In eukaryotic cells, mitochondria are closely tethered to the endoplasmic reticulum (ER) at sites called mitochondria‐associated ER membranes (MAMs). Ca2+ ion and phospholipid transfer occurs at MAMs ...to support diverse cellular functions. Unlike those in yeast, the protein complexes involved in phospholipid transfer at MAMs in humans have not been identified. Here, we determine the crystal structure of the tetratricopeptide repeat domain of PTPIP51 (PTPIP51_TPR), a mitochondrial protein that interacts with the ER‐anchored VAPB protein at MAMs. The structure of PTPIP51_TPR shows an archetypal TPR fold, and an electron density map corresponding to an unidentified lipid‐like molecule probably derived from the protein expression host is found in the structure. We reveal functions of PTPIP51 in phospholipid binding/transfer, particularly of phosphatidic acid, in vitro. Depletion of PTPIP51 in cells reduces the mitochondrial cardiolipin level. Additionally, we confirm that the PTPIP51–VAPB interaction is mediated by the FFAT‐like motif of PTPIP51 and the MSP domain of VAPB. Our findings suggest that PTPIP51 is a phospholipid transfer protein with a MAM‐tethering function.
SYNOPSIS
The crystal structure and biochemical analyses of PTPIP51, a mitochondrial protein localized at the mitochondria‐associated ER membrane (MAM), revealed its phospholipid binding and transfer activity.
The crystal structure of the TPR domain of PTPIP51 at 1.45 Å resolution revealed the presence of a lipid‐like serpentine electron density.
PTPIP51 has phospholipid (especially phosphatidic acid) binding and transfer functions in vitro.
Mitochondrial cardiolipin levels are affected by PTPIP51.
The crystal structure and biochemical analyses of PTPIP51, a mitochondrial protein localized at the mitochondria‐associated ER membrane (MAM), revealed its phospholipid binding and transfer activity.
To determine prospectively the diagnostic performance of unenhanced computed tomography (CT) in the assessment of macrovesicular steatosis in potential donors for living donor liver transplantation ...by using same-day biopsy as a reference standard.
Institutional review board approval and informed consent were obtained. A total of 154 candidates, including 104 men (mean age, 30.2 years +/- 10.3 standard deviation) and 50 women (mean age, 31.8 years +/- 11.2), underwent same-day unenhanced CT and ultrasonography-guided liver biopsy. Histologic degree of macrovesicular steatosis was determined. Three liver attenuation indices were derived: liver-to-spleen attenuation ratio (CT(L)(/S)), difference between hepatic and splenic attenuation (CT(L)(-S)), and blood-free hepatic parenchymal attenuation (CT(LP)). Regression equations were used to quantitatively estimate the degree of macrovesicular steatosis. Limits of agreement between estimated macrovesicular steatosis and the reference standard were calculated. Receiver operating characteristic analyses were used to determine the performance of each index for qualitative diagnosis of macrovesicular steatosis of 30% or greater. The cutoff value that provided a balance between sensitivity and specificity and the highest cutoff value that yielded 100% specificity were determined.
Limits of agreement were -14% to 14% for CT(L)(/S) and CT(L)(-S) and -13% to 13% for CT(LP). Performance in diagnosing macrovesicular steatosis of 30% or greater was not significantly different among indices (P > .05). Cutoff values of 0.9, -7, and 58 were determined for CT(L)(/S), CT(L)(-S), and CT(LP), respectively, and provided a balance between sensitivity and specificity. Cutoff values of 0.8, -9, and 42 were determined for CT(L)(/S), CT(L)(-S), and CT(LP), respectively, and yielded 100% specificity for all indices, with corresponding sensitivities of 82%, 82%, and 73% for CT(L)(/S), CT(L)(-S), and CT(LP), respectively.
Diagnostic performance of unenhanced CT for quantitative assessment of macrovesicular steatosis is not clinically acceptable. Unenhanced CT, however, provides high performance in qualitative diagnosis of macrovesicular steatosis of 30% or greater.
Abstract
When primary cancer faces limited oxygen and nutrient supply, it undergoes an epithelial–mesenchymal transition, which increases cancer cell motility and invasiveness. The migratory and ...invasive cancer cells often exert aggressive cancer development or even cancer metastasis. In this study, we investigated a novel compound, 3-acetyl-5,8-dichloro-2-((2,4-dichlorophenyl)amino)quinolin-4(1H)-one (ADQ), that showed significant suppression of wound healing and cellular invasion. This compound also inhibited anchorage-independent cell growth, multicellular tumor spheroid survival/invasion, and metalloprotease activities. The anti-proliferative effects of ADQ were mediated by inhibition of the Akt pathway. In addition, ADQ reduced the expression of mesenchymal markers of cancer cells, which was associated with the suppressed expression of Twist1. In conclusion, ADQ successfully suppressed carcinogenic activity by inhibiting the Akt signaling pathway and Twist1, which suggests that ADQ may be an efficient candidate for cancer drug development.
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Structure based virtual screening attempts to discover DUSP1 inhibitors have yielded a scaffold featuring benzoxazole and acylthiourea pharmacophore. A series of its analogues were ...synthesized to explore structure activity relationship (SAR) of DUSP1 inhibition.
Cdc25 phosphatases have been considered as attractive drug targets for anticancer therapy because of the correlation of their overexpression with a wide variety of cancers. We have been able to ...identify five novel Cdc25 phosphatase inhibitors with micromolar activity by means of a computer-aided drug design protocol involving the homology modeling of Cdc25A and the virtual screening with the automated AutoDock program implementing the effects of ligand solvation in the scoring function. Because the newly discovered inhibitors are structurally diverse and reveal a significant potency with IC50 values lower than 10 μM, they can be considered for further development by structure−activity relationship studies or de novo design methods. The differences in binding modes of the identified inhibitors in the active sites of Cdc25A and B are discussed in detail.
We have identified novel PTPMT1 inhibitors based on the virtual screening with docking simulations and in vitro enzyme assay.
Dual-specificity protein tyrosine phosphatase localized to mitochondrion ...1 (PTPMT1) has recently proved to be a promising therapeutic target for the treatment of type II diabetes. Herein we report the first example for a successful application of the structure-based virtual screening to identify the novel inhibitors of human PTPMT1. These inhibitors were computationally screened for having desirable physicochemical properties as a drug candidate and reveal a high potency with IC50 values ranging from 0.7 to 17.3μM. Therefore, they deserve consideration for further development by structure–activity relationship studies to optimize the antidiabetic activities. Structural features relevant to the stabilization of the newly identified inhibitors in the active site of PTPMT1 are addressed in detail.
Oxidative stress is one of the major causes of neuronal cell death in disorders such as perinatal hypoxia and ischemia. Protein phosphorylation is the most significant PTM of proteins and plays an ...important role in stress-induced signal transduction. Thus, the analysis of alternative protein phosphorylation states which occur during oxidative stress-induced cell death could provide valuable information regarding cell death. In this study, a reference phosphoproteome map of the mouse hippocampal cell line HT22 was constructed based on 125 spots that were identified by MALDI-TOF or LC-ESI-Q-TOF-MS analysis. In addition, proteins of HT22 cells at various stages of oxidative stress-induced cell death were separated by 2-DE and alterations in phosphoproteins were detected by Pro-Q Diamond staining. A total of 17 spots showing significant quantitative changes and seven newly appearing spots were identified after glutamate treatment. Splicing factor 2, peroxiredoxin 2, S100 calcium binding protein A11, and purine nucleoside phosphorylase were identified as up- or down-regulated proteins. CDC25A, caspase-8, and cyp51 protein appeared during oxidative stress-induced cell death. The data in this study from phosphoproteomic analysis provide a valuable resource for the understanding of HT22 cell death mechanisms mediated by oxidative stress.
The PRL phosphatases, which constitute a subfamily of the protein tyrosine phosphatases (PTPs), are implicated in oncogenic and metastatic processes. Here, we report the crystal structure of human ...PRL-1 determined at 2.7
Å resolution. The crystal structure reveals the shallow active-site pocket with highly hydrophobic character. A structural comparison with the previously determined NMR structure of PRL-3 exhibits significant differences in the active-site region. In the PRL-1 structure, a sulfate ion is bound to the active-site, providing stabilizing interactions to maintain the canonically found active conformation of PTPs, whereas the NMR structure exhibits an open conformation of the active-site. We also found that PRL-1 forms a trimer in the crystal and the trimer exists in the membrane fraction of cells, suggesting the possible biological regulation of PRL-1 activity by oligomerization. The detailed structural information on the active enzyme conformation and regulation of PRL-1 provides the structural basis for the development of potential inhibitors of PRL enzymes.
Insulinoma-associated protein-2 (IA-2) is a major autoantigen in type 1 diabetes that occurs through autoimmune-mediated beta-cell destruction. We present here the crystal structure of the protein ...tyrosine phosphatase (PTP)-like domain of human IA-2. The structure reveals a canonical PTP domain with the closed WPD loop over the active site pocket, explaining the lack of enzyme activity in the native protein. The structural interpretation of previous mutagenesis studies indicates that the B-cell epitopes are concentrated on two distinctive regions on peripheral loops of the central beta-sheet surrounding T-cell epitopes within the sheet. The detailed structural information on immune epitopes provides a framework for the future development of immune intervention strategies against diabetes.
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