Potent inhibitors of lipoprotein-associated phospholipase A(2): benzaldehyde O-heterocycle-4-carbonyloxime

A series of multi-substituted oximes were prepared and their potencies for inhibiting lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) activity were evaluated in vitro. Among them, compounds 3a, 3b, and 3m were identified to display a micromolar potency for inhibiting Lp-PLA(2) in whole human plasma and isolated human LDL. Based on these results, structure-activity relationship was studied on modification of three parts of R(1), R(2), and R(3) to identify a potent pharmacophore for Lp-PLA(2). In an attempt to introduce various functional groups at R(2) and R(3), we discovered that replacement of less lipophilic groups led to an increase of inhibitory activity. Among the tested oxime derivatives, cyano- and morpholino-substituted analogue 4f at R(2) and R(3) had the highest potency with an IC(50) value of 0.05 microM in whole human plasma.     

The PDZ-binding motif of the avian NS1 protein affects transmission of the 2009 influenza A(H1N1) virus

By nature of their segmented RNA genome, influenza A viruses (IAVs) have the potential to generate variants through a reassortment process. The influenza nonstructural (NS) gene is critical for a virus to counteract the antiviral responses of the host. Therefore, a newly acquired NS segment potentially determines the replication efficiency of the reassortant virus in a range of different hosts. In addition, the C-terminal PDZ-binding motif (PBM) has been suggested as a pathogenic determinant of IAVs. To gauge the pandemic potential from human and avian IAV reassortment, we assessed the replication properties of NS-reassorted viruses in cultured cells and in the lungs of mice and determined their transmissibility in guinea pigs. Compared with the recombinant A/Korea/01/2009 virus (rK09; 2009 pandemic H1N1 strain), the rK09/VN:NS virus, in which the NS gene was adopted from the A/Vietnam/1203/2004 virus (a human isolate of the highly pathogenic avian influenza H5N1 virus strains), exhibited attenuated virulence and reduced transmissibility. However, the rK09/VN:NS-PBM virus, harboring the PBM in the C-terminus of the NS1 protein, recovered the attenuated virulence of the rK09/VN:NS virus. In a guinea pig model, the rK09/VN:NS-PBM virus showed even greater transmission efficiency than the rK/09 virus. These results suggest that the PBM in the NS1 protein may determine viral persistence in the human and avian IAV interface.     

Acetylation changes tau interactome to degrade tau in Alzheimer’s disease animal and organoid models

Alzheimer's disease (AD) is an age‐related neurodegenerative disease. The most common pathological hallmarks are amyloid plaques and neurofibrillary tangles in the brain. In the brains of patients with AD, pathological tau is abnormally accumulated causing neuronal loss, synaptic dysfunction, and cognitive decline. We found a histone deacetylase 6 (HDAC6) inhibitor, CKD‐504, changed the tau interactome dramatically to degrade pathological tau not only in AD animal model (ADLP^APT) brains containing both amyloid plaques and neurofibrillary tangles but also in AD patient‐derived brain organoids. Acetylated tau recruited chaperone proteins such as Hsp40, Hsp70, and Hsp110, and this complex bound to novel tau E3 ligases including UBE2O and RNF14. This complex degraded pathological tau through proteasomal pathway. We also identified the responsible acetylation sites on tau. These dramatic tau‐interactome changes may result in tau degradation, leading to the recovery of synaptic pathology and cognitive decline in the ADLP^APT mice.     

TGF-β1 signaling plays a dominant role in the crosstalk between TGF-β1 and the aryl hydrocarbon receptor ligand in prostate epithelial cells

Crosstalk between the aryl hydrocarbon receptor (AhR) and transforming growth factor-β1 (TGF-β1) signaling has been observed in various experimental models. However, both molecular mechanism underlying this crosstalk and tissue-specific context of this interaction are still only partially understood. In a model of human non-tumorigenic prostate epithelial cells BPH-1, derived from the benign prostatic hyperplasia, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) persistently activates the AhR signaling pathway and induces expression of xenobiotic metabolizing enzymes, such as CYP1A1 or CYP1B1. Here we demonstrate that TGF-β1 suppresses the AhR-mediated gene expression through multiple mechanisms, involving inhibition of AhR expression and down-regulation of nuclear AhR, via a SMAD4-dependent pathway. In contrast, TCDD-induced AhR signaling does not affect either TGF-β1-regulated gene expression or epithelial-to-mesenchymal transition. These observations suggest that, in the context of prostate epithelium, TGF-β1 signaling plays a dominant role in the crosstalk with AhR signaling pathway. Given the importance of TGF-β1 signaling in regulation of prostate epithelial tissue homeostasis, as well as the recently revealed role of AhR in prostate development and tumorigenesis, the above findings contribute to our understanding of the mechanisms underlying the crosstalk between the two signaling pathways in the prostate-specific context.     

Apoptosis inhibitor 5 is an endogenous inhibitor of caspase‐2

Caspases are key enzymes responsible for mediating apoptotic cell death. Across species, caspase‐2 is the most conserved caspase and stands out due to unique features. Apart from cell death, caspase‐2 also regulates autophagy, genomic stability and ageing. Caspase‐2 requires dimerization for its activation which is primarily accomplished by recruitment to high molecular weight protein complexes in cells. Here, we demonstrate that apoptosis inhibitor 5 (API5/AAC11) is an endogenous and direct inhibitor of caspase‐2. API5 protein directly binds to the caspase recruitment domain (CARD) of caspase‐2 and impedes dimerization and activation of caspase‐2. Interestingly, recombinant API5 directly inhibits full length but not processed caspase‐2. Depletion of endogenous API5 leads to an increase in caspase‐2 dimerization and activation. Consistently, loss of API5 sensitizes cells to caspase‐2‐dependent apoptotic cell death. These results establish API5/AAC‐11 as a direct inhibitor of caspase‐2 and shed further light onto mechanisms driving the activation of this poorly understood caspase.     

Crystal Structure of Human Myotubularin-Related Protein 1 Provides Insight into the Structural Basis of Substrate Specificity

Myotubularin-related protein 1 (MTMR1) is a phosphatase that belongs to the tyrosine/dual-specificity phosphatase superfamily. MTMR1 has been shown to use phosphatidylinositol 3-monophosphate (PI(3)P) and/or phosphatidylinositol 3,5-bisphosphate (PI(3,5)P₂) as substrates. Here, we determined the crystal structure of human MTMR1. The refined model consists of the Pleckstrin homology (PH)-GRAM and phosphatase (PTP) domains. The overall structure was highly similar to the previously reported MTMR2 structure. Interestingly, two phosphate molecules were coordinated by strictly conserved residues located in the C(X)₅R motif of the active site. Additionally, our biochemical studies confirmed the substrate specificity of MTMR1 for PI(3)P and PI(3,5)P₂ over other phosphatidylinositol phosphates. Our structural and enzymatic analyses provide insight into the catalytic mechanism and biochemical properties of MTMR1.     

ABC transporters affect the detection of intracellular oxidants by fluorescent probes

Intracellular production of reactive oxygen species (ROS) plays an important role in the control of cell physiology. For the assessment of intracellular ROS production, a plethora of fluorescent probes is commonly used. Interestingly, chemical structures of these probes imply they could be substrates of plasma membrane efflux pumps, called ABC transporters. This study tested whether the determination of intracellular ROS production and mitochondrial membrane potential by selected fluorescent probes is modulated by the expression and activity of ABC transporters. The sub-clones of the HL-60 cell line over-expressing MDR1, MRP1 and BCRP transporters were employed. ROS production measured by luminol- and L-012-enhaced chemiluminescence and cytochrome c reduction assay showed similar levels of ROS production in all the employed cell lines. It was proved that dihydrorhodamine 123, dihexiloxocarbocyanine iodide, hydroethidine, tetrachloro-tetraethylbenzimidazolocarbo-cyanine iodide and tetramethylrhodamine ethyl ester perchlorate are substrates for MDR1; dichlorodihydrofluoresceine, hydroethidine and tetramethylrhodamine ethyl ester perchlorate are substrates for MRP1; dichlorodihydrofluoresceine, dihydrorhodamine 123, hydroethidine and tetrachloro-tetraethylbenzimidazolocarbo-cyanine iodide are substrates for BCRP. Thus, the determination of intracellular ROS and mitochondrial potential by the selected probes is significantly altered by ABC transporter activities. The activity of these transporters must be considered when employing fluorescent probes for the assessment of ROS production or mitochondrial membrane potential.