Kiran Kumar Singarapu

Regulation of Estrogen Receptor α N-Terminus Conformation and Function by Peptidyl Prolyl Isomerase Pin1

ABSTRACT Estrogen receptor alpha (ERα), a key driver of growth in the majority of breast cancers, contains an unstructured transactivation domain (AF1) in its N terminus that is a convergence point for growth factor and hormonal activation. This domain is controlled by phosphorylation, but how phosphorylation impacts AF1 structure and function is unclear. We found that serine 118 (S118) phosphorylation of the ERα AF1 region in response to estrogen (agonist), tamoxifen (antagonist), and growth factors results in recruitment of the peptidyl prolyl cis/trans isomerase Pin1. Phosphorylation of S118 is critical for Pin1 binding, and mutation of S118 to alanine prevents this association. Importantly, Pin1 isomerizes the serine118-proline119 bond from a cis to trans isomer, with a concomitant increase in AF1 transcriptional activity. Pin1 overexpression promotes ligand-independent and tamoxifen-inducible activity of ERα and growth of tamoxifen-resistant breast cancer cells. Pin1 expression correlates with proliferation in ERα-positive rat mammary tumors. These results establish phosphorylation-coupled proline isomerization as a mechanism modulating AF1 functional activity and provide insight into the role of a conformational switch in the functional regulation of the intrinsically disordered transactivation domain of ERα.

Ligand-specific structural changes in the vitamin D receptor in solution.

Vitamin D receptor (VDR) is a member of the nuclear hormone receptor superfamily. When bound to a variety of vitamin D analogues, VDR manifests a wide diversity of physiological actions. The molecular mechanism by which different vitamin D analogues cause specific responses is not understood. The published crystallographic structures of the ligand binding domain of VDR (VDR-LBD) complexed with ligands that have differential biological activities have exhibited identical protein conformations. Here we report that rat VDR-LBD (rVDR-LBD) in solution exhibits differential chemical shifts when bound to three ligands that cause diverse responses: the natural hormone, 1,25-dihydroxyvitamin D(3) [1,25(OH)₂D₃], a potent agonist analogue, 2-methylene-19-nor-(20S)-1,25-dihydroxyvitamin D₃ [2MD], and an antagonist, 2-methylene-(22E)-(24R)-25-carbobutoxy-26,27-cyclo-22-dehydro-1α,24-dihydroxy-19-norvitamin D₃ [OU-72]. Ligand-specific chemical shifts mapped not only to residues at or near the binding pocket but also to residues remote from the ligand binding site. The complexes of rVDR-LBD with native hormone and the potent agonist 2MD exhibited chemical shift differences in signals from helix-12, which is part of the AF2 transactivation domain that appears to play a role in the selective recruitment of coactivators. By contrast, formation of the complex of rVDR-LBD with the antagonist OU-72 led to disappearance of signals from residues in helices-11 and -12. We present evidence that disorder in this region of the receptor in the antagonist complex prevents the attachment of coactivators.

One-pot, three-component approach to the synthesis of 3,4,5-trisubstituted pyrazoles.

An operationally simple and high yielding protocol for the synthesis of polyfunctional pyrazoles has been developed through one-pot, three-component coupling of aldehydes, 1,3-dicarbonyls, and diazo compounds as well as tosyl hydrazones. The reaction proceeds through a tandem Knoevenagel condensation, 1,3-dipolar cycloaddition, and transition metal-free oxidative aromatization reaction sequence utilizing molecular oxygen as a green oxidant. The scope of the reaction was studied by varying the aldehyde, 1,3-dicarbonyl, and diazo component individually.

Prins cascade cyclization for the synthesis of 1,9-dioxa-4-azaspiro[5.5]undecane derivatives.

A novel Prins cascade process for the synthesis of 1,9-dioxa-4- azaspiro[5.5]undecane derivatives by the coupling of aldehydes with N-(4-hydroxy-2-methylenebutyl)-N-(2-hydroxyethyl)-4-methylbenzenesulfonamide has been developed. This is the first report of the synthesis of spiromorpholinotetrahydropyran derivatives through a Prins bicyclization.

Stereoselective Synthesis of Hexahydro-1H-pyrano- and thiopyrano[3,4-c]quinoline Derivatives through a Prins Cascade Cyclization

A cascade reaction of (E)-5-(arylamino)pent-3-en-1-ols and thiols with various aldehydes in the presence of 30 mol % BF3·OEt2 in 1,2-dichloroethane at 80 °C affords a novel class of trans-fused hexahydro-1H-pyrano[3,4-c]quinolines and hexahydro-1H-thiopyrano[3,4-c]quinolines in good to excellent yields with high selectivity. The condensation of (Z)-5-(arylamino)pent-3-en-1-ol with aldehydes provides the corresponding cis-fused products under similar conditions.

Structural characterization of Hsp12, the heat shock protein from Saccharomyces cerevisiae, in aqueous solution where it is intrinsically disordered and in detergent micelles where it is locally α-helical.

Background: Heat shock protein 12 (Hsp12) is produced in response to stress. Results: We determined structures of Hsp12 in aqueous solution and in the presence of dodecylphosphocholine (DPC) and SDS micelles. Conclusion: Hsp12 is disordered in water, but forms one helix with DPC and three additional helices with SDS micelles. Significance: Interaction with a membrane-like surface induces local structure in Hsp12. Hsp12 (heat shock protein 12) belongs to the small heat shock protein family, partially characterized as a stress response, stationary phase entry, late embryonic abundant-like protein located at the plasma membrane to protect membrane from desiccation. Here, we report the structural characterization of Hsp12 by NMR and biophysical techniques. The protein was labeled uniformly with nitrogen-15 and carbon-13 so that its conformation could be determined in detail both in aqueous solution and in two membrane-mimetic environments, SDS and dodecylphosphocholine (DPC) micelles. Secondary structural elements determined from assigned chemical shifts indicated that Hsp12 is dynamically disordered in aqueous solution, whereas it gains four helical stretches in the presence of SDS micelles and a single helix in presence of DPC. These conclusions were reinforced by circular dichroism spectra of the protein in all three environments. The lack of long range interactions in NOESY spectra indicated that the helices present in SDS micelles do not pack together. R1 and R2, relaxation and heteronuclear NOE measurements showed that the protein is disordered in aqueous solution but becomes more ordered in presence of detergent micelles. NMR spectra collected in presence of paramagnetic spin relaxation agents (5DSA, 16DSA, and Gd(DTPA-BMA)) indicated that the amphipathic α-helices of Hsp12 in SDS micelles lie on the membrane surface. These observations are in agreement with studies suggesting that Hsp12 functions to protect the membrane from desiccation.

Regioselective Synthesis of Substituted Arenes via Aerobic Oxidative [3 + 3] Benzannulation Reactions of α,β-Unsaturated Aldehydes and Ketones

Facile conversion of α,β-unsaturated aldehydes and ketones into highly substituted arenes via a base-mediated, completely regioselective, air-oxidative [3 + 3] benzannulation reaction with readily available 4-sulfonylcrotonates or 1,3-bisphenylsulfonylpropene is reported. The reaction can also be carried out as a one-pot, three-component operation using 4-bromocrotonates, aryl sulfinates, and cinnamaldehyde. This open-flask, metal-free reaction does not require anhydrous solvents, proceeds under mild conditions, and uses atmospheric oxygen as the oxidant to afford high yields of the 3-(arylsulfonyl)benzoic acid esters.

Robust, integrated computational control of NMR experiments to achieve optimal assignment by ADAPT-NMR.

ADAPT-NMR (Assignment-directed Data collection Algorithm utilizing a Probabilistic Toolkit in NMR) represents a groundbreaking prototype for automated protein structure determination by nuclear magnetic resonance (NMR) spectroscopy. With a [13C,15N]-labeled protein sample loaded into the NMR spectrometer, ADAPT-NMR delivers complete backbone resonance assignments and secondary structure in an optimal fashion without human intervention. ADAPT-NMR achieves this by implementing a strategy in which the goal of optimal assignment in each step determines the subsequent step by analyzing the current sum of available data. ADAPT-NMR is the first iterative and fully automated approach designed specifically for the optimal assignment of proteins with fast data collection as a byproduct of this goal. ADAPT-NMR evaluates the current spectral information, and uses a goal-directed objective function to select the optimal next data collection step(s) and then directs the NMR spectrometer to collect the selected data set. ADAPT-NMR extracts peak positions from the newly collected data and uses this information in updating the analysis resonance assignments and secondary structure. The goal-directed objective function then defines the next data collection step. The procedure continues until the collected data support comprehensive peak identification, resonance assignments at the desired level of completeness, and protein secondary structure. We present test cases in which ADAPT-NMR achieved results in two days or less that would have taken two months or more by manual approaches.

Solution NMR Structures of Pyrenophora tritici-repentis ToxB and Its Inactive Homolog Reveal Potential Determinants of Toxin Activity

Background: ToxB is a proteinaceous toxin but its homolog toxb has no toxic activity. Results: Both adopt a β-sandwich fold stabilized by two disulfide bonds but differ in the dynamics of one sandwich half. Conclusion: Toxicity is correlated with decreased compactness, increased flexibility, and polymorphism in an active site loop. Significance: ToxB activity depends on interplay between internal dynamics and interactions with putative targets. Pyrenophora tritici-repentis Ptr ToxB (ToxB) is a proteinaceous host-selective toxin produced by Pyrenophora tritici-repentis (P. tritici-repentis), a plant pathogenic fungus that causes the disease tan spot of wheat. One feature that distinguishes ToxB from other host-selective toxins is that it has naturally occurring homologs in non-pathogenic P. tritici-repentis isolates that lack toxic activity. There are no high-resolution structures for any of the ToxB homologs, or for any protein with >30% sequence identity, and therefore what underlies activity remains an open question. Here, we present the NMR structures of ToxB and its inactive homolog Ptr toxb. Both proteins adopt a β-sandwich fold comprising three strands in each half that are bridged together by two disulfide bonds. The inactive toxb, however, shows higher flexibility localized to the sequence-divergent β-sandwich half. The absence of toxic activity is attributed to a more open structure in the vicinity of one disulfide bond, higher flexibility, and residue differences in an exposed loop that likely impacts interaction with putative targets. We propose that activity is regulated by perturbations in a putative active site loop and changes in dynamics distant from the site of activity. Interestingly, the new structures identify AvrPiz-t, a secreted avirulence protein produced by the rice blast fungus, as a structural homolog to ToxB. This homology suggests that fungal proteins involved in either disease susceptibility such as ToxB or resistance such as AvrPiz-t may have a common evolutionary origin.

Diastereoselective synthesis of spiro[cyclopropane-1,3′-indolin]-2′-ones through metal-free cyclopropanation using tosylhydrazone salts

Transition metal-free diastereoselective cyclopropanation of 3-methyleneindolin-2-ones using tosylhydrazone salts as a safe alternative to diazo-compounds was achieved in high yields. All the synthesized compounds were evaluated for their biological activity against three different human cancer cell lines DU-145 (prostate cancer), Hela (cervical cancer) and A-549 (lung cancer). Compounds 3b and 3i exhibited promising anticancer activity (IC50 < 10 μM) against the studied cell lines.