The potential role of aquaporin 1 on aristolochic acid I induced epithelial mesenchymal transition on HK-2 cells.

Aristolochic acid I (AA-I), one of the main active components in Aristolochaia herbs, may induce aristolochic acid nephropathy (AAN). Renal interstitial fibrosis is one of the most typical features of AAN. To investigate the mechanism of Aristolochic acid I (AA-I) -induced renal epithelial-mesenchymal transition (EMT) and determine the role of aquaporin-1 (AQP1) in this process, we established an AA-I-induced EMT model in human proximal tubular epithelial cells (HK-2 cells). Morphological examination, MTT assay, and Western blot analysis were performed. Aquaporin 1 (AQP1) and several EMT-related proteins were detected, thereby suggesting the occurrence of AA-I-induced EMT. Two main pathways of transforming growth factor-ß (TGF-ß) signaling, namely, Smad-dependent and Smad-independent signaling pathways, were also detected. The results showed that the TGF-ß / Smad-independent signaling pathways (ß-catenin, Ras-Raf-Erk1/2 signaling pathways) were activated, and AQP1 expression was decreased during the AA-I induced EMT on HK-2 cells. With the presence of TGF-ß1 receptor inhibitor (LY364947) and Erk1/2 inhibitor (PD98059), AQP1 expression was altered by PD98059, suggested that AQP1 could be adjusted by Erk1/2 signaling. Moreover, the inhibitory effect of AA-I on AQP1 was stronger than that of TGF-ß1, suggested that AQP1 may be an important target on AAN clinical therapy.

The UCP2-related mitochondrial pathway participates in rhein-induced apoptosis in HK-2 cells.

Rhein is one of the main active compounds in total rhubarb anthraquinones (TRAs) that were reported to cause nephrotoxicity. This paper explored the mechanism of how rhein induced apoptosis in human renal proximal tubular epithelial cells (HK-2 cells). In this study, rhein was found to induce apoptosis in HK-2 cells according to the results of annexin V/PI staining assay. The underlying mechanisms were investigated, and the mitochondria-mediated pathway was found to be critical. A series of related biological events were explored, including the disruption of mitochondrial membrane potential (MMP), the decrease of the ATP level, the release of cytochrome c (Cyt-c) from the mitochondrion to the cytosol, and down-regulation of Bcl-2 and up-regulation of Bax. Furthermore, rhein significantly increased the levels of ROS and inhibited the expression of mitochondrial uncoupling protein 2 (UCP2). UCP2 inhibition dramatically boosted oxidative stress and exacerbated rhein-induced apoptosis, whereas co-incubation with an ROS scavenger N-acetylcysteine (NAC) could decrease rhein-induced apoptosis. In conclusion, our results have demonstrated that rhein induced apoptosis in HK-2 cells via the UCP2-related mitochondrial pathway and rhein might be a weak inhibitor of UCP2. Our findings provide new evidence that UCP2 plays an important role in the mitochondrial apoptotic pathway.

Structures and Functions of the Multiple KOW Domains of Transcription Elongation Factor Spt5.

The eukaryotic Spt4-Spt5 heterodimer forms a higher-order complex with RNA polymerase II (and I) to regulate transcription elongation. Extensive genetic and functional data have revealed diverse roles of Spt4-Spt5 in coupling elongation with chromatin modification and RNA-processing pathways. A mechanistic understanding of the diverse functions of Spt4-Spt5 is hampered by challenges in resolving the distribution of functions among its structural domains, including the five KOW domains in Spt5, and a lack of their high-resolution structures. We present high-resolution crystallographic results demonstrating that distinct structures are formed by the first through third KOW domains (KOW1-Linker1 [K1L1] and KOW2-KOW3) of Saccharomyces cerevisiae Spt5. The structure reveals that K1L1 displays a positively charged patch (PCP) on its surface, which binds nucleic acids in vitro, as shown in biochemical assays, and is important for in vivo function, as shown in growth assays. Furthermore, assays in yeast have shown that the PCP has a function that partially overlaps that of Spt4. Synthesis of our results with previous evidence suggests a model in which Spt4 and the K1L1 domain of Spt5 form functionally overlapping interactions with nucleic acids upstream of the transcription bubble, and this mechanism may confer robustness on processes associated with transcription elongation.

A dual interface determines the recognition of RNA polymerase II by RNA capping enzyme.

RNA capping enzyme (CE) is recruited specifically to RNA polymerase II (Pol II) transcription sites to facilitate cotranscriptional 5'-capping of pre-mRNA and other Pol II transcripts. The current model to explain this specific recruitment of CE to Pol II as opposed to Pol I and Pol III rests on the interaction between CE and the phosphorylated C-terminal domain (CTD) of Pol II largest subunit Rpb1 and more specifically between the CE nucleotidyltransferase domain and the phosphorylated CTD. Through biochemical and diffraction analyses, we demonstrate the existence of a distinctive stoichiometric complex between CE and the phosphorylated Pol II (Pol IIO). Analysis of the complex revealed an additional and unexpected polymerase-CE interface (PCI) located on the multihelical Foot domain of Rpb1. We name this interface PCI1 and the previously known nucleotidyltransferase/phosphorylated CTD interface PCI2. Although PCI1 and PCI2 individually contribute to only weak interactions with CE, a dramatically stabilized and stoichiometric complex is formed when PCI1 and PCI2 are combined in cis as they occur in an intact phosphorylated Pol II molecule. Disrupting either PCI1 or PCI2 by alanine substitution or deletion diminishes CE association with Pol II and causes severe growth defects in vivo. Evidence from manipulating PCI1 indicates that the Foot domain contributes to the specificity in CE interaction with Pol II as opposed to Pol I and Pol III. Our results indicate that the dual interface based on combining PCI1 and PCI2 is required for directing CE to Pol II elongation complexes.

Structure of the 12-subunit RNA polymerase II refined with the aid of anomalous diffraction data.

RNA polymerase II (Pol II) is the central enzyme of eukaryotic gene expression machinery. Complete definition of the three-dimensional structure of Pol II is essential for understanding the mechanisms that regulate transcription via protein-protein interactions within the Pol II apparatus. To date a series of Pol II-related crystal structures have been reported. However, certain peptide regions, including several that are implicated to interact with regulatory factors, remain obscure. Here we describe conformations for two such regions that are close to the Pol II surface and assume seemingly flexible loop structures. One is located in the TFIIF-interacting Protrusion domain, whereas the other is in the TFIIE-interacting Clamp domain. This structural definition was aided by the application of an advanced crystallographic refinement approach that utilizes the single anomalous diffraction (SAD) from zinc ions bound intrinsically in Pol II. The SAD-based strategy allowed the 12-subunit Pol II model to be fully refined up to 3.8 A with excellent stereochemical properties, demonstrating the effectiveness of the SAD approach for the refinement of large structures at low-to-moderate resolutions. Our results also define additional components of the free Pol II, including the functionally critical Fork Loop-1 and Fork Loop-2 elements. As such, this refined Pol II model provides the most complete structural reference for future analyses of complex structures formed between Pol II and its regulatory factors.

Phasing RNA polymerase II using intrinsically bound Zn atoms: an updated structural model.

Macromolecular assemblies as large as RNA polymerase II (Pol II) can be phased by a few intrinsically bound Zn atoms, by using MAD experiments as described here. A phasing effectiveness of 570 aa/Zn is attained for Pol II. The resulting experimental, unbiased electron density map is of such quality that it confirms the existing crystallographic model and further reveals structural regions not shown by model phases, thus updating the Pol II model at three sites. The mechanistically important fork loop-1 element is observed to be ordered in the absence of nucleic acids, suggesting additional insights into the mechanisms that maintain the stability of the transcription ternary complex and allow its release. Furthermore, a computational experiment with simulated MAD data sets demonstrates that 1 Zn site is able to provide adequate experimental phase information for as many as 1100 amino acids of polypeptide, under the conditions of the current synchrotron and detector technologies.

Fcp1 directly recognizes the C-terminal domain (CTD) and interacts with a site on RNA polymerase II distinct from the CTD.

Fcp1 is an essential protein phosphatase that hydrolyzes phosphoserines within the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II). Fcp1 plays a major role in the regulation of CTD phosphorylation and, hence, critically influences the function of Pol II throughout the transcription cycle. The basic understanding of Fcp1-CTD interaction has remained ambiguous because two different modes have been proposed: the "dockingsite" model versus the "distributive" mechanism. Here we demonstrate biochemically that Fcp1 recognizes and dephosphorylates the CTD directly, independent of the globular non-CTD part of the Pol II structure. We point out that the recognition of CTD by the phosphatase is based on random access and is not driven by Pol II conformation. Results from three different types of experiments reveal that the overall interaction between Fcp1 and Pol II is not stable but dynamic. In addition, we show that Fcp1 also interacts with a region on the polymerase distinct from the CTD. We emphasize that this non-CTD site is functionally distinct from the docking site invoked previously as essential for the CTD phosphatase activity of Fcp1. We speculate that Fcp1 interaction with the non-CTD site may mediate its stimulatory effect on transcription elongation reported previously.

An agarose-acrylamide composite native gel system suitable for separating ultra-large protein complexes.

An agarose-acrylamide composite native gel (CNG) system has been developed for separating protein complexes of ultra-large molecular sizes (over 500kDa) and for analyzing protein-protein interactions in their native states. Various native gel conditions were explored and techniques were improved to facilitate the formation and performance of the CNG system. We demonstrate here that the CNG technique is capable of resolving a complex of RNA polymerase II and an associated factor from the free components, which had not been previously achieved with other methods. Furthermore, this CNG electrophoresis can be conveniently coupled to second-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis for identification of protein components within discrete complexes separated during the CNG run. The CNG technique is particularly suitable for capturing dynamic protein-protein interactions as exemplified here by the formation and demonstration of RNA polymerase II-Fcp1 complex.