ABSTRACT
A 27-year-old female patient presented with a 3-month history of bilateral orbital and facial edema accompanied by skin erythema and heliotrope rash. The left lesion was more critical than the right. Limb muscles were occasionally sore. On physical examination, scattered hemorrhagic rashes were observed on the patient's face and neck. Upon laboratory testing, creatine kinase (CK) was markedly elevated at 1,543.2 U/l, while CK-MB isoenzyme (27.4 U/l), α-hydroxybutyric dehydrogenase (188.4 U/l) and aspartate aminotransferase (65.3 U/l) were marginally elevated. The patient was diagnosed with dermatomyositis due to the dermatological features and elevated CK. Cancer marker analysis revealed positivity for carbohydrate antigen 125 (68.15 U/ml). Magnetic resonance imaging revealed thickening of the soft tissue of the left eyelid; furthermore, concurrent long T1- and T2-weighted signals (fat saturation) were compatible with inflammatory infiltration. Non-enhanced computed tomography identified that the right accessory region (right ovary and fallopian tube) had a cystic mass with a significant fat component and thickening of the anterior uterine wall. Gynecological ultrasound findings indicated an ovarian teratoma (8.7×7.8×9.3 cm) and uterine myoma (3.6×3.1 cm). The patient's dermatological symptoms and laboratory results were significantly relieved one week after surgical removal of the teratoma with the aid of hydrocortisone, methylprednisolone and methotrexate therapy.
ABSTRACT
The forkhead-associated (FHA) domain recognizes phosphothreonine (pT) with high specificity and functional diversity. TIFA (TRAF-interacting protein with an FHA domain) is the smallest FHA-containing human protein. Its overexpression was previously suggested to provoke NF-κB activation, yet its exact roles in this signaling pathway and the underlying molecular mechanism remain unclear. Here we identify a novel threonine phosphorylation site on TIFA and show that this phosphorylated threonine (pT) binds with the FHA domain of TIFA, leading to TIFA oligomerization and TIFA-mediated NF-κB activation. Detailed analysis indicated that unphosphorylated TIFA exists as an intrinsic dimer and that the FHA-pT9 binding occurs between different dimers of TIFA. In addition, silencing of endogenous TIFA resulted in attenuation of tumor necrosis factor alpha (TNF-α)-mediated downstream signaling. We therefore propose that the TIFA FHA-pT9 binding provides a previously unidentified link between TNF-α stimulation and NF-κB activation. The intermolecular FHA-pT9 binding between dimers also represents a new mechanism for the FHA domain.
Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/metabolism , NF-kappa B/metabolism , Tumor Necrosis Factor-alpha/metabolism , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/genetics , Amino Acid Sequence , Amino Acid Substitution , Antibodies, Monoclonal , Forkhead Transcription Factors/chemistry , Forkhead Transcription Factors/metabolism , HEK293 Cells , Humans , Models, Biological , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Phosphothreonine/chemistry , Protein Interaction Domains and Motifs , Protein Multimerization , RNA Interference , RNA, Small Interfering/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Signal TransductionABSTRACT
This study demonstrated that pyrrolidine dithiocarbamate (PDTC), a potent nuclear factor-κB inhibitor, showed stronger inhibitory effects on nuclear factor-κB activation in endometriotic stromal cells than in normal endometrial stromal cells as determined by electrophoretic mobility shift assay and Western blot analysis. Pretreatment of endometriotic stromal cells with PDTC attenuated tumor necrosis factor-α-induced expressions of CD44s, matrix metalloproteinase-9, and vascular endothelial growth factor whereas reversed tumor necrosis factor-α-reduced expressions of tissue inhibitor of metalloproteinase-1 revealed by reverse transcriptase polymerase chain reaction and Western blot analysis, suggesting that PDTC may represent a novel therapeutic strategy for treatment of endometriosis.
Subject(s)
Endometriosis/drug therapy , NF-kappa B/antagonists & inhibitors , Peritoneal Diseases/drug therapy , Pyrrolidines/therapeutic use , Thiocarbamates/therapeutic use , Antioxidants/pharmacology , Antioxidants/therapeutic use , Case-Control Studies , Cells, Cultured , Drug Evaluation, Preclinical , Endometriosis/genetics , Endometriosis/metabolism , Endometriosis/pathology , Female , Gene Expression/drug effects , Humans , Hyaluronan Receptors/genetics , Hyaluronan Receptors/metabolism , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , NF-kappa B/metabolism , Ovarian Cysts/pathology , Peritoneal Diseases/genetics , Peritoneal Diseases/metabolism , Peritoneal Diseases/pathology , Pyrrolidines/pharmacology , Stromal Cells/drug effects , Stromal Cells/metabolism , Stromal Cells/pathology , Thiocarbamates/pharmacology , Tissue Inhibitor of Metalloproteinase-1/genetics , Tissue Inhibitor of Metalloproteinase-1/metabolismABSTRACT
Human Chk2 is a newly identified tumor suppressor protein involved in signaling pathways in response to DNA damage. The protein consists of a forkhead-associated (FHA) domain and a kinase domain. Identification of binding partners of the Chk2FHA domain is important in understanding the roles of Chk2 in signaling. We report development of an approach involving the use of combinatorial libraries, pull-down assays, surface plasmon resonance (SPR), and nuclear magnetic resonance (NMR) methods to identify possible candidates for the binding sites of Chk2FHA. The approach has been used to identify Thr329 of p53 and Thr1852 of breast cancer type 1 susceptibility protein (BRCA1) as very likely biological binding sites of Chk2FHA. The results provide useful leads for further biological analyses of cell signaling involving the FHA domain of Chk2 protein.
Subject(s)
Carrier Proteins/chemistry , Nuclear Proteins/chemistry , Peptide Library , Protein Serine-Threonine Kinases/chemistry , Transcription Factors/chemistry , Tumor Suppressor Protein p53/chemistry , Binding Sites , Checkpoint Kinase 2 , Forkhead Transcription Factors , Humans , Protein Binding , Protein Serine-Threonine Kinases/classification , Protein Structure, Tertiary , Structure-Activity Relationship , Ubiquitin-Protein LigasesABSTRACT
The p16(INK4A)/CDKN2A tumor suppressor gene is known to be inactivated in up to 98% of human pancreatic cancer specimens and represents a potential target for novel therapeutic intervention. Chemically induced pancreatic tumors in Syrian golden hamsters have been demonstrated to share many morphologic and biological similarities with human pancreatic tumors and this model may be appropriate for studying therapies targeting p16(INK4A)/CDKN2A. The purpose of this study was to investigate the fundamental biochemistry of hamster P16 protein. Using both in vivo and in vitro approaches, the CDK4 binding affinity, kinase inhibitory activity, and thermodynamic stability of hamster and human P16 proteins were evaluated. Furthermore, a structural model of hamster P16 protein was generated. These studies demonstrate that hamster P16 protein is biochemically indistinguishable from human P16 protein. From a biochemical perspective, these data strongly support the study of p16-related pancreatic oncogenesis and cancer therapies in the hamster model.
