Protein engineering and design in ion channels and receptors.

Acetylcholine receptors (AChRs) expressed at the neuromuscular junction synapses are typical allosteric proteins that shuttle between at least two stable conformational states: Closed (C) and Open (O). Agonist binding to their target sites on the receptor in the extracellular domain triggers a global C→O conformational change that results in an open channel pore that allows ion conduction. How the receptor senses the chemical signal of an agonist and communicates it to the channel pore, located ~50Šaway, are key to understanding the receptor function. AChRs are indispensable for muscle contraction and their neuronal homologues play critical roles in the nervous system function. In this chapter, using a combination of single channel patch-clamp, computational approaches, and genetic engineering, we elucidate the principles of design and engineering to quantify the fundamental thermodynamic parameters of AChRs that regulate ligand binding and channel opening. The receptor engineering principles outlined here for the neuromuscular AChRs are applicable to the broader class of ligand-gated ion channel proteins.

Efficiency measures the conversion of agonist binding energy into receptor conformational change.

Receptors alternate between resting↔active conformations that bind agonists with low↔high affinity. Here, we define a new agonist attribute, energy efficiency (η), as the fraction of ligand-binding energy converted into the mechanical work of the activation conformational change. η depends only on the resting/active agonist-binding energy ratio. In a plot of activation energy versus binding energy (an "efficiency" plot), the slope gives η and the y intercept gives the receptor's intrinsic activation energy (without agonists; ΔG0). We used single-channel electrophysiology to estimate η for eight different agonists and ΔG0 in human endplate acetylcholine receptors (AChRs). From published equilibrium constants, we also estimated η for agonists of KCa1.1 (BK channels) and muscarinic, γ-aminobutyric acid, glutamate, glycine, and aryl-hydrocarbon receptors, and ΔG0 for all of these except KCa1.1. Regarding AChRs, η is 48-56% for agonists related structurally to acetylcholine but is only ∼39% for agonists related to epibatidine; ΔG0 is 8.4 kcal/mol in adult and 9.6 kcal/mol in fetal receptors. Efficiency plots for all of the above receptors are approximately linear, with η values between 12% and 57% and ΔG0 values between 2 and 12 kcal/mol. Efficiency appears to be a general attribute of agonist action at receptor binding sites that is useful for understanding binding mechanisms, categorizing agonists, and estimating concentration-response relationships.

BASP1 interacts with oestrogen receptor α and modifies the tamoxifen response.

Tamoxifen binds to oestrogen receptor α (ERα) to elicit distinct responses that vary by cell/tissue type and status, but the factors that determine these differential effects are unknown. Here we report that the transcriptional corepressor BASP1 interacts with ERα and in breast cancer cells, this interaction is enhanced by tamoxifen. We find that BASP1 acts as a major selectivity factor in the transcriptional response of breast cancer cells to tamoxifen. In all, 40% of the genes that are regulated by tamoxifen in breast cancer cells are BASP1 dependent, including several genes that are associated with tamoxifen resistance. BASP1 elicits tumour-suppressor activity in breast cancer cells and enhances the antitumourigenic effects of tamoxifen treatment. Moreover, BASP1 is expressed in breast cancer tissue and is associated with increased patient survival. Our data have identified BASP1 as an ERα cofactor that has a central role in the transcriptional and antitumourigenic effects of tamoxifen.

AP1 transcription factors are required to maintain the peripheral taste system.

The sense of taste is used by organisms to achieve the optimal nutritional requirement and avoid potentially toxic compounds. In the oral cavity, taste receptor cells are grouped together in taste buds that are present in specialized taste papillae in the tongue. Taste receptor cells are the cells that detect chemicals in potential food items and transmit that information to gustatory nerves that convey the taste information to the brain. As taste cells are in contact with the external environment, they can be damaged and are routinely replaced throughout an organism's lifetime to maintain functionality. However, this taste cell turnover loses efficiency over time resulting in a reduction in taste ability. Currently, very little is known about the mechanisms that regulate the renewal and maintenance of taste cells. We therefore performed RNA-sequencing analysis on isolated taste cells from 2 and 6-month-old mice to determine how alterations in the taste cell-transcriptome regulate taste cell maintenance and function in adults. We found that the activator protein-1 (AP1) transcription factors (c-Fos, Fosb and c-Jun) and genes associated with this pathway were significantly downregulated in taste cells by 6 months and further declined at 12 months. We generated conditional c-Fos-knockout mice to target K14-expressing cells, including differentiating taste cells. c-Fos deletion caused a severe perturbation in taste bud structure and resulted in a significant reduction in the taste bud size. c-Fos deletion also affected taste cell turnover as evident by a decrease in proliferative marker, and upregulation of the apoptotic marker cleaved-PARP. Thus, AP1 factors are important regulators of adult taste cell renewal and their downregulation negatively impacts taste maintenance.

Regulation of AURORA B function by mitotic checkpoint protein MAD2.

Cell cycle checkpoint signaling stringently regulates chromosome segregation during cell division. MAD2 is one of the key components of the spindle and mitotic checkpoint complex that regulates the fidelity of cell division along with MAD1, CDC20, BUBR1, BUB3 and MAD3. MAD2 ablation leads to erroneous attachment of kinetochore-spindle fibers and defective chromosome separation. A potential role for MAD2 in the regulation of events beyond the spindle and mitotic checkpoints is not clear. Together with active spindle assembly checkpoint signaling, AURORA B kinase activity is essential for chromosome condensation as cells enter mitosis. AURORA B phosphorylates histone H3 at serine 10 and serine 28 to facilitate the formation of condensed metaphase chromosomes. In the absence of functional AURORA B cells escape mitosis despite the presence of misaligned chromosomes. In this study we report that silencing of MAD2 results in a drastic reduction of metaphase-specific histone H3 phosphorylation at serine 10 and serine 28. We demonstrate that this is due to mislocalization of AURORA B in the absence of MAD2. Conversely, overexpression of MAD2 concentrated the localization of AURORA B at the metaphase plate and caused hyper-phosphorylation of histone H3. We find that MAD1 plays a minor role in influencing the MAD2-dependent regulation of AURORA B suggesting that the effects of MAD2 on AURORA B are independent of the spindle checkpoint complex. Our findings reveal that, in addition to its role in checkpoint signaling, MAD2 ensures chromosome stability through the regulation of AURORA B.

Methods to study histone chaperone function in nucleosome assembly and chromatin transcription.

Histone chaperones are histone interacting proteins that are involved in various stages of histone metabolism in the cell such as histone storage, transport, nucleosome assembly and disassembly. Histone assembly and disassembly are essential processes in certain DNA-templated phenomena such as replication, repair and transcription in eukaryotes. Since the first histone chaperone Nucleoplasmin was discovered in Xenopus, a plethora of histone chaperones have been identified, characterized and their functional significance elucidated in the last 35 years or so. Some of the histone chaperone containing complexes such as FACT have been described to play a significant role in nucleosome disassembly during transcription elongation. We have reported earlier that human Nucleophosmin (NPM1), a histone chaperone belonging to the Nucleoplasmin family, is a co-activator of transcription. In this chapter, we describe several methods that are used to study the histone chaperone activity of proteins and their role in transcription.

A role of WT1 in cell division and genomic stability.

Wilms' tumor-1 protein (WT1) is a transcription factor that can either activate or repress genes to regulate cell growth, apoptosis and differentiation. WT1 can act as either a tumor suppressor or an oncogene. The cellular functions of WT1 are predominantly regulated by its various interacting partners. Recently we have found that WT1 can regulate the fidelity of chromosome segregation through its interaction with the spindle assembly checkpoint protein, Mitotic arrest deficient-2 (MAD2). WT1 delays anaphase entry by inhibiting the ubiquitination activity of the Anaphase promoting complex/cyclosome (APC/C). Our findings have revealed an important role of WT1 in the regulation of mitotic checkpoint and genomic stability.

WT1 interacts with MAD2 and regulates mitotic checkpoint function.

Tumour suppressors safeguard the fidelity of the mitotic checkpoint by transcriptional regulation of genes that encode components of the mitotic checkpoint complex (MCC). Here we report a new role for the tumour suppressor and transcription factor, WT1, in the mitotic checkpoint. We show that WT1 regulates the MCC by directly interacting with the spindle assembly checkpoint protein, MAD2. WT1 colocalizes with MAD2 during mitosis and preferentially binds to the functionally active, closed-conformer, C-MAD2. Furthermore, WT1 associates with the MCC containing MAD2, BUBR1 and CDC20, resulting in prolonged inhibition of the anaphase-promoting complex/cyclosome (APC/C) and delayed degradation of its substrates SECURIN and CYCLIN B1. Strikingly, RNA interference-mediated depletion of WT1 leads to enhanced turnover of SECURIN, decreased lag time to anaphase and defects in chromosome segregation. Our findings identify WT1 as a regulator of the mitotic checkpoint and chromosomal stability.

Phosphorylation of multifunctional nucleolar protein nucleophosmin (NPM1) by aurora kinase B is critical for mitotic progression.

The functional association of NPM1 with Aurora kinases is well documented. Surprisingly, although NPM1 is a well characterized phosphoprotein, it is unknown whether it is a substrate of Aurora kinases. We have found that Aurora kinases A and B can phosphorylate NPM1 at a single serine residue, Ser125, in vitro and in vivo. Phosphorylated-S125-NPM1 (pS125-NPM1) localizes to the midbody region during late cytokinesis where it colocalizes with Aurora B. The overexpression of mutant (S125A) NPM1 resulted in the deregulation of centrosome duplication and mitotic defects possibly due to cytokinesis failure. These data suggest that Aurora kinase B-mediated phosphorylation of NPM1 plays a critical role during mitosis, which could have wider implications in oncogenesis.

Centromeric histone variant CENP-A represses acetylation-dependent chromatin transcription that is relieved by histone chaperone NPM1.

Mammalian centromeric histone H3 variant, CENP-A, is involved in maintaining the functional integrity and epigenetic inheritance of the centromere. CENP-A causes transcriptional repression of centromeric chromatin through an unknown mechanism. Here, we report that reconstituted CENP-A nucleosomes are amenable to ATP-dependent SWI/SNF-mediated remodelling but are less permissive to acetylation and acetylation-dependent in vitro chromatin transcription. Remarkably, the transcriptional repression of the CENP-A chromatinized template could be relieved by the ectopic addition of histone chaperone, nucleophosmin.

Presenilin influences glycogen synthase kinase-3 ß (GSK-3ß) for kinesin-1 and dynein function during axonal transport.

Within axons, molecular motors transport essential components required for neuronal growth and viability. Although many levels of control and regulation must exist for proper anterograde and retrograde transport of vital proteins, little is known about these mechanisms. We previously showed that presenilin (PS), a gene involved in Alzheimer's disease (AD), influences kinesin-1 and dynein function in vivo. Here, we show that these PS-mediated effects on motor protein function are via a pathway that involves glycogen synthase kinase-3ß (GSK-3ß). PS genetically interacts with GSK-3ß in an activity-dependent manner. Excess of active GSK-3ß perturbed axonal transport by causing axonal blockages, which were enhanced by reduction of kinesin-1 or dynein. These GSK-3ß-mediated axonal defects do not appear to be caused by disruptions or alterations in microtubules (MTs). Excess of non-functional GSK-3ß did not affect axonal transport. Strikingly, GSK-3ß-activity-dependent axonal transport defects were enhanced by reduction of PS. Collectively, our findings suggest that PS and GSK-3ß are required for normal motor protein function. Our observations propose a model, in which PS likely plays a role in regulating GSK-3ß activity during transport. These findings have important implications for our understanding of the complex regulatory machinery that must exist in vivo and how this system is coordinated during the motility of vesicles within axons.

Minor groove binder distamycin remodels chromatin but inhibits transcription.

The condensed structure of chromatin limits access of cellular machinery towards template DNA. This in turn represses essential processes like transcription, replication, repair and recombination. The repression is alleviated by a variety of energy dependent processes, collectively known as "chromatin remodeling". In a eukaryotic cell, a fine balance between condensed and de-condensed states of chromatin helps to maintain an optimum level of gene expression. DNA binding small molecules have the potential to perturb such equilibrium. We present herein the study of an oligopeptide antibiotic distamycin, which binds to the minor groove of B-DNA. Chromatin mobility assays and circular dichroism spectroscopy have been employed to study the effect of distamycin on chromatosomes, isolated from the liver of Sprague-Dawley rats. Our results show that distamycin is capable of remodeling both chromatosomes and reconstituted nucleosomes, and the remodeling takes place in an ATP-independent manner. Binding of distamycin to the linker and nucleosomal DNA culminates in eviction of the linker histone and the formation of a population of off-centered nucleosomes. This hints at a possible corkscrew type motion of the DNA with respect to the histone octamer. Our results indicate that distamycin in spite of remodeling chromatin, inhibits transcription from both DNA and chromatin templates. Therefore, the DNA that is made accessible due to remodeling is either structurally incompetent for transcription, or bound distamycin poses a roadblock for the transcription machinery to advance.

Characterization of nucleolin K88 acetylation defines a new pool of nucleolin colocalizing with pre-mRNA splicing factors.

Nucleolin is a multifunctional protein that carries several post-translational modifications. We characterized nucleolin acetylation and developed antibodies specific to nucleolin K88 acetylation. Using this antibody we show that nucleolin is acetylated in vivo and is not localized in the nucleoli, but instead is distributed throughout the nucleoplasm. Immunofluorescence studies indicate that acetylated nucleolin is co-localized with the splicing factor SC35 and partially with Y12. Acetylated nucleolin is expressed in all tested proliferating cell types. Our findings show that acetylation defines a new pool of nucleolin which support a role for nucleolin in the regulation of mRNA maturation and transcription by RNA polymerase II.

Control of epigenetic states by WT1 via regulation of de novo DNA methyltransferase 3A.

Although tumour suppressor gene hypermethylation is a universal feature of cancer cells, little is known about the necessary molecular triggers. Here, we show that Wilms' tumour 1 (WT1), a developmental master regulator that can also act as a tumour suppressor or oncoprotein, transcriptionally regulates the de novo DNA methyltransferase 3A (DNMT3A) and that cellular WT1 levels can influence DNA methylation of gene promoters genome-wide. Specifically, we demonstrate that depletion of WT1 by short-interfering RNAs leads to reduced DNMT3A in Wilms' tumour cells and human embryonal kidney-derived cell lines. Chromatin immunoprecipitation assays demonstrate WT1 recruitment to the DNMT3A promoter region and reporter assays confirm that WT1 directly transactivates DNMT3A expression. Consistent with this regulatory role, immunohistochemical analysis shows co-expression of WT1 and DNMT3A proteins in nuclei of blastemal cells in human fetal kidney and Wilms' tumours. Using genome-wide promoter methylation arrays, we show that human embryonal kidney cells over-expressing WT1 acquire DNA methylation changes at specific gene promoters where DNMT3A recruitment is increased, with hypermethylation being associated with silencing of gene expression. Elevated DNMT3A is also demonstrated at hypermethylated genes in Wilms' tumour cells, including a region of long-range epigenetic silencing. Finally, we show that depletion of WT1 in Wilms' tumour cells can lead to reactivation of gene expression from methylated promoters, such as TGFB2, a key modulator of epithelial-mesenchymal transitions. Collectively, our work defines a new regulatory modality for WT1 involving elicitation of epigenetic alterations which is most likely crucial to its functions in development and disease.

TFIIB dephosphorylation links transcription inhibition with the p53-dependent DNA damage response.

The general transcription factor II B (TFIIB) plays a central role in both the assembly of the transcription complex at gene promoters and also in the events that lead to transcription initiation. TFIIB is phosphorylated at serine-65 at the promoters of several endogenous genes, and this modification is required to drive the formation of gene promoter-3' processing site contacts through the cleavage stimulation factor 3' (CstF 3')-processing complex. Here we demonstrate that TFIIB phosphorylation is dispensable for the transcription of genes activated by the p53 tumor suppressor. We find that the kinase activity of TFIIH is critical for the phosphorylation of TFIIB serine-65, but it is also dispensable for the transcriptional activation of p53-target genes. Moreover, we demonstrate that p53 directly interacts with CstF independent of TFIIB phosphorylation, providing an alternative route to the recruitment of 3'-processing complexes to the gene promoter. Finally, we show that DNA damage leads to a reduction in the level of phospho-ser65 TFIIB that leaves the p53 transcriptional response intact, but attenuates transcription at other genes. Our data reveal a mode of phospho-TFIIB-independent transcriptional regulation that prioritizes the transcription of p53-target genes during cellular stress.

Repression of transcription by WT1-BASP1 requires the myristoylation of BASP1 and the PIP2-dependent recruitment of histone deacetylase.

The Wilms' tumor 1 protein WT1 is a transcriptional regulator that is involved in cell growth and differentiation. The transcriptional corepressor BASP1 interacts with WT1 and converts WT1 from a transcriptional activator to a repressor. Here, we demonstrate that the N-terminal myristoylation of BASP1 is required in order to elicit transcriptional repression at WT1 target genes. We show that myristoylated BASP1 binds to nuclear PIP2, which leads to the recruitment of PIP2 to the promoter regions of WT1-dependent target genes. BASP1's myristoylation and association with PIP2 are required for the interaction of BASP1 with HDAC1, which mediates the recruitment of HDAC1 to the promoter and elicits transcriptional repression. Our findings uncover a role for myristoylation in transcription, as well as a critical function for PIP2 in gene-specific transcriptional repression through the recruitment of histone deacetylase.

The transcription cycle in eukaryotes: from productive initiation to RNA polymerase II recycling.

The cycle of eukaryotic transcription, from initiation to elongation and termination is regulated at multiple steps. Coordinated action of regulatory factors keeps in check the transcriptional competence of RNA polymerase II (RNAPII) at different stages. Productive transcription requires the escape of the paused RNAPII from the promoter and transition to rapid elongation of the transcript. Numerous studies have identified diverse mechanisms of initiating transcription by overriding inhibitory signals at the gene promoter. The general theme that has emerged is that the balance between positive and negative regulatory factors determines the overall rate of transcription. Recently transcription termination has emerged as an important area of transcriptional regulation that is coupled with the efficient recycling of RNAPII. The factors associated with transcription termination can also mediate gene looping and thereby determine the efficiency of re-initiation. This review highlights these regulatory steps, the key modulators involved in transcription dynamics, and the emerging tools to analyze them.

HIV-1 infection induces acetylation of NPM1 that facilitates Tat localization and enhances viral transactivation.

Human immunodeficiency virus type 1 (HIV-1) following integration hijacks host cell machineries where chromatinization of the viral genome regulates its latency, transcription, and replication. The cooperation among ATP-dependent chromatin remodeling factors, posttranslational modifying enzymes, and histone chaperones is well established during transcriptional activation in eukaryotes. However, the role of histone chaperones in transcription of the HIV promoter is poorly understood. Previous studies from our group have established the role of the human histone chaperone nucleophosmin (NPM1) in the acetylation-dependent chromatin transcription. NPM1 is known to interact with HIV-Tat. Here, we report that infection by HIV-1 induces the acetylation of histone chaperone NPM1. Acetylation of NPM1 was found to be critical for nuclear localization of Tat as well as Tat-mediated transcription alluding to the critical role for the host factor towards viral pathogenesis. Furthermore, knockdown experiments mediated by small interfering RNA identified the critical role played by the chaperone NPM1 in transcriptional activation of the integrated provirus. These results shed further insights into the possible role of histone chaperone NPM1 acetylation in viral gene transcription, which could be a potential therapeutic target.

The multifunctional protein nucleophosmin (NPM1) is a human linker histone H1 chaperone.

Linker histone H1 plays an essential role in chromatin organization. Proper deposition of linker histone H1 as well as its removal is essential for chromatin dynamics and function. Linker histone chaperones perform this important task during chromatin assembly and other DNA-templated phenomena in the cell. Our in vitro data show that the multifunctional histone chaperone NPM1 interacts with linker histone H1 through its first acidic stretch (residues 120-132). Association of NPM1 with linker histone H1 was also observed in cells in culture. NPM1 exhibited remarkable linker histone H1 chaperone activity, as it was able to efficiently deposit histone H1 onto dinucleosomal templates. Overexpression of NPM1 reduced the histone H1 occupancy on the chromatinized template of HIV-1 LTR in TZM-bl cells, which led to enhanced Tat-mediated transactivation. These data identify NPM1 as an important member of the linker histone chaperone family in humans.