The propeptide of the transforming growth factor-beta superfamily member, macrophage inhibitory cytokine-1 (MIC-1), is a multifunctional domain that can facilitate protein folding and secretion

Macrophage inhibitory cytokine-1 (MIC-1) is a divergent member of the transforming growth factor-beta (TGF-beta) superfamily. While it is synthesized in a pre-pro form, it is unique among superfamily members because it does not require its propeptide for correct folding or secretion of the mature peptide. To investigate factors that enable these propeptide independent events to occur, we constructed MIC-1/TGF-beta1 chimeras, both with and without a propeptide. All chimeras without a propeptide secreted less efficiently compared with the corresponding constructs with propeptide. Folding and secretion were most affected after replacement of the predicted major alpha-helix in the mature protein, residues 56-68. Exchanging the human propeptide in this chimera with either the murine MIC-1 or TGF-beta1 propeptide resulted in secretion of the unprocessed, monomeric chimera, suggesting a specific interaction between the human MIC-1 propeptide and mature peptide. Propeptide deletion mutants enabled identification of a region between residues 56 and 78, which is important for the interaction between the propeptide and the mature peptide. Cotransfection experiments demonstrated that the propeptide must be in cis with the mature peptide for this phenomenon to occur. These results suggest a model for TGF-beta superfamily protein folding.     

Antibody-based approach to high-volume genotyping for MIC-1 polymorphism

Macrophage inhibitory cytokine-1 (MIC-1) is a divergent member of the TGF-beta superfamily. There are at least two known alleles of MIC-1 that are due to a G-->C point substitution at position 6 of the mature protein, which alters a histidine to an aspartic acid (MIC-1 H and MIC-1 D). We have determined the phenotype of MIC-1 circulating in serum by exploiting the differences in the affinity of the two monoclonal antibodies to the H and D alleles of MIC-1. A PCR-RFLP-based method for genotyping MIC-1 is also described. We validate these two assays using DNA sequencing of 19 subjects as the standard. We then used the validated assay to determine the frequency of the two MIC-1 alleles in a population of 261 adult blood donors. Inter-assay and sequencing concordance was 100%. The frequency of the three common MIC-1 genotypes was homozygous (HH), 54%; heterozygous (HD), 39%; and homozygous (DD), 7%. This novel antibody-based assay confidently determines the genotype of MIC-1. It offers the advantages of an ELISA-ease of automation, high-volume throughput of samples, and ease of use in a routine, clinical laboratory.     

Expression of a novel member of the TGF-β superfamily, growth/differentiation factor-15/macrophage-inhibiting cytokine-1 (GDF-15/MIC-1) in adult rat tissues

We have cloned a novel member of the transforming growth factor-β (TGF-β) superfamily from a human placental cDNA library. The sequence is identical to five very recently published sequences, of which only one (macrophage inhibitory cytokine-1, MIC-1) has been characterized in terms of function. In light of the present data demonstrating the wide distribution of the mRNA and putative multifunctionality, we propose to name this molecule growth/differentiation factor-15/MIC-1 (GDF-15/MIC-1). The deduced amino acid sequence reveals typical features of a secreted molecule. The epithelium of the choroid plexus is the only site in the adult brain expressing detectable levels of GDF-15/MIC-1 mRNA. Many epithelia of non-neural tissues including those of the prostate and intestinal mucosa, bronchi and bronchioli, secretory tubuli of the submandibular gland, and lactating mammary gland are prominent sites of GDF-15/MIC-1 synthesis. GDF-15/MIC-1 is also strongly expressed by macrophages in the adrenal gland. Thus, GDF-15/MIC-1, like many other members of the TGF-β superfamily, is widely distributed in adult tissues, being most strongly expressed in epithelial cells and macrophages. Received: 25 January 1999 / Accepted: 17 March 1999     

ER stress induces alternative nonproteasomal degradation of ER proteins but not of cytosolic ones

Inhibition of protein folding in the endoplasmic reticulum (ER) causes ER stress, which triggers the unfolded protein response (UPR). To decrease the biosynthetic burden on the ER, the UPR inhibits in its initial stages protein synthesis. At later stages it upregulates components of ER-associated degradation (ERAD) and of the ubiquitin/proteasome system, which targets ER as well as cytosolic proteins for disposal. Here we report that, at later stages, the UPR also activates an alternative nonproteasomal pathway of degradation, which is resistant to proteasome inhibitors and is specific for ER substrates (assessed with uncleaved precursor of asialoglycoprotein receptor H2a and unassembled CD3delta) and not for cytosolic ones (p53). To mimic the initial inhibition of translation during UPR, we incubated cells with cycloheximide. After this treatment, degradation of ERAD substrates was no longer effected by proteasomal inhibition, similarly to the observed outcome of UPR. The degradation also became insensitive to abrogation of ubiquitination in a cell line carrying a thermosensitive E1 ubiquitin activating enzyme mutant. Of all protease inhibitors tested, only the metal chelator o-phenanthroline could block this nonproteasomal degradation. Preincubation of o-phenanthroline with Mn2+ or Co2+, but not with other cations, reversed the inhibition. Our results suggest that, upon inhibition of translation, an alternative nonproteasomal pathway is activated for degradation of proteins from the ER. This involves a Mn2+/Co2+-dependent metalloprotease or other metalloprotein. The alternative pathway selectively targets ERAD substrates to reduce the ER burden, but does not affect p53, the levels of which remain dependent on proteasomal control.     

TGF-b Superfamily Cytokine MIC-1/GDF15 Is a Physiological Appetite and Body Weight Regulator

The TGF-b superfamily cytokine MIC-1/GDF15 circulates in all humans and when overproduced in cancer leads to anorexia/cachexia, by direct action on brain feeding centres. In these studies we have examined the role of physiologically relevant levels of MIC-1/GDF15 in the regulation of appetite, body weight and basal metabolic rate. MIC-1/GDF15 gene knockout mice (MIC-1^−/−) weighed more and had increased adiposity, which was associated with increased spontaneous food intake. Female MIC-1^−/− mice exhibited some additional alterations in reduced basal energy expenditure and physical activity, possibly owing to the associated decrease in total lean mass. Further, infusion of human recombinant MIC-1/GDF15 sufficient to raise serum levels in MIC-1^−/− mice to within the normal human range reduced body weight and food intake. Taken together, our findings suggest that MIC-1/GDF15 is involved in the physiological regulation of appetite and energy storage.     

Phenylenediamine derivatives induce GDF-15/MIC-1 and inhibit adipocyte differentiation of mouse 3T3-L1 cells

Phenylenediamine derivatives can function as a hydrogen donor and reportedly exert various biological actions including cytoprotective effects against oxidative stress, possibly by acting as an antioxidant. Previous studies showed that feeding of such compounds to mice reduced their body weight, but the precise mechanism remains unknown at present. Here, we found that these compounds inhibited the in vitro differentiation of mouse preadipocytes, 3T3-L1 cells, into adipocytes, suggesting that, at least in part, reduced generation of adipocytes might contribute to the observed weight loss in mice. Next, we performed array analysis and found that the expression of GDF-15/MIC-1, which is a TGFβ superfamily cytokine, and Trib 3, an intracellular downstream effector of the cytokines, was up-regulated by these derivatives. Thus, we identified the compounds as inducers of GDF-15/MIC-1 and suggest that such induction may have led to inhibition of adipocyte differentiation, which could account for the weight-loss effect of these compounds.     

Latent TGF-beta binding proteins

Latent TGF-beta binding proteins are multidomain proteins with a common, highly repetitive structural organization and partially overlapping expression patterns. Latent TGF-beta binding protein-1, -3 and -4 bind latent TGF-beta. TGF-betas are normally secreted as latent complexes, consisting of the mature TGF-beta dimer non-covalently bound to its processed propeptide dimer plus a latent TGF-beta binding protein. The latent TGF-beta binding protein is covalently bound to the propeptide. These binding proteins may perform at least two functions: structural, as components of the matrix, and regulatory, as modulators of TGF-beta availability.     

Epitope mapping of the transforming growth factor-beta superfamily protein, macrophage inhibitory cytokine-1 (MIC-1): identification of at least five distinct epitope specificities

Macrophage inhibitory cytokine-1 (MIC-1) is a divergent member of the transforming growth factor-beta (TGF-beta) superfamily whose increased expression is associated with macrophage activation and which is expressed highly in placenta as compared to other tissues. There are two known allelic forms of human MIC-1 due an amino acid substitution at position 6 of the mature protein. We have raised four monoclonal antibodies (MAbs) and one polyclonal antiserum to the mature protein region of human MIC-1 and have used an extensive panel of MIC-1 relatives, mutants, and chimeras to map their epitopes. None of the MAbs were able to cross-react with either the murine homologue of MIC-1 or with hTGF-beta1, and all of the MAb epitopes were conformation-dependent. A distinct cross-reactivity pattern with the various antigens was observed for each of the monoclonal and polyclonal antibodies suggesting the presence of at least five immunogenic regions on the MIC-1 surface. One of the MAbs is directed against the amino terminus of the protein and can distinguish between the two allelic forms of MIC-1. The epitopes for the other three MAbs were located near the tips of the so-called "fingers" of the protein and appeared to be partially overlapping as each involved amino acids in the region 24-37. In one case, it was possible to mutate murine MIC-1 so that it could be recognized by one of the MAbs. Finally, the use of another mutant in which Cys 77 was replaced by serine enabled confirmation of the location of the MIC-1 interchain disulfide bond.     

beta-Subunit appendages promote 20S proteasome assembly by overcoming an Ump1-dependent checkpoint

Proteasomes are responsible for most intracellular protein degradation in eukaryotes. The 20S proteasome comprises a dyad-symmetric stack of four heptameric rings made from 14 distinct subunits. How it assembles is not understood. Most subunits in the central pair of beta-subunit rings are synthesized in precursor form. Normally, the beta5 (Doa3) propeptide is essential for yeast proteasome biogenesis, but overproduction of beta7 (Pre4) bypasses this requirement. Bypass depends on a unique beta7 extension, which contacts the opposing beta ring. The resulting proteasomes appear normal but assemble inefficiently, facilitating identification of assembly intermediates. Assembly occurs stepwise into precursor dimers, and intermediates contain the Ump1 assembly factor and a novel complex, Pba1-Pba2. beta7 incorporation occurs late and is closely linked to the association of two half-proteasomes. We propose that dimerization is normally driven by the beta5 propeptide, an intramolecular chaperone, but beta7 addition overcomes an Ump1-dependent assembly checkpoint and stabilizes the precursor dimer.     

Oligosaccharide trimming plays a role in the endoplasmic reticulum-associated degradation of tyrosinase

The effect of glucosidase and mannosidase inhibitors on the ER-associated degradation of tyrosinase was assessed in transiently transfected COS-7 cells. We found that the glucosidase inhibitors castanospermine and deoxynojirimycin had very little effect on tyrosinase degradation, whereas the mannosidase inhibitors deoxymannojirimycin and kifunensine significantly delayed the rate of tyrosinase degradation as measured by pulse-chase analysis. In addition, we show that tyrosinase degradation is sensitive to the proteasome inhibitor lactacystin and that tyrosinase associates with endogenous calnexin in COS-7 cells. Our data support a model of tyrosinase degradation that involves mannose trimming, calnexin association, and the retrograde transport of tyrosinase from the ER to the cytosol for proteasomal degradation. The pathways of tyrosinase degradation have important ramifications with regard to the exact types of antigenic epitopes that are presented to the immune system.     

Synphilin-1 degradation by the ubiquitin-proteasome pathway and effects on cell survival

Parkinson's disease is characterized by loss of nigral dopaminergic neurons and the presence of cytoplasmic inclusions known as Lewy bodies. alpha-Synuclein and its interacting partner synphilin-1 are among constituent proteins in these aggregates. The presence of ubiquitin and proteasome subunits in these inclusions supports a role for this protein degradation pathway in the processing of proteins involved in this disease. To begin elucidating the kinetics of synphilin-1 in cells, we studied its degradation pathway in HEK293 cells that had been engineered to stably express FLAG-tagged synphilin-1. Pulse-chase experiments revealed that this protein is relatively stable with a half-life of about 16 h. Treatment with proteasome inhibitors resulted in attenuation of degradation and the accumulation of high molecular weight ubiquitinated synphilin-1 in immunoprecipitation/immunoblot experiments. Additionally, proteasome inhibitors stimulated the formation of peri-nuclear inclusions which were immunoreactive for synphilin-1, ubiquitin and alpha-synuclein. Cell viability studies revealed increased susceptibility of synphilin-1 over-expressing cells to proteasomal dysfunction. These observations indicate that synphilin-1 is ubiquitinated and degraded by the proteasome. Accumulation of ubiquitinated synphilin-1 due to impaired clearance results in its aggregation as peri-nuclear inclusions and in poor cell survival.     

Transforming growth factor-beta differentially inhibits MyD88-dependent, but not TRAM- and TRIF-dependent, lipopolysaccharide-induced TLR4 signaling

Transforming growth factor-beta1 (TGF-beta1) is a multifunctional, potent anti-inflammatory cytokine produced by many cell types that regulates cell proliferation, apoptosis, and immune responses. Toll-like receptors (TLRs) recognize various pathogen-associated molecular patterns and are therefore a pivotal component of the innate immune system. In this study we show that TGF-beta1 blocks the NF-kappaB activation and cytokine release that is stimulated by ligands for TLRs 2, 4, and 5. We further show that TGF-beta1 can specifically interfere with TLR2, -4, or -5 ligand-induced responses involving the adaptor molecule MyD88 (myeloid differentiation factor 88) but not the TRAM/TRIF signaling pathway by decreasing MyD88 protein levels in a dose- and time-dependent manner without altering its mRNA expression. The proteasome inhibitor epoxomicin abolished the MyD88 degradation induced by TGF-beta1. Furthermore, TGF-beta1 resulted in ubiquitination of MyD88 protein, suggesting that TGF-beta1 facilitates ubiquitination and proteasomal degradation of MyD88 and thereby attenuates MyD88-dependent signaling by decreasing cellular levels of MyD88 protein. These findings importantly contribute to our understanding of molecular mechanisms mediating anti-inflammatory modulation of immune responses by TGF-beta1.     

The human ubiquitin conjugating enzyme UBE2J2 (Ubc6) is a substrate for proteasomal degradation

The human Ube2J2 enzyme functions in the ubiquitination of proteins at the ER. Here we demonstrate that it, and a second ubiquitin conjugating (Ubc) enzyme Ube2G2, are unstable, and incubation of transfected cells with proteasome inhibitors increased steady-state protein levels. For Ube2J2, pharmacological induction of the unfolded protein response (UPR) did not significantly alter ectopic protein levels, however the effect of proteasomal inhibition was abolished if the enzyme was inactivated or truncated to disrupt its ER-localization. These results suggest for the first time that the steady state expression of Ubcs’ may be important in regulating the degradation of ER proteins in mammalian cells.     

The glycan domain of thrombopoietin (TPO) acts in trans to enhance secretion of the hormone and other cytokines

Thrombopoietin (TPO), the primary regulator of platelet production, is composed of an amino-terminal 152 amino acids, sufficient for activity, and a carboxyl-terminal region rich in carbohydrates (183 residues) that enhances secretion of the molecule. Full-length TPO is secreted at levels 10-20-fold greater than truncated TPO. By introducing into mammalian cells a novel cDNA encoding the TPO secretory leader linked to its carboxyl-terminal domain (TPO glycan domain (TGD)), we tested whether TGD could function in trans to enhance secretion of TPO. The artificial TGD was secreted, inactive in proliferation assays, and did not inhibit TPO activity. However, when co-transfected with a cDNA encoding truncated TPO, TGD enhanced secretion 4-fold, measured by specific bioassay and immunoassay. TGD also enhanced secretion of granulocyte monocyte colony-stimulating factor and stem cell factor but did not affect the production of erythropoietin, interleukin-3, growth hormone, or of full-length TPO. To localize TGD function, we added an endoplasmic reticulum (ER) retention signal to TGD and, separately, deleted the secretory leader. Deletion of the secretory leader attenuated the secretory function of TGD, whereas addition of the ER retention signal did not alter its function. To investigate the physiologic role of TGD in folding and proteasomal protection, we tested full-length and truncated TPO in assays of protein refolding, and we examined protein stability in the presence of proteasome inhibitors. We found that truncated TGD re-folds readily and that proteasome-mediated degradation contributes to the poor secretion of truncated TPO. We conclude that TGD enhances secretion of TPO and can additionally function as an inter-molecular chaperone, in part because of its ability to prevent degradation of the hormone. The cellular location of TGD action is likely to be within the ER or earlier in the secretory pathway.