Progress in precision medicine is limited by insufficient knowledge of transcriptomic or proteomic features in involved tissues that define pathobiological differences between patients. Here, ...myectomy tissue from patients with obstructive hypertrophic cardiomyopathy and heart failure is analyzed using RNA-Seq, and the results are used to develop individualized protein-protein interaction networks. From this approach, hypertrophic cardiomyopathy is distinguished from dilated cardiomyopathy based on the protein-protein interaction network pattern. Within the hypertrophic cardiomyopathy cohort, the patient-specific networks are variable in complexity, and enriched for 30 endophenotypes. The cardiac Janus kinase 2-Signal Transducer and Activator of Transcription 3-collagen 4A2 (JAK2-STAT3-COL4A2) expression profile informed by the networks was able to discriminate two hypertrophic cardiomyopathy patients with extreme fibrosis phenotypes. Patient-specific network features also associate with other important hypertrophic cardiomyopathy clinical phenotypes. These proof-of-concept findings introduce personalized protein-protein interaction networks (reticulotypes) for characterizing patient-specific pathobiology, thereby offering a direct strategy for advancing precision medicine.
Imatinib mesylate (Gleevec) is a small-molecule inhibitor of the fusion protein Bcr-Abl, the causal agent in chronic myelogenous leukemia. Here we report ten individuals who developed severe ...congestive heart failure while on imatinib and we show that imatinib-treated mice develop left ventricular contractile dysfunction. Transmission electron micrographs from humans and mice treated with imatinib show mitochondrial abnormalities and accumulation of membrane whorls in both vacuoles and the sarco- (endo-) plasmic reticulum, findings suggestive of a toxic myopathy. With imatinib treatment, cardiomyocytes in culture show activation of the endoplasmic reticulum (ER) stress response, collapse of the mitochondrial membrane potential, release of cytochrome c into the cytosol, reduction in cellular ATP content and cell death. Retroviral gene transfer of an imatinib-resistant mutant of c-Abl, alleviation of ER stress or inhibition of Jun amino-terminal kinases, which are activated as a consequence of ER stress, largely rescues cardiomyocytes from imatinib-induced death. Thus, cardiotoxicity is an unanticipated side effect of inhibition of c-Abl by imatinib.
A concern in the field of genomics is the proper interpretation of large, high-throughput sequencing datasets. The use of DNA FISH followed by high-content microscopy is a valuable tool for ...validation and contextualization of frequently occurring gene pairing events at the single-cell level identified by deep sequencing. However, these techniques possess certain limitations. Firstly, they do not permit the study of colocalization of many gene loci simultaneously. Secondly, the direct assessment of the relative position of many clustered gene loci within their respective chromosome territories is impossible. Thus, methods are required to advance the study of higher-order nuclear and cellular organization. Here, we describe a multiplexed DNA FISH technique combined with indirect immunofluorescence to study the relative position of 6 distinct genomic or cellular structures. This can be achieved in a single hybridization step using spectral imaging during image acquisition and linear unmixing. Here, we detail the use of this method to quantify gene pairing between highly expressed spliceosomal genes and compare these data to randomly positioned in silico simulated gene clusters. This is a potentially universally applicable approach for the validation of 3C-based technologies, deep imaging of spatial organization within the nucleus and global cellular organization.
Certain hydroxylated polychlorinated biphenyls (OH-PCBs) inhibit the human estrogen sulfotransferase (hEST) at subnanomolar concentrations, suggesting a possible pathway for PCB toxicity due to ...environmental exposure in humans. To address the structural basis of the inhibition, we have determined the crystal structure of hEST in the presence of the sulfuryl donor product 3′-phosphoadenosine 5′-phosphate and the OH-PCB 4,4′-OH 3,5,3′,5′-tetraCB. The OH-PCB binds in the estrogen binding site with the position of the first phenolic ring in an orientation similar to the phenolic ring of 17β-estradiol. Interestingly, the OH-PCB does not bind in a planar conformation, but rather with a 30-degree twist between the phenyl rings. The crystal structure of hEST with the OH-PCB bound gives physical evidence that certain OH-PCBs can mimic binding of estrogenic compounds in biological systems.
Structure and Function of Sulfotransferases Negishi, Masahiko; Pedersen, Lee G.; Petrotchenko, Evgeniy ...
Archives of Biochemistry and Biophysics,
06/2001, Letnik:
390, Številka:
2
Book Review, Journal Article
Recenzirano
Odprti dostop
Sulfotransferases (STs) catalyze the transfer reaction of the sulfate group from the ubiquitous donor 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to an acceptor group of numerous substrates. This ...reaction, often referred to as sulfuryl transfer, sulfation, or sulfonation, is widely observed from bacteria to humans and plays a key role in various biological processes such as cell communication, growth and development, and defense. The cytosolic STs sulfate small molecules such as steroids, bioamines, and therapeutic drugs, while the Golgi-membrane counterparts sulfate large molecules including glucosaminylglycans and proteins. We have now solved the X-ray crystal structures of four cytosolic and one membrane ST. All five STs are globular proteins composed of a single α/β domain with the characteristic five-stranded β-sheet. The β-sheet constitutes the core of the Paps-binding and catalytic sites. Structural analysis of the PAPS-, PAP-, substrate-, and/or orthovanadate (VO3−4)-bound enzymes has also revealed the common molecular mechanism of the transfer reaction catalyzed by sulfotransferses. The X-ray crystal structures have opened a new era for the study of sulfotransferases.
Estrogen sulfotransferase (EST) transfers the sulfate group from 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to estrogenic steroids. Here we report the crystal structure of human EST (hEST) in the ...context of the V269E mutant-PAPS complex, which is the first structure containing the active sulfate donor for any sulfotransferase. Superimposing this structure with the crystal structure of hEST in complex with the donor product 3′-phosphoadenosine 5′-phosphate (PAP) and the acceptor substrate 17β-estradiol, the ternary structure with the PAPS and estradiol molecule, is modeled. These structures have now provided a more complete view of the SN2-like in-line displacement reaction catalyzed by sulfotransferases. In the PAPS-bound structure, the side chain nitrogen of the catalytic Lys47 interacts with the side chain hydroxyl of Ser137 and not with the bridging oxygen between the 5′-phosphate and sulfate groups of the PAPS molecule as is seen in the PAP-bound structures. This conformational change of the side chain nitrogen indicates that the interaction of Lys47 with Ser137 may regulate PAPS hydrolysis in the absences of an acceptor substrate. Supporting the structural data, the mutations of Ser137 to cysteine and alanine decrease gradually kcat for PAPS hydrolysis and transfer activity. Thus, Ser137 appears to play an important role in regulating the side chain interaction of Lys47 with the bridging oxygen between the 5′-phosphate and the sulfate of PAPS.
In cells capable of entering the cell cycle, including cancer cells, {beta}-catenin has been termed a master switch, driving proliferation over differentiation. However, its role as a transcriptional ...activator in terminally differentiated cells is relatively unknown. Herein we utilize conditional, cardiac-specific deletion of the {beta}-catenin gene and cardiac-specific expression of a dominant inhibitory mutant of Lef-1 (Lef-1 Delta 20), one of the members of the T-cell factor/lymphocyte enhancer factor (Tcf/Lef) family of transcription factors that functions as a coactivator with {beta}-catenin, to demonstrate that {beta}-catenin/Tcf/Lef-dependent gene expression regulates both physiologic and pathological growth (hypertrophy) of the heart. Indeed, the profound nature of the growth impairment of the heart in the Lef-1 Delta 20 mouse, which leads to very early development of heart failure and premature death, suggests {beta}-catenin/Tcf/Lef targets are dominant regulators of cardiomyocyte growth. Thus, our studies, employing complementary models in vivo, implicate {beta}-catenin/Tcf/Lef signaling as an essential growth-regulatory pathway in terminally differentiated cells.
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