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Kuo-Liang Su Ya-Fan Liao Hui-Chih Hung Guang-Yaw Liu 《The Journal of biological chemistry》2009,284(39):26768-26777
Ornithine decarboxylase (ODC) is the first enzyme involved in polyamine biosynthesis, and it catalyzes the decarboxylation of ornithine to putrescine. ODC is a dimeric enzyme, whereas antizyme inhibitor (AZI), a positive regulator of ODC that is homologous to ODC, exists predominantly as a monomer and lacks decarboxylase activity. The goal of this paper was to identify the essential amino acid residues that determine the dimerization of AZI. The nonconserved amino acid residues in the putative dimer interface of AZI (Ser-277, Ser-331, Glu-332, and Asp-389) were substituted with the corresponding residues in the putative dimer interface of ODC (Arg-277, Tyr-331, Asp-332, and Tyr-389, respectively). Analytical ultracentrifugation analysis was used to determine the size distribution of these AZI mutants. The size-distribution analysis data suggest that residue 331 may play a major role in the dimerization of AZI. Mutating Ser-331 to Tyr in AZI (AZI-S331Y) caused a shift from a monomer configuration to a dimer. Furthermore, in comparison with the single mutant AZI-S331Y, the AZI-S331Y/D389Y double mutant displayed a further reduction in the monomer-dimer Kd, suggesting that residue 389 is also crucial for AZI dimerization. Analysis of the triple mutant AZI-S331Y/D389Y/S277R showed that it formed a stable dimer (Kd value = 1.3 μm). Finally, a quadruple mutant, S331Y/D389Y/S277R/E332D, behaved as a dimer with a Kd value of ∼0.1 μm, which is very close to that of the human ODC enzyme. The quadruple mutant, although forming a dimer, could still be disrupted by antizyme (AZ), further forming a heterodimer, and it could rescue the AZ-inhibited ODC activity, suggesting that the AZ-binding ability of the AZI dimer was retained.Polyamines (putrescine, spermidine, and spermine) have been shown to have both structural and regulatory roles in protein and nucleic acid biosynthesis and function (1–3). Ornithine decarboxylase (ODC,3 EC 4.1.1.17) is a central regulator of cellular polyamine synthesis (reviewed in Refs. 1, 4, 5). This enzyme catalyzes the pyridoxal 5-phosphate (PLP)-dependent decarboxylation of ornithine to putrescine, and it is the first and rate-limiting enzyme in polyamine biosynthesis (2, 3, 6, 7). ODC and polyamines play important roles in a number of biological functions, including embryonic development, cell cycle, proliferation, differentiation, and apoptosis (8–15). They also have been associated with human diseases and a variety of cancers (16–26). Because the regulation of ODC and polyamine content is critical to cell proliferation (11), as well as in the origin and progression of neoplastic diseases (23, 24), ODC has been identified as an oncogenic enzyme, and the inhibitors of ODC and the polyamine pathway are important targets for therapeutic intervention in many cancers (6, 11).ODC is ubiquitously found in organisms ranging from bacteria to humans. It contains 461 amino acid residues in each monomer and is a 106-kDa homodimer with molecular 2-fold symmetry (27, 28). Importantly, ODC activity requires the formation of a dimer (29–31). X-ray structures of the ODC enzyme reveal that this dimer contains two active sites, both of which are formed at the interface between the N-terminal domain of one monomer, which provides residues involved in PLP interactions, and the C-terminal domain of the other subunit, which provides the residues that interact with substrate (27, 32–41).ODC undergoes a unique ubiquitin-independent proteasomal degradation via a direct interaction with the regulatory protein antizyme (AZ). Binding of AZ promotes the dissociation of the ODC homodimers and targets ODC for degradation by the 26 S proteasome (42–46). Current models of antizyme function indicate that increased polyamine levels promote the fidelity of the AZ mRNA translational frameshift, leading to increased concentrations of AZ (47). The AZ monomer selectively binds to dimeric ODC, thereby inactivating ODC by forming inactive AZ-ODC heterodimers (44, 48–50). AZ acts as a regulator of polyamine metabolism that inhibits ODC activity and polyamine transport, thus restricting polyamine levels (4, 5, 51, 52). When antizymes are overexpressed, they inhibit ODC and promote ubiquitin-independent proteolytic degradation of ODC. Because elevated ODC activity is associated with most forms of human malignancies (1), it has been suggested that antizymes may function as tumor suppressors.In contrast to the extensive studies on the oncogene ODC, the endogenous antizyme inhibitor (AZI) is less well understood. AZI is homologous to the enzyme ODC. It is a 448-amino acid protein with a molecular mass of 50 kDa. However, despite the homology between these proteins, AZI does not possess any decarboxylase activity. It binds to antizyme more tightly than does ODC and releases ODC from the ODC-antizyme complex (53, 54). Both the AZI and AZ proteins display rapid ubiquitin-dependent turnover within a few minutes to 1 h in vivo (5). However, AZ binding actually stabilizes AZI by inhibiting its ubiquitination (55).AZI, which inactivates all members of the AZ family (53, 56), restores ODC activity (54), and prevents the proteolytic degradation of ODC, may play a role in tumor progression. It has been reported that down-regulation of AZI is associated with the inhibition of cell proliferation and reduced ODC activity, presumably through the modulation of AZ function (57). Moreover, overexpression of AZI has been shown to increase cell proliferation and promote cell transformation (58–60). Furthermore, AZI is capable of direct interaction with cyclin D1, preventing its degradation, and this effect is at least partially independent of AZ function (60, 61). These results demonstrate a role for AZI in the positive regulation of cell proliferation and tumorigenesis.It is now known that ODC exists as a dimer and that AZI may exist as a monomer physiologically (62). Fig. 1 shows the dimeric structures of ODC (Fig. 1A) and AZI (Fig. 1B). Although structural studies indicate that both ODC and AZI crystallize as dimers, the dimeric AZI structure has fewer interactions at the dimer interface, a smaller buried surface area, and a lack of symmetry of the interactions between residues from the two monomers, suggesting that the AZI dimer may be nonphysiological (62). In this study, we identify the critical amino acid residues governing the difference in dimer formation between ODC and AZI. Our preliminary studies using analytical ultracentrifugation indicated that ODC exists as a dimer, whereas AZI exists in a concentration-dependent monomer-dimer equilibrium. Multiple sequence alignments of ODC and AZI from various species have shown that residues 277, 331, 332, and 389 are not conserved between ODC and AZI (Open in a separate windowFIGURE 1.Crystal structure and the amino acid residues at the dimer interface of human ornithine decarboxylase (hODC) and mouse antizyme inhibitor (mAZI). A, homodimeric structure of human ODC with the cofactor PLP analog, LLP (Protein Data Bank code 1D7K). B, putative dimeric structure of mouse AZI (Protein Data Bank code 3BTN). The amino acid residues in the dimer interface are shown as a ball-and-stick model. The putative AZ-binding site is colored in cyan. This figure was generated using PyMOL (DeLano Scientific LLC, San Carlos, CA).
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TABLE 1
Amino acid residues at the dimer interface of human ODC and AZIHuman ODC | Residue | Human AZI |
---|---|---|
Nonconserved | ||
Arg | 277 | Ser |
Tyr | 331 | Ser |
Asp | 332 | Glu |
Tyr | 389 | Asp |
Conserved | ||
Asp | 134 | Asp |
Lys | 169 | Lys |
Lys | 294 | Lys |
Tyr | 323 | Tyr |
Asp | 364 | Asp |
Gly | 387 | Gly |
Phe | 397 | Phe |
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Raheleh Masoudi Maria S. Ioannou Michael D. Coughlin Promila Pagadala Kenneth E. Neet Oliver Clewes Shelley J. Allen David Dawbarn Margaret Fahnestock 《The Journal of biological chemistry》2009,284(27):18424-18433
Nerve growth factor (NGF) is produced as a precursor called pro-nerve growth factor (proNGF), which is secreted by many tissues and is the predominant form of NGF in the central nervous system. In Alzheimer disease brain, cholinergic neurons degenerate and can no longer transport NGF as efficiently, leading to an increase in untransported NGF in the target tissue. The protein that accumulates in the target tissue is proNGF, not the mature form. The role of this precursor is controversial, and both neurotrophic and apoptotic activities have been reported for recombinant proNGFs. Differences in the protein structures, protein expression systems, methods used for protein purification, and methods used for bioassay may affect the activity of these proteins. Here, we show that proNGF is neurotrophic regardless of mutations or tags, and no matter how it is purified or in which system it is expressed. However, although proNGF is neurotrophic under our assay conditions for primary sympathetic neurons and for pheochromocytoma (PC12) cells, it is apoptotic for unprimed PC12 cells when they are deprived of serum. The ratio of tropomyosin-related kinase A to p75 neurotrophin receptor is low in unprimed PC12 cells compared with primed PC12 cells and sympathetic neurons, altering the balance of proNGF-induced signaling to favor apoptosis. We conclude that the relative level of proNGF receptors determines whether this precursor exhibits neurotrophic or apoptotic activity.Nerve growth factor (NGF)3 regulates neuronal survival, neurite outgrowth, and differentiation in the peripheral and central nervous systems (1). The mature form of NGF forms a non-covalent homodimer and binds with high affinity (kd ≈ 10−11 m) to tropomyosin-related kinase A (TrkA) and with low affinity (kd ≈ 10−9 m) to the common neurotrophin receptor p75NTR (p75 neurotrophin receptor) (2). NGF promotes cell survival and growth in cells expressing TrkA through activation of the phosphatidylinositol 3-kinase/AKT pathway and the Ras/mitogen-activated protein kinase (MAPK) pathway (3, 4). p75NTR plays diverse roles, ranging from cell survival to cell death depending on the cellular context in which it is expressed. Through activation of the NF-κB pathway, p75NTR can contribute to cell survival in sensory neurons (5), it is involved in axonal growth via regulation of Rho activity (6), and it can interact with Trks to enhance neurotrophin affinity (at low concentration of ligand) and specificity of binding to Trks (7–9). High levels of p75NTR expression can induce apoptosis when there are low levels of Trk or when Trk is absent (10, 11). Apoptosis occurs through increased ceramide production (12), activation of c-Jun N-terminal kinase (JNK1), and p53 (10, 13). p75NTR requires a co-receptor called sortilin to induce cell death (14).NGF is produced as a precursor called pro-nerve growth factor (proNGF) (15). ProNGF is secreted by many tissues such as prostate cells, spermatids, hair follicles, oral mucosal keratinocytes, sympathetic neurons, cortical astrocytes, heart, and spleen (16–20). ProNGF is the predominant form of NGF in the central and peripheral nervous systems, whereas little or no mature NGF can be detected (21–24). In Alzheimer disease brain, retrograde transport from the cortex and hippocampus to basal forebrain cholinergic neurons is reduced as these neurons degenerate, with concomitant proNGF accumulation in the cortex and hippocampus (21, 23). This suggested that proNGF mediates biological activity besides its prodomain function of promoting protein folding and regulation of neurotrophin secretion (25–28). To study the role of proNGF protein in vitro, point mutations were inserted at the cleavage site used by furin, a proprotein convertase known to cleave proNGF (29), to minimize the conversion of proNGF to mature NGF. The resulting recombinant, cleavage-resistant proNGFs reportedly exhibit either apoptotic activity (30, 31) or neurotrophic activity (32, 33). These recombinant proteins differ in several ways (ProNGF(R−1G) ProNGFhis ProNGFE ProNGF123 WT-NGFhis Mutations −1 (R to G) −2 and −1 (RR to AA), 118 and 119 (RR to AA) −1 and +1 (RS to AA) −73 and −72 (RR to AA), −43 and −42 (KKRR to KAAR), −2 and −1 (KR to AA) None: cleavable proNGF Tag No tag Histidine tag No tag No tag Histidine tag Expression system Insect cells Insect cells, mammalian cells Bacteria Insect cells Insect cells, mammalian cells Purification No purification Nickel column Refolded from inclusion bodies, FPLC Cation exchange chromatography, immunoaffinity chromatography Nickel column