Biomedical subjects
Publications and source records attributed to Rui Hong.
Place exchange reactions were studied using dye displacement: subtle changes in ligand structure greatly affected both the rate of displacement and the stability of the monolayer.
We demonstrate the patterning of silica substrates with thymine (Thy-PS) and positively charged N-methylpyridinium (PVMP) polymers using photolithography and the subsequent orthogonal modification of these surfaces using diaminopyridine-functionalized polystyrene (DAP-PS) and carboxylate-derivatized CdSe/ZnS core-shell nanoparticles (COO-NP) through diamidopyridine-thymine three-point hydrogen bonding and pyridinium-carboxylate electrostatic interactions, respectively. This two-component orthogonal surface modification was accomplished in a self-sorting, single-step fashion, providing a versatile tool for the rapid and efficient creation of complex materials.
We demonstrate here the effective delivery of a dye payload into cells using 2-nm core gold nanoparticles, with release occurring via place exchange of glutathione onto the particle surface. In vitro experiments demonstrate effective release of drug analogues upon addition of glutathione. Cell culture experiments show rapid uptake of nanoparticle and effective release of payload. The role of glutathione in the release process was demonstrated through improved payload release upon transient increase in glutathione levels achieved via introduction of glutathione ethyl ester into the cell.
A family of nanoparticles featuring surfaces of varying hydrophobicity was synthesized. The efficiency of DNA-binding was determined, demonstrating in a fivefold modulation in binding a 37-mer DNA strand. Nanoparticle-binding causes a reversible conformational change in the DNA structure, as demonstrated by circular dichroism and fluorescence experiments. Furthermore, the affinity of the nanoparticle for the DNA can be regulated by external agents, though stability of the complex is observed at relatively high ionic strengths.
We report here on a new amphiphilic homopolymer that binds noncovalently to proteins. This polymer not only binds to the target protein chymotrypsin with submicromolar affinity but also stabilizes the native structure of the protein. Since the polymer-protein binding process is based on electrostatic interaction, the bound protein can be released from the polymer surface and reactivated either by increasing the ionic strength or by adding complementary cationic surfactants. The electrostatic binding of polymer to the protein results in a marked change in the substrate specificity of chymotrypsin.
Electrostatic interactions were used to noncovalently conjugate chymotrypsin to gold nanoparticles featuring hybrid tetraethylene(glycol)alkanethiol monolayers terminated with carboxylate groups. This conjugation process greatly alters the substrate selectivity of the adsorbed chymotrypsin, inhibiting the hydrolysis of anionic subtrates without affecting the hydrolysis rate of cationic analogues.
The versatile phosphoprotein pp32 is involved in important physiological processes, including cell proliferation, apoptosis, mRNA transport, and transcription. We have previously reported that pp32, through histone masking, inhibits histone acetylation and transcriptional activation by histone acetyltransferases. However, how pp32 itself is regulated remained largely unknown. Although pp32 is a phosphoprotein, neither the phosphorylation sites nor the cellular kinase has been identified. In this report, utilizing an in vitro kinase assay and a biochemical purification scheme, we identify casein kinase II as a cellular pp32-kinase. Our deletion and site-specific mutagenesis studies identify serines 158 and 204 as the sites of phosphorylation. Generation and utilization of antibodies with higher affinity for phospho-pp32 demonstrate that pp32 is indeed phosphorylated in vivo at these two sites. Mutagenesis studies on pp32 suggest a role for serines 158 and 204 in its function. The identification of the pp32 kinase and the sites of pp32 phosphorylation as well as the generation of antibodies with higher affinity for phospho-pp32 should now provide key information and tools for future studies on pp32 regulation.
Various post-translational modifications of histones significantly influence gene transcription. Although un- or hypoacetylated histones are tightly linked to transcriptional repression, the mechanisms and identities of chromatin signal transducer proteins integrating histone hypoacetylation into repression in humans have remained largely unknown. Here we show that the mammalian histone-binding proteins and inhibitor of acetyltransferases (INHAT) complex subunits, Set/template-activating factor-Ibeta (TAF-Ibeta) and pp32, specifically bind to unacetylated, hypoacetylated, and repressively marked histones but not to hyperacetylated histones. Additionally, Set/TAF-Ibeta and pp32 associate with histone deacetylases in vitro and in vivo and repress transcription from a chromatin-integrated template in vivo. Finally, Set/TAF-Ibeta and pp32 associate with an endogenous estrogen receptor-regulated gene, EB1, in the hypoacetylated transcriptionally inactive state but not with the hyperacetylated transcriptionally active form. Together, these data define a novel in vivo mechanistic role for the mammalian Set/TAF-Ibeta and pp32 proteins as transducers of chromatin signaling by integrating chromatin hypoacetylation and transcriptional repression.
Thioalkyl and thioalkylated oligo(ethylene glycol) (OEG) ligands with chain-end functionality were used to fabricate water-soluble CdSe nanoparticle scaffolds. Surface recognition of chymotrypsin (ChT) was achieved using these functionalized nanoparticle scaffolds, with three levels of interaction demonstrated: no interaction (OEG terminated with hydroxyl group), inhibition with denaturation (carboxylate-terminated thioalkyl ligands), and inhibition with retention of structure (carboxylate-terminated OEG). The latter process was reversible upon an increase in ionic strength, with essentially complete restoration of enzymatic activity.
Chromatin structure plays an important role in DNA replication, repair, and transcription. p300 is a transcriptional coactivator with protein acetyltransferase activity, and proliferating cell nuclear antigen (PCNA) plays important roles in DNA replication and repair. It has been shown recently that p300 is necessary for DNA synthesis and repair. However, it is not known whether human PCNA, in a reciprocal manner, can regulate the enzymatic activity and transcriptional regulatory properties of p300. Here we show that human PCNA associates with p300 and potently inhibits the acetyltransferase activity and transcriptional activation properties of p300. Surprisingly, PCNA fails to inhibit p300/CBP-associated factor (PCAF) acetyltransferase function as well as PCAF-dependent transcription. Additionally, PCNA potently represses transcription when targeted to chromatin in vivo. Consistent with these observations, using chromatin immunoprecipitation assays, we demonstrate that PCNA recruitment to promoters causes hypoacetylation of chromatin. Together, our results demonstrate for the first time a novel role for human PCNA in transcriptional repression and in modulating chromatin modification. The reciprocal modulation of p300 and PCNA activities by each other provides an example of integrative regulatory cross-talk among chromatin-based processes such as DNA transcription, repair, and synthesis.
Cytotoxic T lymphocytes release granzymes (Gzm) A and B to induce apoptosis or programmed cell death of virally infected or tumor cells. In this issue of Cell, Fan et al. identify the tumor metastasis suppressor NM23-H1 as a GzmA-activated, apoptosis-inducing DNase and the oncoprotein SET as its inhibitor. Work from the Lieberman and Wang groups indicates a surprising role for a group of acidic nucleo-cytoplasmic proteins in regulating apoptosis.
Affinity chromatography was used to identify cellular proteins that interact with the herpes simplex virus (HSV) tegument protein VP22. Among a small set of proteins that bind specifically to VP22, we identified TAF-I (template-activating factor I), a chromatin remodelling protein and close homologue of the histone chaperone protein NAP-1. TAF-I has been shown previously to promote more ordered transfer of histones to naked DNA through a direct interaction with histones. TAF-I, as a subunit of the INHAT (inhibitor of acetyltransferases) protein complex, also binds to histones and masks them from being substrates for the acetyltransferases p300 and PCAF. Using in vitro assays for TAF-I activity in chromatin assembly, we show that VP22 inhibits nucleosome deposition on DNA by binding to TAF-I. We also observed that VP22 binds non-specifically to DNA, an activity that is abolished by TAF-I. However, the presence of VP22 does not affect the property of INHAT in inhibiting the histone acetyltransferase activity of p300 or PCAF in vitro. We speculate that this interaction could be relevant to HSV DNA organization early in infection, for example, by interfering with nucleosomal deposition on the genome. Consistent with this possibility was the observation that overexpression of TAF-I in transfected cells interferes with the progression of HSV-1 infection.
Histone acetylation by p300/CBP and PCAF coactivators is considered to be a key mechanism of chromatin modification and transcriptional regulation. A multiprotein cellular complex, INHAT (inhibitor of acetyltransferases), containing the Set/TAF-Ibeta oncoprotein and pp32 strongly inhibits the HAT activity of p300/CBP and PCAF by histone masking. Here we report that the INHAT complex and its subunits have overlapping but distinct HAT inhibitory and histone binding characteristics. We provide evidence suggesting that the histone binding and INHAT activity of pp32 can be regulated by its physical association with other INHAT subunits. In vivo colocalization and transfection studies show that pp32 INHAT domains are responsible for histone binding, HAT inhibitory activity, and repression of transcription. We propose that INHAT and its subunits may function by modulating histone acetyltransferases through a histone-masking mechanism and may play important regulatory roles in the establishment and maintenance of the newly proposed "histone code" of chromatin.
Butyrolactone I (BL) is a competitive inhibitor of ATP for binding and activation of cyclin-dependent kinases and is a potent inhibitor of cell cycle progression. Treatment of H460 human lung and SW480 human colon cancer cells with doses of BL that exceed the Ki for CDK inhibition but which are much lower than doses required to inhibit MAPK, PKA, PKC, or EGFR lead to a rapid significant reduction of endogenous p21 protein expression. BL-dependent inhibition of p21 expression appears to be p53-independent. BL-dependent p21 degradation was blocked by lactacystin, consistent with the hypothesis that there is accelerated p21 proteasomal degradation in the presence of BL. BL also inhibited the p53-dependent increase of p21 protein expression in cells exposed to the DNA damag-ing agent etoposide, and favored a greater G2/M arrest as compared to the non-BL exposed cells. BL accelerated the degradation of exogenously expressed p21 that was not observed with a C-terminal truncated form of p21. Degradation of exogenous p21 led to a shift to G2 accumulation in the cells exposed to BL. We conclude that BL has effects on the cell cycle beyond its role as a CDK inhibitor and can be used as a novel tool to study the mechanism of p21 degradation and the consequences towards p21- dependent checkpoints.
Positively charged trimethylammonium-functionalized mixed monolayer protected clusters (MMPCs) bind DNA through complementary electrostatic interactions, resulting in complete inhibition of DNA transcription of T7 RNA polymerase. DNA was released from the nanoparticle by intracellular concentrations of glutathione, resulting in efficient transcription. The restoration of RNA production was dose-dependent in terms of GSH, with considerable control of the release process possible through variation in monolayer structure. This work presents a new approach to controlled release of DNA, with potential applications in the creation of transfection vectors and gene regulation systems.