Human cytomegalovirus microRNA miR-US4-1 inhibits CD8(+) T cell responses by targeting the aminopeptidase ERAP1.

Major histocompatibility complex (MHC) class I molecules present peptides on the cell surface to CD8(+) T cells, which is critical for the killing of virus-infected or transformed cells. Precursors of MHC class I-presented peptides are trimmed to mature epitopes by the aminopeptidase ERAP1. The US2-US11 genomic region of human cytomegalovirus (HCMV) is dispensable for viral replication and encodes three microRNAs (miRNAs). We show here that HCMV miR-US4-1 specifically downregulated ERAP1 expression during viral infection. Accordingly, the trimming of HCMV-derived peptides was inhibited, which led to less susceptibility of infected cells to HCMV-specific cytotoxic T lymphocytes (CTLs). Our findings identify a previously unknown viral miRNA-based CTL-evasion mechanism that targets a key step in the MHC class I antigen-processing pathway.

Analysis of Brain and Cerebrospinal Fluid from Mouse Models of the Three Major Forms of Neuronal Ceroid Lipofuscinosis Reveals Changes in the Lysosomal Proteome.

Treatments are emerging for the neuronal ceroid lipofuscinoses (NCLs), a group of similar but genetically distinct lysosomal storage diseases. Clinical ratings scales measure long-term disease progression and response to treatment but clinically useful biomarkers have yet to be identified in these diseases. We have conducted proteomic analyses of brain and cerebrospinal fluid (CSF) from mouse models of the most frequently diagnosed NCL diseases: CLN1 (infantile NCL), CLN2 (classical late infantile NCL) and CLN3 (juvenile NCL). Samples were obtained at different stages of disease progression and proteins quantified using isobaric labeling. In total, 8303 and 4905 proteins were identified from brain and CSF, respectively. We also conduced label-free analyses of brain proteins that contained the mannose 6-phosphate lysosomal targeting modification. In general, we detect few changes at presymptomatic timepoints but later in disease, we detect multiple proteins whose expression is significantly altered in both brain and CSF of CLN1 and CLN2 animals. Many of these proteins are lysosomal in origin or are markers of neuroinflammation, potentially providing clues to underlying pathogenesis and providing promising candidates for further validation.

Endoplasmic Reticulum Aminopeptidase 1 beyond Antigenic Peptide-Processing Enzyme in the Endoplasmic Reticulum.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is well known as a processing enzyme of antigenic peptides, which are presented to major histocompatibility complex (MHC) class I molecules in the lumen of endoplasmic reticulum. Besides antigen processing, ERAP1 performs multiple functions in various cells depending on its intracellular and extracellular localization. Of note is the secretion of ERAP1 into the extracellular milieu in response to inflammatory stimuli, which further activates immune cells including macrophages and natural killer cells. Furthermore, secreted ERAP1 enhances the expression of pro-inflammatory cytokines like tumor necrosis factor-α, interleukin-1ß, and interleukin-6. Such findings indicate that ERAP1 plays a significant role in the field of innate and acquired immunity. This review summarizes the functional analyses of ERAP1 that support our current understanding of its role as more than an antigenic peptide-processing enzyme, specifically emphasizing on its secretory form.

The proteasome immunosubunits, PA28 and ER-aminopeptidase 1 protect melanoma cells from efficient MART-126-35 -specific T-cell recognition.

The immunodominant MART-1(26(27)-35) epitope, liberated from the differentiation antigen melanoma antigen recognized by T cells/melanoma antigen A (MART-1/Melan-A), has been frequently targeted in melanoma immunotherapy, but with limited clinical success. Previous studies suggested that this is in part due to an insufficient peptide supply and epitope presentation, since proteasomes containing the immunosubunits ß5i/LMP7 (LMP, low molecular weight protein) or ß1i/LMP2 and ß5i/LMP7 interfere with MART-1(26-35) epitope generation in tumor cells. Here, we demonstrate that in addition the IFN-γ-inducible proteasome subunit ß2i/MECL-1 (multicatalytic endopeptidase complex-like 1), proteasome activator 28 (PA28), and ER-resident aminopeptidase 1 (ERAP1) impair MART-1(26-35) epitope generation. ß2i/MECL-1 and PA28 negatively affect C- and N-terminal cleavage and therefore epitope liberation from the proteasome, whereas ERAP1 destroys the MART-1(26-35) epitope by overtrimming activity. Constitutive expression of PA28 and ERAP1 in melanoma cells indicate that both interfere with MART-1(26-35) epitope generation even in the absence of IFN-γ. In summary, our results provide first evidence that activities of different antigen-processing components contribute to an inefficient MART-1(26-35) epitope presentation, suggesting the tumor cell's proteolytic machinery might have an important impact on the outcome of epitope-specific immunotherapies.

Endoplasmic reticulum aminopeptidase 1 and rheumatic disease: functional variation.

PURPOSE OF REVIEW: To review the recent developments in our understanding of endoplasmic reticulum (ER) aminopeptidase 1 (ERAP1) function in relation to its role in major histocompatibility complex (MHC) class I peptide presentation and human leukocyte antigen (HLA) class I-associated diseases. RECENT FINDINGS: ERAP1 polymorphisms exhibiting loss-of-function have been associated with protection from AS. The aminopeptidase function of ERAP1 optimizes peptides for binding and presentation by MHC class I. Most of the studies have revealed reduced MHC class I expression in situations of reduced ERAP1 function. Under these circumstances, the presented peptides are often N-terminally extended, and cell surface complexes are unstable and fall apart more readily. In contrast, peptides presented by HLA-B*27 : 05 when ERAP1 is silenced are frequently extended on the C-terminus. Recent work has emphasized on the importance of assessing the function of allotypes encoded by ERAP1 haplotypes, rather than effects of single amino acid substitutions. The allotypes found in a series of AS patients were poorer at restoring HLA-B27 expression than allotypes found in unaffected controls, which may seem contrary to the genetic data linking loss-of-function to protection. SUMMARY: More work is needed to understand how ERAP1 variants associated with risk and protection influence the quality and quantity of peptides available for binding to HLA class I molecules in the ER. Moreover, we need to determine allele-specific effects of ERAP1 variants in the context of HLA-B*51 and HLA-Cw*6, which are associated with Behçet's disease and psoriasis, respectively.

Identifying a novel role for X-prolyl aminopeptidase (Xpnpep) 2 in CrVI-induced adverse effects on germ cell nest breakdown and follicle development in rats.

Environmental exposure to endocrine-disrupting chemicals (EDCs) is one cause of premature ovarian failure (POF). Hexavalent chromium (CrVI) is a heavy metal EDC widely used in more than 50 industries, including chrome plating, welding, wood processing, and tanneries. Recent data from U.S. Environmental Protection Agency indicate increased levels of Cr in drinking water from several American cities, which potentially predispose residents to various health problems. Recently, we demonstrated that gestational exposure to CrVI caused POF in F1 offspring. The current study was performed to identify the molecular mechanism behind CrVI-induced POF. Pregnant rats were treated with 25 ppm of potassium dichromate from Gestational Day (GD) 9.5 to GD 14.5 through drinking water, and the fetuses were exposed to CrVI through transplacental transfer. Ovaries were removed from the fetuses or pups on Embryonic Day (ED) 15.5, ED 17.5, Postnatal Day (PND) 1, PND 4, or PND 25, and various analyses were performed. Results showed that gestational exposure to CrVI: 1) increased germ cell/oocyte apoptosis and advanced germ cell nest (GCN) breakdown; 2) increased X-prolyl aminopeptidase (Xpnpep) 2, a POF marker in humans, during GCN breakdown; 3) decreased Xpnpep2 during postnatal follicle development; and 4) increased colocalization of Xpnpep2 with Col3 and Col4. We also found that Xpnpep2 inversely regulated the expression of Col1, Col3, and Col4 in all the developmental stages studied. Thus, CrVI advanced GCN breakdown and increased follicle atresia in F1 female progeny by targeting Xpnpep2.

Endoplasmic reticulum aminopeptidases in the pathogenesis of ankylosing spondylitis.

There has been significant progress in our understanding of the pathogenesis of AS. The advent of genome-wide association studies has increased the known loci associated with AS to more than 40. The endoplasmic reticulum resident aminopeptidases (ERAP) 1 and 2 were identified in this manner and are of particular interest. There appears to be a genetic as well as a functional interaction of ERAP1 and 2 with HLA-B27 based on the known functions of these molecules. Recent studies on the structure, immunological effects and the peptide-trimming properties of ERAP 1 and 2 have helped to provide insight into their pathogenic potential in AS. In this review, we explore the role of ERAP 1 and 2 in the pathogenesis of AS.

A zebrafish model of CLN2 disease is deficient in tripeptidyl peptidase 1 and displays progressive neurodegeneration accompanied by a reduction in proliferation.

Tripeptidyl peptidase 1 (TPP1) deficiency causes CLN2 disease, late infantile (or classic late infantile neuronal ceroid lipofuscinosis), a paediatric neurodegenerative disease of autosomal recessive inheritance. Patients suffer from blindness, ataxia, epilepsy and cognitive defects, with MRI indicating widespread brain atrophy, and profound neuron loss is evident within the retina and brain. Currently there are no effective therapies for this disease, which causes premature death in adolescence. Zebrafish have been successfully used to model a range of neurological and behavioural abnormalities. The aim of this study was to characterize the pathological and functional consequences of Tpp1 deficiency in zebrafish and to correlate these with human CLN2 disease, thereby providing a platform for drug discovery. Our data show that homozygous tpp1(sa0011) mutant (tpp1(sa0011)(-/-)) zebrafish display a severe, progressive, early onset neurodegenerative phenotype, characterized by a significantly small retina, a small head and curved body. The mutant zebrafish have significantly reduced median survival with death occurring 5 days post-fertilization. As in human patients with CLN2 disease, mutant zebrafish display storage of subunit c of mitochondrial ATP-synthase, hypertrophic lysosomes as well as localized apoptotic cell death in the retina, optic tectum and cerebellum. Further neuropathological phenotypes of these mutants provide novel insights into mechanisms of pathogenesis in CLN2 disease. Secondary neurogenesis in the retina, optic tectum and cerebellum is impaired and axon tracts within the spinal cord, optic nerve and the posterior commissure are disorganized, with the optic nerve failing to reach its target. This severe neurodegenerative phenotype eventually results in functional motor impairment, but this is preceded by a phase of hyperactivity that is consistent with seizures. Importantly, both of these locomotion phenotypes can be assayed in an automated manner suitable for high-throughput studies. Our study provides proof-of-principle that tpp1(sa0011)(-/-) mutants can utilize the advantages of zebrafish for understanding pathogenesis and drug discovery in CLN2 disease and other epilepsies.

Novel highly sensitive, specific, and straightforward strategy for comprehensive N-terminal proteomics reveals unknown substrates of the mitochondrial peptidase Icp55.

We present a novel straightforward method for enrichment of N-terminal peptides, utilizing charge-based fractional diagonal chromatography (ChaFRADIC). Our method is robust, easy to operate, fast, specific, and more sensitive than existing methods, enabling the differential quantitation of 1459 nonredundant N-terminal peptides between two S. cerevisiae samples within 10 h of LC-MS, starting from only 50 µg of protein per condition and analyzing only 40% of the obtained fractions. Using ChaFRADIC we compared mitochondrial proteins from wild-type and icp55Δ yeast (30 µg each). Icp55 is an intermediate cleaving peptidase, which, following mitochondrial processing peptidase (MPP)-dependent cleavage of signal sequences, removes a single amino acid from a specific set of proteins according to the N-end rule. Using ChaFRADIC we identified 36 icp55 substrates, 14 of which were previously unknown, expanding the set of known icp55 substrates to a total of 52 proteins. Interestingly, a novel substrate, Isa2, is likely processed by Icp55 in two consecutive steps and thus might represent the first example of a triple processing event in a mitochondrial precursor protein. Thus, ChaFRADIC is a powerful and practicable tool for protease and peptidase research, providing the sensitivity to characterize even samples that can be obtained only in small quantities.

Structure and function of tripeptidyl peptidase II, a giant cytosolic protease.

Tripeptidyl peptidase II is the largest known eukaryotic peptidase. It has been described as a multi-purpose peptidase, which, in addition to its house-keeping function in intracellular protein degradation, plays a role in several vital cellular processes such as antigen processing, apoptosis, or cell division, and is involved in diseases like muscle wasting, obesity, and in cancer. Biochemical studies and bioinformatics have identified TPPII as a subtilase, but its structure is very unusual: it forms a large homooligomeric complex (6 MDa) with a spindle-like shape. Recently, the high-resolution structure of TPPII homodimers (300 kDa) was solved and a hybrid structure of the holocomplex built of 20 dimers was obtained by docking it into the EM-density. Here, we summarize our current knowledge about TPPII with a focus on structural aspects. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.

Methionine aminopeptidase 2 is required for HSC initiation and proliferation.

In a chemical screening, we tested the antiangiogenic effects of fumagillin derivatives and identified fumagillin as an inhibitor of definitive hematopoiesis in zebrafish embryos. Fumagillin is known to target methionine aminopeptidase II (MetAP2), an enzyme whose function in hematopoiesis is unknown. We investigated the role of MetAP2 in hematopoiesis by using zebrafish embryo and human umbilical cord blood models. Zebrafish metap2 was expressed ubiquitously during early embryogenesis and later in the somitic region, the caudal hematopoietic tissue, and pronephric duct. metap2 was inhibited by morpholino and fumagillin treatment, resulting in increased mpo expression at 18 hours postfertilization and reduced c-myb expression along the ventral wall of dorsal aorta at 36 hours postfertilization. It also disrupted intersegmental vessels in Tg(fli1:gfp) embryos without affecting development of major axial vasculatures. Inhibition of MetAP2 in CB CD34(+) cells by fumagillin had no effect on overall clonogenic activity but significantly reduced their engraftment into immunodeficient nonobese diabetes/severe combined immunodeficiency mice. metap2 knock-down in zebrafish and inhibition by fumagillin in zebrafish and human CB CD34(+) cells inhibited Calmodulin Kinase II activity and induced ERK phosphorylation. This study demonstrated a hitherto-undescribed role of MetAP2 in definitive hematopoiesis and a possible link to noncanonical Wnt and ERK signaling.

Cutting Edge: Coding single nucleotide polymorphisms of endoplasmic reticulum aminopeptidase 1 can affect antigenic peptide generation in vitro by influencing basic enzymatic properties of the enzyme.

ER aminopeptidase 1 (ERAP1) customizes antigenic peptide precursors for MHC class I presentation and edits the antigenic peptide repertoire. Coding single nucleotide polymorphisms (SNPs) in ERAP1 were recently linked with predisposition to autoimmune disease, suggesting a link between pathogenesis of autoimmunity and ERAP1-mediated Ag processing. To investigate this possibility, we analyzed the effect that disease-linked SNPs have on Ag processing by ERAP1 in vitro. Michaelis-Menten analysis revealed that the presence of SNPs affects the Michaelis constant and turnover number of the enzyme. Strikingly, specific ERAP1 allele-substrate combinations deviate from standard Michaelis-Menten behavior, demonstrating substrate-inhibition kinetics; to our knowledge, this phenomenon has not been described for this enzyme. Cell-based Ag-presentation analysis was consistent with changes in the substrate inhibition constant K(i), further supporting that ERAP1 allelic composition may affect Ag processing in vivo. We propose that these phenomena should be taken into account when evaluating the possible link between Ag processing and autoimmunity.

The crystal structure of human endoplasmic reticulum aminopeptidase 2 reveals the atomic basis for distinct roles in antigen processing.

Endoplasmic reticulum aminopeptidases ERAP1 and ERAP2 cooperate to trim a vast variety of antigenic peptide precursors to generate mature epitopes for binding to major histocompatibility class I molecules. We report here the first structure of ERAP2 determined at 3.08 Å by X-ray crystallography. On the basis of residual electron density, a lysine residue has been modeled in the active site of the enzyme; thus, the structure corresponds to an enzyme-product complex. The overall domain organization is highly similar to that of the recently determined structure of ERAP1 in its closed conformation. A large internal cavity adjacent to the catalytic site can accommodate large peptide substrates. The ERAP2 structure provides a structural explanation for the different peptide N-terminal specificities between ERAP1 and ERAP2 and suggests that such differences extend throughout the whole peptide sequence. A noncrystallographic dimer observed may constitute a model for a proposed ERAP1-ERAP2 heterodimer. Overall, the structure helps explain how two homologous aminopeptidases cooperate to process a large variety of sequences, a key property of their biological role.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) exhibits functionally significant interaction with HLA-B27 and relates to subtype specificity in ankylosing spondylitis.

OBJECTIVES: The functional interaction of endoplasmic reticulum aminopeptidase 1 (ERAP1) with human leucocyte antigen (HLA)-B*27 could be important in the pathogenesis of ankylosing spondylitis (AS). AS is associated with B*27:04 and B*27:05, but not with B*27:06 and B*27:09. The authors studied the surface expression of peptide-HLA(pHLA)-B27 complexes and HLA class-I free heavy chains (FHCs) on peripheral blood mononuclear cells of patients with AS with different ERAP1 single nucleotide polymorphisms. The effects of ERAP1 suppression on HLA-B*27 subtypes were tested. METHODS: Peripheral blood mononuclear cells were collected from Caucasian patients with AS for flow cytometry and were stained for pHLA and FHCs. Genotyping was performed for two ERAP1 single nucleotide polymorphisms (rs27044(C/G) and rs30187(C/T)). C1R cells transfected with different HLA-B27 subtypes (B*27:04, B*27:05, B*27:06 and B*27:09) were subjected to ERAP1 suppression by small interfering RNA and stained using the monoclonal antibody (mAb) MARB4 as well as antibodies for pHLA, FHC, intracellular FHC (IC-FHC). MARB4 has been reported to bind to HLA-B27 with extended peptides. RESULTS: The authors found variations in FHC expression on the monocytes of patients with AS, depending on different ERAP1 variants. Subsequently, using Hmy2.C1R cells in vitro, the authors show that ERAP1 suppression leads to increased IC-FHC and surface pHLA that react with the monoclonal antibody MARB4. The functional interaction between ERAP1 and HLA-B27 molecules appears to be subtype-specific, since ERAP1 suppression leads to changes only in cells expressing B*27:04 or B*27:05, but not B*27:06 or B*27:09. CONCLUSIONS: Direct or indirect alterations in the ERAP1-HLA-B27 interaction could be crucial by causing changes in peptide presentation or FHC formation by HLA-B27 molecules, as well as by contributing to differential subtype association in spondyloarthropathies.

Characterizing the specificity and cooperation of aminopeptidases in the cytosol and endoplasmic reticulum during MHC class I antigen presentation.

Many MHC class I-binding peptides are generated as N-extended precursors during protein degradation by the proteasome. These peptides can subsequently be trimmed by aminopeptidases in the cytosol and/or the endoplasmic reticulum (ER) to produce mature epitope. However, the contribution and specificity of each of these subcellular compartments in removing N-terminal amino acids for Ag presentation is not well defined. In this study, we investigated this issue for antigenic precursors that are expressed in the cytosol. By systematically varying the N-terminal flanking sequences of peptides, we show that the amino acids upstream of an epitope precursor are a major determinant of the amount of Ag presentation. In many cases, MHC class I-binding peptides are produced through sequential trimming in the cytosol and ER. Trimming of flanking residues in the cytosol contributes most to sequences that are poorly trimmed in the ER. Because N-terminal trimming has different specificity in the cytosol and ER, the cleavage of peptides in both of these compartments serves to broaden the repertoire of sequences that are presented.

Role of endoplasmic reticulum aminopeptidases in health and disease: from infection to cancer.

Endoplasmic reticulum (ER) aminopeptidases ERAP1 and ERAP2 (ERAPs) are essential for the maturation of a wide spectrum of proteins involved in various biological processes. In the ER, these enzymes work in concert to trim peptides for presentation on MHC class I molecules. Loss of ERAPs function substantially alters the repertoire of peptides presented by MHC class I molecules, critically affecting recognition of both NK and CD8(+) T cells. In addition, these enzymes are involved in the modulation of inflammatory responses by promoting the shedding of several cytokine receptors, and in the regulation of both blood pressure and angiogenesis. Recent genome-wide association studies have identified common variants of ERAP1 and ERAP2 linked to several human diseases, ranging from viral infections to autoimmunity and cancer. More recently, inhibition of ER peptide trimming has been shown to play a key role in stimulating innate and adaptive anti-tumor immune responses, suggesting that inhibition of ERAPs might be exploited for the establishment of innovative therapeutic approaches against cancer. This review summarizes data currently available for ERAP enzymes in ER peptide trimming and in other immunological and non-immunological functions, paying attention to the emerging role played by these enzymes in human diseases.

Accumulation of polyubiquitylated proteins in response to Ala-Ala-Phe-chloromethylketone is independent of the inhibition of Tripeptidyl peptidase II.

In the present study we have addressed the issue of proteasome independent cytosolic protein degradation. Tripeptidyl peptidase II (TPPII) has been suggested to compensate for a reduced proteasome activity, partly based on evidence using the inhibitor Ala-Ala-Phe-chloromethylketone (AAF-cmk). Here we show that AAF-cmk induces the formation of polyubiquitin-containing accumulations in osteosarcoma and Burkitt's lymphoma cell lines. These accumulations meet many of the landmarks of the aggresomes that form after proteasome inhibition. Using a combination of experiments with chemical inhibitors and interference of gene expression, we show that TPPII inhibition is not responsible for these accumulations. Our evidence suggests that the relevant target(s) is/are in the ubiquitin-proteasome pathway, most likely upstream the proteasome. We obtained evidence supporting this model by inhibition of Hsp90, which also acts upstream the proteasome. Although our data suggest that Hsp90 is not a target of AAF-cmk, its inhibition resulted in accumulations similar to those obtained with AAF-cmk. Therefore, our results question the proposed role for TPPII as a prominent alternative to the proteasome in cellular proteolysis.

The catalytic and protein-protein interaction domains are required for APM1 function.

Aminopeptidase M1 (APM1) is essential for embryonic, vegetative, and reproductive development in Arabidopsis (Arabidopsis thaliana). Here, we show that, like mammalian M1 proteases, APM1 appears to have distinct enzymatic and protein-protein interaction domains and functions as a homodimer. Arabidopsis seedlings treated with ezetimibe, an inhibitor of M1 protein-protein interactions, mimicked a subset of apm1 phenotypes distinct from those resulting from treatment with PAQ-22, an inhibitor of M1 catalytic activity, suggesting that the APM1 catalytic and interaction domains can function independently. apm1-1 knockdown mutants transformed with catalytically inactive APM1 did not prevent seedling lethality. However, apm1-2 has a functional enzymatic domain but lacks the carboxyl (C) terminus, and transformation with catalytically inactive APM1 rescued the mutant. Overexpression of human insulin-responsive aminopeptidase/oxytocinase rescued all apm1 phenotypes, suggesting that the catalytic activity was sufficient to compensate for loss of APM1 function, while overexpression of catalytically inactive insulin-responsive aminopeptidase/oxytocinase only rescued apm1-2. Increased catalytic activity alone is not sufficient to compensate for loss of APM1 function, as overexpression of another Arabidopsis M1 family member lacking an extended C terminus did not rescue apm1-1. The protein interactions facilitating enzymatic activity appear to be dependent on the C terminus of APM1, as transformation with an open reading frame containing an internal deletion of a portion of the C terminus or a point mutation in a dileucine motif did not rescue the mutant. These results suggest that both the catalytic and interaction domains are necessary for APM1 function but that APM1 function and dimerization do not require these domains to be present in the same linear molecule.

Metalloaminopeptidases of the Protozoan Parasite Plasmodium falciparum as Targets for the Discovery of Novel Antimalarial Drugs.

Malaria poses a significant threat to approximately half of the world's population with an annual death toll close to half a million. The emergence of resistance to front-line antimalarials in the most lethal human parasite species, Plasmodium falciparum (Pf), threatens progress made in malaria control. The prospect of losing the efficacy of antimalarial drugs is driving the search for small molecules with new modes of action. Asexual reproduction of the parasite is critically dependent on the recycling of amino acids through catabolism of hemoglobin (Hb), which makes metalloaminopeptidases (MAPs) attractive targets for the development of new drugs. The Pf genome encodes eight MAPs, some of which have been found to be essential for parasite survival. In this article, we discuss the biological structure and function of each MAP within the Pf genome, along with the drug discovery efforts that have been undertaken to identify novel antimalarial candidates of therapeutic value.

p67/MetAP2 suppresses K-RasV12-mediated transformation of NIH3T3 mouse fibroblasts in culture and in athymic mice.

In many tumor cells, the activation and activity of extracellular signal-regulated kinases (ERK1/2) are very high because of the constitutive activation of the Ras-mediated signaling pathway. Here, we ectopically expressed the human homologue of rat eukaryotic initiation factor 2-associated glycoprotein, p67/MetAP2, in EGF-treated mouse embryonic NIH3T3 fibroblasts and C2C12 myoblasts and NIH3T3 cell lines expressing the constitutively active form of MAP kinase kinase (MEK) to inhibit the activation and activity of ERK1/2 MAP kinases. In addition, we also ectopically expressed rat p67/MetAP2 in oncogenic Ras-induced transformed NIH3T3 fibroblasts and inhibited their transformed phenotype both in culture and in athymic nude mice possibly by inhibiting angiogenesis. This inhibition of ERK1/2 MAP kinases is due to the direct binding with rat p67/MetAP2, and this leads to the inhibition of activity of ERK1/2 MAP kinases both in vitro and in vivo. Furthermore, expression of p67/MetAP2 siRNA in both NIH3T3 fibroblasts and C2C12 myoblasts causes activation and activity of ERK1/2 MAP kinases. Our results thus suggest that ectopic expression of rat p67/MetAP2 in transformed cells can inhibit the tumorigenic phenotype by inhibiting the activation and activity of ERK1/2 MAP kinases and, thus, that p67/MetAP2 has tumor suppression activity.