCA in this population.     

Autosomal-Recessive Hypophosphatemic Rickets Is Associated with an Inactivation Mutation in the ENPP1 Gene

Human disorders of phosphate (Pi) handling and hypophosphatemic rickets have been shown to result from mutations in PHEX, FGF23, and DMP1, presenting as X-linked recessive, autosomal-dominant, and autosomal-recessive patterns, respectively. We present the identification of an inactivating mutation in the ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene causing autosomal-recessive hypophosphatemic rickets (ARHR) with phosphaturia by positional cloning. ENPP1 generates inorganic pyrophosphate (PPi), an essential physiologic inhibitor of calcification, and previously described inactivating mutations in this gene were shown to cause aberrant ectopic calcification disorders, whereas no aberrant calcifications were present in our patients. Our surprising result suggests a different pathway involved in the generation of ARHR and possible additional functions for ENPP1.     

20S proteasomal degradation of ornithine decarboxylase is regulated by NQO1

Ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines, is a very labile protein. ODC is a homodimeric enzyme that undergoes ubiquitin-independent proteasomal degradation via direct interaction with antizyme, a polyamine-induced protein. Binding of antizyme promotes the dissociation of ODC homodimers and marks ODC for degradation by the 26S proteasomes. We describe here an alternative pathway for ODC degradation that is regulated by NAD(P)H quinone oxidoreductase 1 (NQO1). We show that NQO1 binds and stabilizes ODC. Dicoumarol, an inhibitor of NQO1, dissociates ODC-NQO1 interaction and enhances ubiquitin-independent ODC proteasomal degradation. We further show that dicoumarol sensitizes ODC monomers to proteasomal degradation in an antizyme-independent manner. This process of NQO1-regulated ODC degradation was recapitulated in vitro by using purified 20S proteasomes. Finally, we show that the regulation of ODC stability by NQO1 is especially prominent under oxidative stress. Our findings assign to NQO1 a role in regulating ubiquitin-independent degradation of ODC by the 20S proteasomes.     

Protein interactions within the TcZFP zinc finger family members of Trypanosoma cruzi: implications for their functions

The small zinc finger proteins tbZFP1 and tbZFP2 have been implicated in the control of Trypanosoma brucei differentiation to the procyclic form. Here, we report that the complete ZFP family in Trypanosoma cruzi is composed by four members, ZFP1A and B, and ZFP2A and B. ZFP1B is a paralog specific gene restricted to T. cruzi, while the ZFP2A and B paralogs diverged prior to the trypanosomatid lineage separation. Moreover, we demonstrate that TcZFP1 and TcZFP2 members interact with each other and that this interaction is mediated by a WW domain in TcZFP2. Also, TcZFP2B strongly homodimerizes by a glycine rich region absent in TcZFP2A. We propose a model to discuss the relevance of these protein-protein interactions in terms of the functions of these proteins.     

A deleterious mutation in SAMD9 causes normophosphatemic familial tumoral calcinosis

Familial tumoral calcinosis (FTC) is a rare autosomal recessive disorder characterized by the progressive deposition of calcified masses in cutaneous and subcutaneous tissues, which results in painful ulcerative lesions and severe skin and bone infections. Two major types of FTC have been recognized: hyperphosphatemic FTC (HFTC) and normophosphatemic FTC (NFTC). HFTC was recently shown to result from mutations in two different genes: GALNT3, which codes for a glycosyltransferase, and FGF23, which codes for a potent phosphaturic protein. To determine the molecular cause of NFTC, we performed homozygosity mapping in five affected families of Jewish Yemenite origin and mapped NFTC to 7q21-7q21.3. Mutation analysis revealed a homozygous mutation in the SAMD9 gene (K1495E), which was found to segregate with the disease in all families and to interfere with the protein expression. Our data suggest that SAMD9 is involved in the regulation of extraosseous calcification, a process of considerable importance in a wide range of diseases as common as atherosclerosis and autoimmune disorders.