Identification and structure of AIMP2-DX2 for therapeutic perspectives.

Regulation of cell fate and lung cell differentiation is associated with Aminoacyl-tRNA synthetases (ARS)-interacting multifunctional protein 2 (AIMP2), which acts as a non-enzymatic component required for the multi-tRNA synthetase complex. In response to DNA damage, a component of AIMP2 separates from the multi-tRNA synthetase complex, binds to p53, and prevents its degradation by MDM2, inducing apoptosis. Additionally, AIMP2 reduces proliferation in TGF-ß and Wnt pathways, while enhancing apoptotic signaling induced by tumor necrosis factor-ß. Given the crucial role of these pathways in tumorigenesis, AIMP2 is expected to function as a broad-spectrum tumor suppressor. The full-length AIMP2 transcript consists of four exons, with a small section of the pre-mRNA undergoing alternative splicing to produce a variant (AIMP2-DX2) lacking the second exon. AIMP2-DX2 binds to FBP, TRAF2, and p53 similarly to AIMP2, but competes with AIMP2 for binding to these target proteins, thereby impairing its tumor-suppressive activity. AIMP2-DX2 is specifically expressed in a diverse range of cancer cells, including breast cancer, liver cancer, bone cancer, and stomach cancer. There is growing interest in AIMP2-DX2 as a promising biomarker for prognosis and diagnosis, with AIMP2-DX2 inhibition attracting significant interest as a potentially effective therapeutic approach for the treatment of lung, ovarian, prostate, and nasopharyngeal cancers. [BMB Reports 2024; 57(7): 318-323].

Influence of the interaction between p53 and ZNF568 on mitochondrial oxidative phosphorylation.

The tumor suppressor p53 plays important roles in suppressing the development and progression of cancer by responding to various stress signals. In addition, p53 can regulate the metabolic pathways of cancer cells by regulating energy metabolism and oxidative phosphorylation. Here, we present a mechanism for the interaction between p53 and ZNF568. Initially, we used X-ray crystallography to determine the irregular loop structure of the ZNF568 KRAB domain; this loop plays an important role in the interaction between p53 and ZNF568. In addition, Cryo-EM was used to examine how the p53 DBD and ZNF568 KRAB domains bind together. The function of ZNF568 on p53-mediated mitochondrial respiration was confirmed by measuring glucose consumption and lactate production. These findings show that ZNF568 can reduce p53-mediated mitochondrial respiratory activity by binding to p53 and inhibiting the transcription of SCO2. SIGNIFICANCE: ZNF568 can directly bind to the p53 DBD and transcriptionally regulate the SCO2 gene. SCO2 transcriptional regulation by interaction between ZNF568 and p53 may regulate the balance between mitochondrial respiration and glycolysis.

Structural identification and comprehension of human ALDH1L1-Gossypol complex.

The folate metabolism enzyme ALDH1L1 catalyzed 10-formyltetrahydrofolate to tetrahydrofolate and CO2. Non-small cell lung cancer cells (NSCLC) strongly express ALDH1L1. Gossypol binds to an allosteric site and disrupts the folate metabolism by preventing NADP+ binding. The Cryo-EM structures of tetrameric C-terminal aldehyde dehydrogenase human ALDH1L1 complex with gossypol were examined. Gossypol-bound ALDH1L1 interfered with NADP+ by shifting the allosteric site of the structural conformation, producing a closed-form NADP+ binding site. In addition, the inhibition activity of ALDH1L1 was targeted with gossypol in NSCLC. The gossypol treatment had anti-cancer effects on NSCLC by blocking NADPH and ATP production. These findings emphasize the structure characterizing ALDH1L1 with gossypol.

Structural basis for T cell immunoglobulin and mucin protein 3 and Toxascaris leonina galectin complex.

T-cell immunoglobulin and mucin protein 3 (Tim-3), also known as Hepatitis A virus cellular receptor 2, has been discovered to have a negative regulatory effect on murine T-cell responses. Galectin-9 exhibits various biological effects, including cell aggregation, eosinophil chemoattraction, activation, and apoptosis, observed in murine thymocytes, T-cells, and human melanoma cells. Such approach demonstrated that Galectin-9 acts as a binding partner on Tim-3 and mediates the T-cell inhibitory effects. Tl-gal is a homologous protein to galectin-9, isolated from the adult stage of the canine gastrointestinal nematode parasite Toxascaris leonina. However, molecular mechanism between Tim-3 and galectin-9 is still remain unknown. Here, we describe the cryo-electron microscopy and X-ray structures and interactions of the Tim-3 and Tl-gal complex as well as their biochemical and biophysical characterization. In the structure, Ser46 residue of Tl-gal NCRD was bound to Asp25 residue of hTim-3. Compared to our previous study, the binding site of the complex is the same as the sugar binding site (the Ser46 residue) of Tl-gal. In addition, analysis of the complex structure revealed that the four Tl-gal molecules were in an open form packing and one mTim-3 peptide was bound to one Tl-gal molecule. These observations suggest that how Tl-gal binds hTim3 is essential to understanding the molecular mechanism for the Tim-3-galectin 9 interaction that regulates immune responses. This could potentially serve as a therapeutic target for inflammatory diseases.

Structural basis of the oncogenic KRAS mutant and GJ101 complex.

KRAS mutations occur in a quarter of all human cancers. When activated in its GTP-bound form, RAS stimulates diverse cellular systems, such as cell division, differentiation, growth, and apoptosis through the activations of various signaling pathways, which include mitogen-activated protein kinase (MAPK), phosphoinositide 3 kinases (PI3K), and RAL-GEFs pathways. We found that GJ101 (65LYDVA69) binds directly to the KRAS mutant (G12V) and showed tumor-suppressive activity. In addition, the GJ101 peptide inhibited KRAS mutant as determined by a [α-32P] guanosine triphosphate (GTP) binding assay and suppressed pancreatic cell line in a cell proliferation assay. Herein, the complex structure of KRAS and GJ101 was clarified by X-ray crystallography. Isothermal titration calorimetry showed that GJ101 binds highly with KRAS mutant and the complex structure of KRAS G12V.GJ101 complex presented that the residue of Q61 directly interacted with L65 of GJ101. Overall, the results suggest GJ101 be considered a developmental starting point for KRAS G12V inhibitor.

DED Interaction of FADD and Caspase-8 in the Induction of Apoptotic Cell Death.

Fas-associated death domain (FADD) is an adapter molecule that bridges the interaction between receptor-interacting protein 1 (RIP1) and aspartate-specific cysteine protease-8 (caspase-8). As the primary mediator of apoptotic cell death, caspase-8 has two N-terminal death-effector domains (DEDs) and it interacts with other proteins in the DED subfamily through several conserved residues. In the tumor necrosis receptor-1 (TNFR-1)-dependent signaling pathway, apoptosis is triggered by the caspase-8/FADD complex by stimulating receptor internalization. However, the molecular mechanism of complex formation by the DED proteins remains poorly understood. Here, we found that direct DED-DED interaction between FADD and caspase-8 and the structure-based mutations (Y8D/I128A, E12A/I128A, E12R/I128A, K39A/I128A, K39D/I128A, F122A/I128A, and L123A/I128A) of caspase-8 disrupted formation of the stable DED complex with FADD. Moreover, the monomeric crystal structure of the caspase-8 DEDs (F122A/I128A) was solved at 1.7 Å. This study will provide new insight into the interaction mechanism and structural characteristics between FADD and caspase-8 DED subfamily proteins.

Oncogenic KRAS: Signaling and Drug Resistance.

RAS proteins play a role in many physiological signals transduction processes, including cell growth, division, and survival. The Ras protein has amino acids 188-189 and functions as GTPase. These proteins are switch molecules that cycle between inactive GDP-bound and active GTP-bound by guanine nucleotide exchange factors (GEFs). KRAS is one of the Ras superfamily isoforms (N-RAS, H-RAS, and K-RAS) that frequently mutate in cancer. The mutation of KRAS is essentially performing the transformation in humans. Since most RAS proteins belong to GTPase, mutated and GTP-bound active RAS is found in many cancers. Despite KRAS being an important molecule in mostly human cancer, including pancreatic and breast, numerous efforts in years past have persisted in cancer therapy targeting KRAS mutant. This review summarizes the biological characteristics of these proteins and the recent progress in the exploration of KRAS-targeted anticancer, leading to new insight.

Molecular Characteristics of RAGE and Advances in Small-Molecule Inhibitors.

Receptor for advanced glycation end-products (RAGE) is a member of the immunoglobulin superfamily. RAGE binds and mediates cellular responses to a range of DAMPs (damage-associated molecular pattern molecules), such as AGEs, HMGB1, and S100/calgranulins, and as an innate immune sensor, can recognize microbial PAMPs (pathogen-associated molecular pattern molecules), including bacterial LPS, bacterial DNA, and viral and parasitic proteins. RAGE and its ligands stimulate the activations of diverse pathways, such as p38MAPK, ERK1/2, Cdc42/Rac, and JNK, and trigger cascades of diverse signaling events that are involved in a wide spectrum of diseases, including diabetes mellitus, inflammatory, vascular and neurodegenerative diseases, atherothrombosis, and cancer. Thus, the targeted inhibition of RAGE or its ligands is considered an important strategy for the treatment of cancer and chronic inflammatory diseases.

Molecular Characteristics of Amyloid Precursor Protein (APP) and Its Effects in Cancer.

Amyloid precursor protein (APP) is a type 1 transmembrane glycoprotein, and its homologs amyloid precursor-like protein 1 (APLP1) and amyloid precursor-like protein 2 (APLP2) are highly conserved in mammals. APP and APLP are known to be intimately involved in the pathogenesis and progression of Alzheimer's disease and to play important roles in neuronal homeostasis and development and neural transmission. APP and APLP are also expressed in non-neuronal tissues and are overexpressed in cancer cells. Furthermore, research indicates they are involved in several cancers. In this review, we examine the biological characteristics of APP-related family members and their roles in cancer.

Understand KRAS and the Quest for Anti-Cancer Drugs.

The KRAS oncogene is mutated in approximately ~30% of human cancers, and the targeting of KRAS has long been highlighted in many studies. Nevertheless, attempts to target KRAS directly have been ineffective. This review provides an overview of the structure of KRAS and its characteristic signaling pathways. Additionally, we examine the problems associated with currently available KRAS inhibitors and discuss promising avenues for drug development.

Structural basis of the p53 DNA binding domain and PUMA complex.

PUMA (p53-upregulated modulator of apoptosis) is localized in mitochondria and a direct target in p53-mediated apoptosis. p53 elicits mitochondrial apoptosis via transcription-dependent and independent mechanisms. p53 is known to induce apoptosis via the transcriptional induction of PUMA, which encodes proapoptotic BH3-only members of the Bcl-2 protein family. However, the transcription-independent mechanisms of human PUMA remain poorly defined. For example, it is not known whether PUMA interacts directly with the DNA binding domain (DBD: residues 92-293) of p53 in vitro. Here, the structure of the complex between the DBD of p53 and PUMA peptide was elucidated by X-ray crystallography. Isothermal titration calorimetry showed that PUMA peptide binds strongly with p53 DBD, and the crystal structure of p53-PUMA peptide complex revealed it contains four molecules of p53 DBD and one PUMA peptide per asymmetric unit in space group P1. PUMA peptide bound to the N-terminal residues of p53 DBD. A cell proliferation assay demonstrated PUMA peptide inhibited the growth of a lung cancer cell line. These results contribute to understanding of the mechanism responsible for p53-mediated apoptosis.

A H-REV107 Peptide Inhibits Tumor Growth and Interacts Directly with Oncogenic KRAS Mutants.

Kirsten-RAS (KRAS) has been the target of drugs because it is the most mutated gene in human cancers. Because of the low affinity of drugs for KRAS mutations, it was difficult to target these tumor genes directly. We found a direct interaction between KRAS G12V and tumor suppressor novel H-REV107 peptide with high binding affinity. We report the first crystal structure of an oncogenic mutant, KRAS G12V-H-REV107. This peptide was shown to interact with KRAS G12V in the guanosine diphosphate (GDP)-bound inactive state and to form a stable complex, blocking the activation function of KRAS. We showed that the peptide acted as an inhibitor of mutant KRAS targets by [α-32P] guanosine triphosphate (GTP) binding assay. The H-REV107 peptide inhibited pancreatic cancer and colon cancer cell lines in cell proliferation assay. Specially, the H-REV107 peptide can suppress pancreatic tumor growth by reduction of tumor volume and weight in xenotransplantation mouse models. Overall, the results presented herein will facilitate development of novel drugs for inhibition of KRAS mutations in cancer patients.

Machilin A Inhibits Tumor Growth and Macrophage M2 Polarization Through the Reduction of Lactic Acid.

Lactate dehydrogenase A (LDHA) is an important enzyme responsible for cancer growth and energy metabolism in various cancers via the aerobic glycolytic pathway. Here, we report that machilin A (MA), which acts as a competitive inhibitor by blocking the nicotinamide adenine dinucleotide (NAD) binding site of LDHA, suppresses growth of cancer cells and lactate production in various cancer cell types, including colon, breast, lung, and liver cancers. Furthermore, MA markedly decreased LDHA activity, lactate production, and intracellular adenosine triphosphate (ATP) levels induced by hypoxia-induced LDHA expression in cancer cells, and significantly inhibited colony formation, leading to reduced cancer cell survival. In mouse models inoculated with murine Lewis lung carcinoma, MA significantly suppressed tumor growth as observed by a reduction of tumor volume and weight; resulting from the inhibition of LDHA activity. Subsequently, the suppression of tumor-derived lactic acid in MA-treated cancer cells resulted in decrease of neovascularization through the regulation of alternatively activated macrophages (M2) polarization in macrophages. Taken together, we suggest that the reduction of lactate by MA in cancer cells directly results in a suppression of cancer cell growth. Furthermore, macrophage polarization and activation of endothelial cells for angiogenesis were indirectly regulated preventing lactate production in MA-treated cancer cells.

Structural basis of the cystein protease inhibitor Clonorchis sinensis Stefin-1.

Cystein protease plays a critical role as a virulence factor in the development and progression of various diseases. Cystatin is a superfamily of cysteine protease inhibitors that participates in various physiological and pathological processes. The cysteine protease inhibitor CsStein-1 isolated from Clonorchis sinensis belongs to the type 1 stefin of cystatins. This inhibitor regulates the activity and processing of CsCF (Cathepsin F of Clonorchis sienesis), which plays an important role in parasite nutrition and host-parasite interaction. CsStefin-1 has also been proposed as a host immune modulator and a participant in the mechanism associated with anti-inflammatory ability. Here, we report the first crystal structure of CsStefin-1 determined by the multi-wavelength anomalous diffraction (MAD) method to 2.3 Å. There are six molecules of CsStefin-1 per asymmetric unit, with a solvent content of 36.5%. The structure of CsStefin-1 is composed of twisted four-stranded antiparallel ß-sheets, a central α-helix, and a short α-helix. We also demonstrate that CsStefin-1 binds to CsCF-8 cysteine protease and inhibits its activity. In addition, a molecular docking model of CsStefin-1 and CsCF-8 was developed using homology modeling based on their structures. The structural information regarding CsStefin-1 and molecular insight into its interaction with CsCF-8 are important to understanding their biological function and to design of inhibitors that modulate cysteine protease activity.

Structure and function of vascular endothelial growth factor and its receptor system.

Vascular endothelial growth factor and its receptor (VEGF-VEGFR) system play a critical role in the regulation of angiogenesis and lymphangiogenesis in vertebrates. Each of the VEGF has specific receptors, which it activates by binding to the extracellular domain of the receptors, and, thus, regulates the angiogenic balance in the early embryonic and adult stages. However, de-regulation of the VEGF-VEGFR implicates directly in various diseases, particularly cancer. Moreover, tumor growth needs a dedicated blood supply to provide oxygen and other essential nutrients. Tumor metastasis requires blood vessels to carry tumors to distant sites, where they can implant and begin the growth of secondary tumors. Thus, investigation of signaling systems related to the human disease, such as VEGF-VEGFR, will facilitate the development of treatments for such illnesses. [BMB Reports 2018; 51(2): 73-78].

Molecular interaction between K-Ras and H-REV107 in the Ras signaling pathway.

Ras proteins are small GTPases that serve as master moderators of a large number of signaling pathways involved in various cellular processes. Activating mutations in Ras are found in about one-third of cancers. H-REV107, a K-Ras binding protein, plays an important role in determining K-Ras function. H-REV107 is a member of the HREV107 family of class II tumor suppressor genes and a growth inhibitory Ras target gene that suppresses cellular growth, differentiation, and apoptosis. Expression of H-REV107 was strongly reduced in about 50% of human carcinoma cell lines. However, the specific molecular mechanism by which H-REV107 inhibits Ras is still unknown. In the present study, we suggest that H-REV107 forms a strong complex with activating oncogenic mutation Q61H K-Ras from various biochemical binding assays and modeled structures. In addition, the interaction sites between K-Ras and H-REV107 were predicted based on homology modeling. Here, we found that some structure-based mutants of the K-Ras disrupted the complex formation with H-REV107. Finally, a novel molecular mechanism describing K-Ras and H-REV107 binding is suggested and insights into new K-Ras effector target drugs are provided.

Structure, signaling and the drug discovery of the Ras oncogene protein.

Mutations in Ras GTPase are among the most common genetic alterations in human cancers. Despite extensive research investigating Ras proteins, their functions still remain a challenge over a long period of time. The currently available data suggests that solving the outstanding issues regarding Ras could lead to development of effective drugs that could have a significant impact on cancer treatment. Developing a better understanding of their biochemical properties or modes of action, along with improvements in their pharmacologic profiles, clinical design and scheduling will enable the development of more effective therapies. [BMB Reports 2017; 50(7): 355-360].

Structural characterization of As-MIF and hJAB1 during the inhibition of cell-cycle regulation.

The biological activities of macrophage migration inhibitory factor (MIF) might be mediated through a classical receptormediated or non-classical endocytic pathway. JAB1 (C-Jun activation domain-binding protein-1) promotes the degradation of the tumor suppressor, p53, and the cyclin-dependent kinase inhibitor, p27. When MIF and JAB1 are bound to each other in various intracellular sites, MIF inhibits the positive regulatory effects of JAB1 on the activity of AP-1. The intestinal parasite, Anisakis simplex, has an immunomodulatory effect. The molecular mechanism of action of As-MIF and human JAB1 are poorly understood. In this study, As-MIF and hJAB1 were expressed and purified with high solubility. The structure of As-MIF and hJAB1 interaction was modeled by homology modeling based on the structure of Ace-MIF. This study provides evidence indicating that the MIF domain of As-MIF interacts directly with the MPN domain of hJAB1, and four structure-based mutants of As-MIF and hJAB1 disrupt the As-MIF-hJAB1 interaction. [BMB Reports 2017; 50(5): 269-274].

Structural insights of homotypic interaction domains in the ligand-receptor signal transduction of tumor necrosis factor (TNF).

Several members of tumor necrosis factor receptor (TNFR) superfamily that these members activate caspase-8 from death-inducing signaling complex (DISC) in TNF ligand-receptor signal transduction have been identified. In the extrinsic pathway, apoptotic signal transduction is induced in death domain (DD) superfamily; it consists of a hexahelical bundle that contains 80 amino acids. The DD superfamily includes about 100 members that belong to four subfamilies: death domain (DD), caspase recruitment domain (CARD), pyrin domain (PYD), and death effector domain (DED). This superfamily contains key building blocks: with these blocks, multimeric complexes are formed through homotypic interactions. Furthermore, each DD-binding event occurs exclusively. The DD superfamily regulates the balance between death and survival of cells. In this study, the structures, functions, and unique features of DD superfamily members are compared with their complexes. By elucidating structural insights of DD superfamily members, we investigate the interaction mechanisms of DD domains; these domains are involved in TNF ligand-receptor signaling. These DD superfamily members play a pivotal role in the development of more specific treatments of cancer. [BMB Reports 2016; 49(3): 159-166].

Structural and Functional Insight into Proliferating Cell Nuclear Antigen.

Proliferating cell nuclear antigen (PCNA) is a critical eukaryotic replication accessory factor that supports DNA binding in DNA processing, such as DNA replication, repair, and recombination. PCNA consists of three toroidal-shaped monomers that encircle doublestranded DNA. The diverse functions of PCNA may be regulated by its interactions with partner proteins. Many of the PCNA partner proteins generally have a conserved PCNAinteracting peptide (PIP) motif, located at the N- or C- terminal region. The PIP motif forms a 310 helix that enters into the hydrophobic groove produced by an interdomain-connecting loop, a central loop, and a C-terminal tail in the PCNA. Post-translational modification of PCNA also plays a critical role in regulation of its function and binding partner proteins. Structural and biochemical studies of PCNA-protein will be useful in designing therapeutic agents, as well as estimating the outcome of anticancer drug development. This review summarizes the characterization of eukaryotic PCNA in relation to the protein structures, functions, and modifications, and interaction with proteins.