岡西 広樹

Proteases play crucial roles in numerous biological processes through specific protein cleavage, and their dysregulation has been implicated in various diseases. To better understand protease specificity, we developed a lauroylation-assisted proteomic identification of protease cleavage sites (PICS) workflow that labels and enriches targeted protease-generated neo-N-termini using economical reagents and standard laboratory equipment. The lauroylation enables both discrimination of the neo-N-termini in LC-MS/MS and efficient enrichment on a C18 StageTip by exploiting its hydrophobicity. Among tested acylations, we found lauroylation to be optimal for PICS and improved enrichment and fractionation conditions. We demonstrated that this method can profile specificities of multiple proteases with high sensitivity. Furthermore, we extended this concept to N-terminomics to examine proteolysis at the protein level. Protein N-terminal dimethylation is used for labeling, and tryptic internal peptides are lauroylated for removal. This approach identified over 1500 cleavages induced by etoposide, including 912 Asp-cleaved sites consistent with caspase-3 motifs and sensitive to inhibition by Z-DEVD-FMK. Additionally, 2286 protein N-termini were identified in untreated cells, including 1794 non-ORF N-termini with 665 previously annotated processing sites. These results demonstrate that our workflow provides a simple, economical, and widely applicable method for characterizing protease cleavage at both peptide and protein levels.

BACKGROUND: L-type amino acid transporter 1 (LAT1; SLC7A5), which preferentially transports large neutral amino acids (LNAAs), is highly upregulated in various cancers and represents a promising therapeutic target. The first-in-class LAT1-specific inhibitor, nanvuranlat (JPH203, KYT-0353), has exhibited potent anti-cancer effects and is under clinical evaluation. However, alterations in the amino acid availability in cancer cells underlying its pharmacological activities remain to be elucidated. METHODS: Amino acids in nanvuranlat-treated cancer cells were measured by high-performance liquid chromatography. LAT1 knockdown was performed using siRNA. To mimic LAT1 inhibition, cancer cells were incubated in culture media lacking specific LNAA(s) reduced by nanvuranlat. The consequences of these treatments were compared by cell-based assays, including analyses of amino acid contents, cell growth, amino acid-related signaling pathways, cell cycle, ATP production rate, and transcriptomes by RNA sequencing. Metabolome of nanvuranlat-treated and untreated cells was compared by mass spectrometry. The effects of nanvuranlat on amino acid composition were also examined in three-dimensional cancer cell spheroids. RESULTS: Both pharmacological and genetic inhibition of LAT1 preferentially and continuously reduced valine, isoleucine, and tryptophan in pancreatic cancer MIA PaCa-2 cells. Nanvuranlat induced similar alterations in intracellular amino acids in multiple cancer cell lines. Depletion of these amino acids from culture media selectively lowered their intracellular concentrations, recapitulating the effects of nanvuranlat on cell growth, amino acid-related mTORC1/GAAC signaling pathways, and cell cycle. Deprivation of valine or isoleucine exhibited more pronounced impacts than tryptophan in all assays. As a novel pharmacological action of nanvuranlat mediated by the reductions in valine and isoleucine, we revealed downregulation of multiple genes in the TCA cycle and respiratory chain, accompanied by a decreased mitochondrial ATP production rate. Consistently, metabolomics revealed broad decreases in the TCA cycle intermediates by LAT1 inhibition. Nanvuranlat also similarly influenced the amino acid levels in cancer cell spheroids. CONCLUSIONS: Reductions in valine and isoleucine in cancer cells primarily account for the multifaceted anti-cancer pharmacological activities of LAT1 inhibition by nanvuranlat. This study establishes the molecular basis for LAT1-targeted therapy and highlights growth-promoting processes in cancer cells that can be exploited pharmacologically by modulating the availability of specific amino acids.

In Thermus thermophilus, an aerobic Gram-negative eubacterium used as a model organism, more than half of the phosphorylation sites identified by proteomic analysis are located near the ligand-binding site, including the active site, of the enzyme in the three-dimensional structure. We investigated the effect of these phosphorylation events on the activity of six enzymes (three nucleoside monophosphate kinases, isocitrate kinase, malate dehydrogenase and inorganic pyrophosphatase) by introducing phosphomimetic mutations, Glu, into the phosphorylation sites. All phosphomimetic mutants showed severely reduced activity compared with the wild-type, particularly in the turnover number. The proteins analyzed in this study belong to different families and have various functions. This suggests that there is a widespread mechanism by which phosphorylation of amino acid residues near the active site reduces enzyme activity independent of the protein family and function.

Bacteria reduce toxic selenium oxyanions, such as selenite, to elemental selenium (Se0), forming selenium nanoparticles (SeNPs) either intracellularly or extracellularly. However, the mechanism through which extracellular SeNPs (Ex-SeNPs) are exported remains unclear. In this study, we characterized Ex-SeNPs biosynthesized by Escherichia coli during the aerobic reduction of selenite. The SeNPs appeared within 2 h of exposure, remained extracellular, and displayed a consistent spherical morphology (∼100 nm). Purified Ex-SeNPs consisted of an Se0 core enveloped by a membrane-like layer containing lipids, proteins, carbohydrates, peptidoglycan, and lipopolysaccharides. Fluorescence microscopy and gas chromatography-mass spectrometry indicated that the encapsulated membrane originates from the E. coli cell membrane. Notably, mutants deficient in the outer membrane proteins OmpC or TolA failed to excrete SeNPs, resulting in intracellular accumulation despite efficient Se0 synthesis. Our findings suggest that E. coli forms SeNPs intracellularly and exports them via an envelope-dependent process, during which the particles may become encapsulated in membrane-like structures. These findings help clarify the mechanism underlying a membrane-dependent pathway for SeNP detoxification and export that had been suggested but not directly demonstrated.

Zinc finger domains are important interaction modules for binding to nucleic acids, proteins, lipids and small molecules. Many small-sized zinc finger proteins are encoded in bacterial genomes, but most of them have not been functionally annotated. We focused on TTHA0897, ZifS, as a small zinc finger protein from the extremely thermophilic eubacterium Thermus thermophilus HB8. In vivo experiments suggested that the cellular function of ZifS is related to the growth transition of T. thermophilus from the lag to the exponential phase under nutritionally limited conditions. In vitro biochemical experiments, including electrophoretic mobility shift assay and pull-down assay, yielded no clues about molecular functions of ZifS. X-ray crystallographic analysis revealed that the dimeric ZifS globally forms a cylinder-like structure, although ZifS dimer has no overall structural similarity to other known zinc finger proteins. The zinc ion-binding manner of ZifS fitted the characteristics of the zinc ribbon fold, which are mostly found in domains from proteins involved in the transcriptional and translational machinery. The crystal structure of ZifS is the first experimental insight into the molecular structure of this protein family, revealing several conserved features that may be functionally relevant.

The cochlea contains two extracellular fluids, perilymph and endolymph. Endolymph exhibits high potential of approximately +80 to +110 mV (depending on species), which sensitizes sensory hair cells. Other properties of this unique fluid remain elusive, owing to its minuscule volume in rodent cochlea. We therefore developed a technique to collect high-purity endolymph from mouse cochleae. Comprehensive proteomic analysis of sampled endolymph using liquid chromatography with mass spectrometry identified 301 proteins, dominated by molecules engaged in immunity and proteostasis. Approximately 30% of these proteins were undetectable in our perilymph. A combination of mass spectrometry and different approaches revealed that, compared to perilymph, endolymph was enriched with α2-macroglobulin, osteopontin, apolipoprotein D, apolipoprotein E, and apolipoprotein J/clusterin. In other cells or tissues, α2-macroglobulin, apolipoprotein E, and apolipoprotein J contribute to the clearance of degraded proteins from extracellular fluid. Altogether, with the proteins described here, endolymph may play a protective role in stabilizing cochlear homeostasis.

L-type amino acid transporter 1 (LAT1) is upregulated in various cancer types and contributes to disease progression. Previous studies have demonstrated or suggested that hypoxia-inducible factors (HIFs), the key transcription factors in hypoxic responses, control the expression of LAT1 gene in several types of cancer cells. However, this regulatory relationship has not been investigated yet in colorectal cancer (CRC), one of the cancer types in which the increased LAT1 expression holds prognostic significance. In this study, we found that neither LAT1 mRNA nor protein is induced under hypoxic condition (1% O2) in CRC HT-29 cells in vitro, regardless of the prominent HIF-1/2α accumulation and HIFs-dependent upregulation of glucose transporter 1. The hypoxic treatment generally did not increase either the mRNA or protein expression of LAT1 in eight CRC cell lines tested, in contrast to the pronounced upregulation by amino acid restriction. Interestingly, knockdown of von Hippel-Lindau ubiquitin ligase to inhibit the proteasomal degradation of HIFs caused an accumulation of HIF-2α and increased the LAT1 expression in certain CRC cell lines. This study contributes to delineating the molecular mechanisms responsible for the pathological expression of LAT1 in CRC cells, emphasizing the ambiguity of HIFs-dependent transcriptional upregulation of LAT1 across cancer cells.

Abstract L-type amino acid transporter 1 (LAT1) is a transmembrane protein responsible for transporting large neutral amino acids. While numerous LAT1-targeted compound delivery for the brain and tumors have been investigated, their LAT1 selectivity often remains ambiguous despite high LAT1 affinity. This study assessed the LAT1 selectivity of phenylalanine (Phe) analogs, focusing on their structure–activity characteristics. We discovered that 2-iodo-l-phenylalanine (2-I-Phe), with an iodine substituent at position 2 in the benzene ring, markedly improves LAT1 affinity and selectivity compared to parent amino acid Phe, albeit at the cost of reduced transport velocity. l-Phenylglycine (Phg), one carbon shorter than Phe, was found to be a substrate for LAT1 with a lower affinity, exhibiting a low level of selectivity for LAT1 equivalent to Phe. Notably, (R)-2-amino-1,2,3,4-tetrahydro-2-naphthoic acid (bicyclic-Phe), with an α-methylene moiety akin to the α-methyl group in α-methyl-l-phenylalanine (α-methyl-Phe), a known LAT1-selective compound, showed similar LAT1 transport maximal velocity to α-methyl-Phe, but with higher LAT1 affinity and selectivity. In vivo studies revealed tumor-specific accumulation of bicyclic-Phe, underscoring the importance of LAT1-selectivity in targeted delivery. These findings emphasize the potential of bicyclic-Phe as a promising LAT1-selective component, providing a basis for the development of LAT1-targeting compounds based on its structural framework.

Hearing loss is a pivotal risk factor for dementia. It has recently emerged that a disruption in the intercommunication between the cochlea and brain is a key process in the initiation and progression of this disease. However, whether the cochlear properties can be influenced by pathological signals associated with dementia remains unclear. In this study, using a mouse model of Alzheimer's disease (AD), we investigated the impacts of the AD-like amyloid β (Aβ) pathology in the brain on the cochlea. Despite little detectable change in the age-related shift of the hearing threshold, we observed quantitative and qualitative alterations in the protein profile in perilymph, an extracellular fluid that fills the path of sound waves in the cochlea. Our findings highlight the potential contribution of Aβ pathology in the brain to the disturbance of cochlear homeostasis.

Metastasis is the leading cause of mortality in cancer patients. L-type amino acid transporter 1 (LAT1, SLC7A5) is a Na+-independent neutral amino acid transporter highly expressed in various cancers to support their growth. Although high LAT1 expression is closely associated with cancer metastasis, its role in this process remains unclear. This study aimed to investigate the effect of LAT1 inhibition on cancer metastasis using B16-F10 melanoma mouse models. Our results demonstrated that nanvuranlat (JPH203), a high-affinity LAT1-selective inhibitor, suppressed B16-F10 cell proliferation, migration, and invasion. Similarly, LAT1 knockdown reduced cell proliferation, migration, and invasion. LAT1 inhibitors and LAT1 knockdown diminished B16-F10 lung metastasis in a lung metastasis model. Furthermore, nanvuranlat and LAT1 knockdown suppressed lung, spleen, and lymph node metastasis in an orthotopic metastasis model. We discovered that the LAT1 inhibitor reduced the cell surface expression of integrin αvβ3. Our findings revealed that the downregulation of the mTOR signaling pathway, induced by LAT1 inhibitors, decreased the expression of integrin αvβ3, contributing to the suppression of metastasis. These results highlight the critical role of LAT1 in cancer metastasis and suggest that LAT1 inhibition may serve as a potential target for anti-metastasis cancer therapy.

Abstract Background Cytotoxic anticancer drugs widely used in cancer chemotherapy have some limitations, such as the development of side effects and drug resistance. Furthermore, monotherapy is often less effective against heterogeneous cancer tissues. Combination therapies of cytotoxic anticancer drugs with molecularly targeted drugs have been pursued to solve such fundamental problems. Nanvuranlat (JPH203 or KYT-0353), an inhibitor for L-type amino acid transporter 1 (LAT1; SLC7A5), has novel mechanisms of action to suppress the cancer cell proliferation and tumor growth by inhibiting the transport of large neutral amino acids into cancer cells. This study investigated the potential of the combined use of nanvuranlat and cytotoxic anticancer drugs. Methods The combination effects of cytotoxic anticancer drugs and nanvuranlat on cell growth were examined by a water-soluble tetrazolium salt assay in two-dimensional cultures of pancreatic and biliary tract cancer cell lines. To elucidate the pharmacological mechanisms underlying the combination of gemcitabine and nanvuranlat, we investigated apoptotic cell death and cell cycle by flow cytometry. The phosphorylation levels of amino acid-related signaling pathways were analyzed by Western blot. Furthermore, growth inhibition was examined in cancer cell spheroids. Results All the tested seven types of cytotoxic anticancer drugs combined with nanvuranlat significantly inhibited the cell growth of pancreatic cancer MIA PaCa-2 cells compared to their single treatment. Among them, the combined effects of gemcitabine and nanvuranlat were relatively high and confirmed in multiple pancreatic and biliary tract cell lines in two-dimensional cultures. The growth inhibitory effects were suggested to be additive but not synergistic under the tested conditions. Gemcitabine generally induced cell cycle arrest at the S phase and apoptotic cell death, while nanvuranlat induced cell cycle arrest at the G0/G1 phase and affected amino acid-related mTORC1 and GAAC signaling pathways. In combination, each anticancer drug basically exerted its own pharmacological activities, although gemcitabine more strongly influenced the cell cycle than nanvuranlat. The combination effects of growth inhibition were also verified in cancer cell spheroids. Conclusions Our study demonstrates the potential of first-in-class LAT1 inhibitor nanvuranlat as a concomitant drug with cytotoxic anticancer drugs, especially gemcitabine, on pancreatic and biliary tract cancers.

Fatty acid kinase is necessary for the incorporation of exogenous fatty acids into membrane phospholipids. Fatty acid kinase consists of two components: a kinase component, FakA, that phosphorylates a fatty acid bound to a fatty acid-binding component, FakB. However, the molecular details underlying the phosphotransfer reaction remain to be resolved. We determined the crystal structure of the N-terminal domain of FakA bound to ADP from Thermus thermophilus HB8. The overall structure of this domain showed that the helical barrel fold is similar to the nucleotide-binding component of dihydroxyacetone kinase. The structure of the nucleotide-binding site revealed the roles of the conserved residues in recognition of ADP and Mg2+, but the N-terminal domain of FakA lacked the ADP-capping loop found in the dihydroxyacetone kinase component. Based on the structural similarity to the two subunits of dihydroxyacetone kinase complex, we constructed a model of the complex of T. thermophilus FakB and the N-terminal domain of FakA. In this model, the invariant Arg residue of FakB occupied a position that was spatially similar to that of the catalytically important Arg residue of dihydroxyacetone kinase, which predicted a composite active site in the Fatty acid kinase complex.

BACKGROUND: Cancer-upregulated L-type amino acid transporter 1 (LAT1; SLC7A5) supplies essential amino acids to cancer cells. LAT1 substrates are not only needed for cancer rapid growth, but involved in cellular signaling. LAT1 has been proposed as a potential target for cancer treatment-its inhibitor, JPH203, is currently in clinical trials and targets biliary tract cancer (BTC). Here, we revealed to what extent LAT1 inhibitor affects intracellular amino acid content and what kind of cellular signals are directly triggered by LAT1 inhibition. METHODS: Liquid chromatography assay combined with o-phthalaldehyde- and 9-fluorenyl-methylchloroformate-based derivatization revealed changes in intracellular amino acid levels induced by LAT1 inhibition with JPH203 treatment in three BTC cell lines. Tandem mass tag-based quantitative phosphoproteomics characterized the effect of JPH203 treatment on BTC cells, and suggested key regulators in LAT1-inhibited cells. We further studied one of the key regulators, CK2 protein kinase, by using Western blot, enzymatic activity assay, and co-immunoprecipitation. We evaluated anticancer effects of combination of JPH203 with CK2 inhibitor using cell growth and would healing assay. RESULTS: JPH203 treatment decreased intracellular levels of LAT1 substrates including essential amino acids of three BTC cell lines, immediately and drastically. We also found levels of some of these amino acids were partially recovered after longer-time treatment. Therefore, we performed phosphoproteomics with short-time JPH203 treatment prior to the cellular compensatory response, and revealed hundreds of differentially phosphorylated sites. Commonly downregulated phosphorylation sites were found on proteins involved in the cell cycle and RNA splicing. Our phosphoproteomics also suggested key regulators immediately responding to LAT1 inhibition. Focusing on one of these regulators, protein kinase CK2, we revealed LAT1 inhibition decreased phosphorylation of CK2 substrate without changing CK2 enzymatic activity. Furthermore, LAT1 inhibition abolished interaction between CK2 and its regulatory protein NOLC1, which suggests regulatory mechanism of CK2 substrate protein specificity controlled by LAT1 inhibition. Moreover, we revealed that the combination of JPH203 with CK2 inhibitor resulted in the enhanced inhibition of proliferation and migration of BTC cells. CONCLUSION: This study provides new perspectives on LAT1-dependent cellular processes and a rationale for therapeutics targeting reprogrammed cancer metabolism.

L-type amino acid transporter 1 (LAT1) is highly expressed in various cancers and plays important roles not only in the amino acid uptake necessary for cancer growth but also in cellular signaling. Recent research studies have reported anticancer effects of LAT1 inhibitors and demonstrated their potential for cancer therapy. Here, we characterized the proteome and phosphoproteome in LAT1-inhibited cancer cells. We used JPH203, a selective LAT1 inhibitor, and performed tandem mass tag-based quantitative proteomics and phosphoproteomics on four biliary tract cancer cell lines sensitive to JPH203. Our analysis identified hundreds to thousands of differentially expressed proteins and phosphorylated sites, demonstrating the broad influence of LAT1 inhibition. Our findings showed various functional pathways altered by LAT1 inhibition, and provided possible regulators and key kinases in LAT1-inhibited cells. Comparison of these changes among cell lines provides insights into general pathways and regulators associated with LAT1 inhibition and particularly suggests the importance of cell cycle-related pathways and kinases. Moreover, we evaluated the anticancer effects of the combinations of JPH203 with cell cycle-related kinase inhibitors and demonstrated their potential for cancer therapy. This is the first study providing the proteome-wide scope of both protein expression and phosphorylation signaling perturbed by LAT1 inhibition in cancer cells.

Halogenated tyrosine/phenylalanine derivatives have been developed for use in tumor imaging and targeted alpha therapy. 3-Fluoro-α-methyl-l-tyrosine (FAMT), targeting amino acid transporter LAT1 (SLC7A5), is a cancer-specific positron emission tomography probe that exhibits high renal accumulation, which is supposed to be mediated by organic anion transporter OAT1 (SLC22A6). In the present study, we investigated the structural requirements of FAMT essential for interaction with OAT1. OAT1 transported FAMT with a Km of 171.9 μM. In structure-activity relationship analyses, removal of either the 3-halogen or 4-hydroxyl group from FAMT or its structural analog 3-iodo-α-methyl-l-tyrosine greatly decreased the interaction with OAT1, reducing the [14C]p-aminohippurate uptake inhibition and the efflux induction. By contrast, the α-methyl group, which is essential for LAT1 specificity, contributed to a lesser degree. In fluorinated tyrosine derivatives, fluorine at any position was accepted by OAT1 when there was a hydroxyl group at the ortho-position, whereas ortho-fluorine was less interactive when a hydroxyl group was at meta- or para-positions. The replacement of the ortho-fluorine with a bulky iodine atom greatly increased the interaction. In in vivo studies, probenecid decreased the renal accumulation (P < 0.001) and urinary excretion (P = 0.0012) of FAMT, whereas the plasma concentration was increased, suggesting the involvement of OAT1-mediated transepithelial organic anion excretion. LAT1-specific 2-fluoro-α-methyltyrosine, which had lower affinity for OAT1, exhibited lower renal accumulation (P = 0.0142) and higher tumor uptake (P = 0.0192) compared with FAMT. These results would provide a basis to design tumor-specific compounds that can avoid renal accumulation for tumor imaging and targeted alpha therapy. SIGNIFICANCE STATEMENT: We revealed the structural characteristics of halogenated tyrosine derivatives essential for interaction with the organic anion transporter responsible for their renal accumulation. We have confirmed that such interactions are important for renal handling and tumor uptake. The critical contribution of hydroxyl and halogen groups and their positions as well as the role of α-methyl group found in the present study may facilitate the development of tumor-specific compounds while avoiding renal accumulation for use in tumor imaging and targeted alpha therapy.

BACKGROUND: Tumor angiogenesis is regarded as a rational anti-cancer target. The efficacy and indications of anti-angiogenic therapies in clinical practice, however, are relatively limited. Therefore, there still exists a demand for revealing the distinct characteristics of tumor endothelium that is crucial for the pathological angiogenesis. L-type amino acid transporter 1 (LAT1) is well known to be highly and broadly upregulated in tumor cells to support their growth and proliferation. In this study, we aimed to establish the upregulation of LAT1 as a novel general characteristic of tumor-associated endothelial cells as well, and to explore the functional relevance in tumor angiogenesis. METHODS: Expression of LAT1 in tumor-associated endothelial cells was immunohistologically investigated in human pancreatic ductal adenocarcinoma (PDA) and xenograft- and syngeneic mouse tumor models. The effects of pharmacological and genetic ablation of endothelial LAT1 were examined in aortic ring assay, Matrigel plug assay, and mouse tumor models. The effects of LAT1 inhibitors and gene knockdown on cell proliferation, regulation of translation, as well as on the VEGF-A-dependent angiogenic processes and intracellular signaling were investigated in in vitro by using human umbilical vein endothelial cells. RESULTS: LAT1 was highly expressed in vascular endothelial cells of human PDA but not in normal pancreas. Similarly, high endothelial LAT1 expression was observed in mouse tumor models. The angiogenesis in ex/in vivo assays was suppressed by abrogating the function or expression of LAT1. Tumor growth in mice was significantly impaired through the inhibition of angiogenesis by targeting endothelial LAT1. LAT1-mediated amino acid transport was fundamental to support endothelial cell proliferation and translation initiation in vitro. Furthermore, LAT1 was required for the VEGF-A-dependent migration, invasion, tube formation, and activation of mTORC1, suggesting a novel cross-talk between pro-angiogenic signaling and nutrient-sensing in endothelial cells. CONCLUSIONS: These results demonstrate that the endothelial LAT1 is a novel key player in tumor angiogenesis, which regulates proliferation, translation, and pro-angiogenic VEGF-A signaling. This study furthermore indicates a new insight into the dual functioning of LAT1 in tumor progression both in tumor cells and stromal endothelium. Therapeutic inhibition of LAT1 may offer an ideal option to potentiate anti-angiogenic therapies.

Thermophilic proteins maintain their structure at high temperatures through a combination of various factors. Here, we report the ligand-induced stabilization of a thermophilic Ser/Thr protein kinase. Thermus thermophilus TpkD unfolds completely at 55 °C despite the optimum growth temperature of 75 °C. Unexpectedly, we found that the TpkD structure is drastically stabilized by its natural ligands ATP and ADP, as evidenced by the increase in the melting temperature to 80 °C. Such a striking effect of a substrate on thermostability has not been reported for other protein kinases. Conformational changes upon ATP binding were observed in fluorescence quenching and limited proteolysis experiments. Urea denaturation of Trp mutants suggested that ATP binding affects not only the ATP-binding site, but also the remote regions. Our findings shed light on thermoadaptation of thermophilic proteins.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease that predisposes individuals to developing benign neurofibromas and malignant peripheral nerve sheath tumors (MPNST). The mechanism of NF1-tumorigenesis or the curatives have not been established. Using unique trascriptome and proteome integration method, iPEACH (1), we previously identified translationally controlled tumor protein (TCTP) as a novel biological target for NF1-associated tumors (2). Here, we identified specific TCTP-interacting proteins by sequential affinity purification and data-independent mass spectrometry acquisition (AP-DIA/SWATH) to investigate the role of TCTP in NF1-associated malignant tumors. TCTP mainly interacts with proteins related to protein synthesis and especially to elongation factor complex components, including EF1A2, EF1B, EF1D, EF1G, and valyl-tRNA synthetase (VARS), in NF1-deficient malignant tumor cells. Interestingly, TCTP preferentially binds to EF1A2 (normally found only in neural and skeletal-muscle cells and several cancer cells), rather than EF1A1 despite the high homologies (98%) in their sequences. The docking simulation and further validations to study the interaction between TCTP and EF1A2 revealed that TCTP directly binds with EF1A2 via the contact areas of EF1A2 dimerization. Using unique and common sequences between EF1A2 and EF1A1 in AP-DIA/SWATH, we quantitatively validated the interaction of EF1A2 and TCTP/other elongation factors and found that TCTP coordinates the translational machinery of elongation factors via the association with EF1A2. These data suggest that TCTP activates EF1A2-dependent translation by mediating complex formation with other elongation factors. Inhibiting the TCTP-EF1A2 interaction with EF1A2 siRNAs or a TCTP inhibitor, artesunate, significantly down-regulated the factors related to protein translation and caused dramatic suppression of growth/translation in NF1-associated tumors. Our findings demonstrate that a specific protein translation machinery related to the TCTP-EF1A2 interaction is functionally implicated in the tumorigenesis and progression of NF1-associated tumors and could represent a therapeutic target.

SP7/Osterix (OSX) is a master regulatory transcription factor that activates a variety of genes during differentiation of osteoblasts. However, the influence of post-translational modifications on the regulation of its transactivation activity is largely unknown. Here, we report that sirtuins, which are NAD(+)-dependent deacylases, regulate lysine deacylation-mediated transactivation of OSX. Germline Sirt7 knockout mice develop severe osteopenia characterized by decreased bone formation and an increase of osteoclasts. Similarly, osteoblast-specific Sirt7 knockout mice showed attenuated bone formation. Interaction of SIRT7 with OSX leads to the activation of transactivation by OSX without altering its protein expression. Deacylation of lysine (K) 368 in the C-terminal region of OSX by SIRT7 promote its N-terminal transactivation activity. In addition, SIRT7-mediated deacylation of K368 also facilitates depropionylation of OSX by SIRT1, thereby increasing OSX transactivation activity. In conclusion, our findings suggest that SIRT7 has a critical role in bone formation by regulating acylation of OSX.