De novo design and molecular assembly of a transmembrane diporphyrin-binding protein complex.

The de novo design of membrane proteins remains difficult despite recent advances in understanding the factors that drive membrane protein folding and association. We have designed a membrane protein PRIME (PoRphyrins In MEmbrane) that positions two non-natural iron diphenylporphyrins (Fe(III)DPP's) sufficiently close to provide a multicentered pathway for transmembrane electron transfer. Computational methods previously used for the design of multiporphyrin water-soluble helical proteins were extended to this membrane target. Four helices were arranged in a D(2)-symmetrical bundle to bind two Fe(II/III) diphenylporphyrins in a bis-His geometry further stabilized by second-shell hydrogen bonds. UV-vis absorbance, CD spectroscopy, analytical ultracentrifugation, redox potentiometry, and EPR demonstrate that PRIME binds the cofactor with high affinity and specificity in the expected geometry.

MYC activity mitigates response to rapamycin in prostate cancer through eukaryotic initiation factor 4E-binding protein 1-mediated inhibition of autophagy.

Loss of PTEN and activation of phosphoinositide 3-kinase are commonly observed in advanced prostate cancer. Inhibition of mammalian target of rapamycin (mTOR), a downstream target of phosphoinositide 3-kinase signaling, results in cell cycle arrest and apoptosis in multiple in vitro and in vivo models of prostate cancer. However, single-agent use of mTOR inhibition has limited clinical success, and the identification of molecular events mitigating tumor response to mTOR inhibition remains a critical question. Here, using genetically engineered human prostate epithelial cells (PrEC), we show that MYC, a frequent target of genetic gain in prostate cancers, abrogates sensitivity to rapamycin by decreasing rapamycin-induced cytostasis and autophagy. Analysis of MYC and the mTOR pathway in human prostate tumors and PrEC showed selective increased expression of eukaryotic initiation factor 4E-binding protein 1 (4EBP1) with gain in MYC copy number or forced MYC expression, respectively. We have also found that MYC binds to regulatory regions of the 4EBP1 gene. Suppression of 4EBP1 expression resulted in resensitization of MYC-expressing PrEC to rapamycin and increased autophagy. Taken together, our findings suggest that MYC expression abrogates sensitivity to rapamycin through increased expression of 4EBP1 and reduced autophagy.

Neuroprotection resulting from insufficiency of RANBP2 is associated with the modulation of protein and lipid homeostasis of functionally diverse but linked pathways in response to oxidative stress.

Oxidative stress is a deleterious stressor associated with a plethora of disease and aging manifestations, including neurodegenerative disorders, yet very few factors and mechanisms promoting the neuroprotection of photoreceptor and other neurons against oxidative stress are known. Insufficiency of RAN-binding protein-2 (RANBP2), a large, mosaic protein with pleiotropic functions, suppresses apoptosis of photoreceptor neurons upon aging and light-elicited oxidative stress, and promotes age-dependent tumorigenesis by mechanisms that are not well understood. Here we show that, by downregulating selective partners of RANBP2, such as RAN GTPase, UBC9 and ErbB-2 (HER2; Neu), and blunting the upregulation of a set of orphan nuclear receptors and the light-dependent accumulation of ubiquitylated substrates, light-elicited oxidative stress and Ranbp2 haploinsufficiency have a selective effect on protein homeostasis in the retina. Among the nuclear orphan receptors affected by insufficiency of RANBP2, we identified an isoform of COUP-TFI (Nr2f1) as the only receptor stably co-associating in vivo with RANBP2 and distinct isoforms of UBC9. Strikingly, most changes in proteostasis caused by insufficiency of RANBP2 in the retina are not observed in the supporting tissue, the retinal pigment epithelium (RPE). Instead, insufficiency of RANBP2 in the RPE prominently suppresses the light-dependent accumulation of lipophilic deposits, and it has divergent effects on the accumulation of free cholesterol and free fatty acids despite the genotype-independent increase of light-elicited oxidative stress in this tissue. Thus, the data indicate that insufficiency of RANBP2 results in the cell-type-dependent downregulation of protein and lipid homeostasis, acting on functionally interconnected pathways in response to oxidative stress. These results provide a rationale for the neuroprotection from light damage of photosensory neurons by RANBP2 insufficiency and for the identification of novel therapeutic targets and approaches promoting neuroprotection.

Distinct functions of POT1 at telomeres.

The mammalian protein POT1 binds to telomeric single-stranded DNA (ssDNA), protecting chromosome ends from being detected as sites of DNA damage. POT1 is composed of an N-terminal ssDNA-binding domain and a C-terminal protein interaction domain. With regard to the latter, POT1 heterodimerizes with the protein TPP1 to foster binding to telomeric ssDNA in vitro and binds the telomeric double-stranded-DNA-binding protein TRF2. We sought to determine which of these functions-ssDNA, TPP1, or TRF2 binding-was required to protect chromosome ends from being detected as DNA damage. Using separation-of-function POT1 mutants deficient in one of these three activities, we found that binding to TRF2 is dispensable for protecting telomeres but fosters robust loading of POT1 onto telomeric chromatin. Furthermore, we found that the telomeric ssDNA-binding activity and binding to TPP1 are required in cis for POT1 to protect telomeres. Mechanistically, binding of POT1 to telomeric ssDNA and association with TPP1 inhibit the localization of RPA, which can function as a DNA damage sensor, to telomeres.

Nucleolar organization, ribosomal DNA array stability, and acrocentric chromosome integrity are linked to telomere function.

The short arms of the ten acrocentric human chromosomes share several repetitive DNAs, including ribosomal RNA genes (rDNA). The rDNA arrays correspond to nucleolar organizing regions that coalesce each cell cycle to form the nucleolus. Telomere disruption by expressing a mutant version of telomere binding protein TRF2 (dnTRF2) causes non-random acrocentric fusions, as well as large-scale nucleolar defects. The mechanisms responsible for acrocentric chromosome sensitivity to dysfunctional telomeres are unclear. In this study, we show that TRF2 normally associates with the nucleolus and rDNA. However, when telomeres are crippled by dnTRF2 or RNAi knockdown of TRF2, gross nucleolar and chromosomal changes occur. We used the controllable dnTRF2 system to precisely dissect the timing and progression of nucleolar and chromosomal instability induced by telomere dysfunction, demonstrating that nucleolar changes precede the DNA damage and morphological changes that occur at acrocentric short arms. The rDNA repeat arrays on the short arms decondense, and are coated by RNA polymerase I transcription binding factor UBF, physically linking acrocentrics to one another as they become fusogenic. These results highlight the importance of telomere function in nucleolar stability and structural integrity of acrocentric chromosomes, particularly the rDNA arrays. Telomeric stress is widely accepted to cause DNA damage at chromosome ends, but our findings suggest that it also disrupts chromosome structure beyond the telomere region, specifically within the rDNA arrays located on acrocentric chromosomes. These results have relevance for Robertsonian translocation formation in humans and mechanisms by which acrocentric-acrocentric fusions are promoted by DNA damage and repair.

Development and Application of Covalent-Labeling Strategies for the Large-Scale Thermodynamic Analysis of Protein Folding and Ligand Binding

Thermodynamic stability measurements on proteins and protein-ligand complexes can offer insights not only into the fundamental properties of protein folding reactions and protein functions, but also into the development of protein-directed therapeutic agents to combat disease. Conventional calorimetric or spectroscopic approaches for measuring protein stability typically require large amounts of purified protein. This requirement has precluded their use in proteomic applications. Stability of Proteins from Rates of Oxidation (SPROX) is a recently developed mass spectrometry-based approach for proteome-wide thermodynamic stability analysis. Since the proteomic coverage of SPROX is fundamentally limited by the detection of methionine-containing peptides, the use of tryptophan-containing peptides was investigated in this dissertation. A new SPROX-like protocol was developed that measured protein folding free energies using the denaturant dependence of the rate at which globally protected tryptophan and methionine residues are modified with dimethyl (2-hydroxyl-5-nitrobenzyl) sulfonium bromide and hydrogen peroxide, respectively. This so-called Hybrid protocol was applied to proteins in yeast and MCF-7 cell lysates and achieved a ~50% increase in proteomic coverage compared to probing only methionine-containing peptides. Subsequently, the Hybrid protocol was successfully utilized to identify and quantify both known and novel protein-ligand interactions in cell lysates. The ligands under study included the well-known Hsp90 inhibitor geldanamycin and the less well-understood omeprazole sulfide that inhibits liver-stage malaria. In addition to protein-small molecule interactions, protein-protein interactions involving Puf6 were investigated using the SPROX technique in comparative thermodynamic analyses performed on wild-type and Puf6-deletion yeast strains. A total of 39 proteins were detected as Puf6 targets and 36 of these targets were previously unknown to interact with Puf6. Finally, to facilitate the SPROX/Hybrid data analysis process and minimize human errors, a Bayesian algorithm was developed for transition midpoint assignment. In summary, the work in this dissertation expanded the scope of SPROX and evaluated the use of SPROX/Hybrid protocols for characterizing protein-ligand interactions in complex biological mixtures.

Thermodynamic analysis of a molecular chaperone binding to unfolded protein substrates.

Molecular chaperones are a highly diverse group of proteins that recognize and bind unfolded proteins to facilitate protein folding and prevent nonspecific protein aggregation. The mechanisms by which chaperones bind their protein substrates have been studied for decades. However, there are few reports about the affinity of molecular chaperones for their unfolded protein substrates. Thus, little is known about the relative binding affinities of different chaperones and about the relative binding affinities of chaperones for different unfolded protein substrates. Here we describe the application of SUPREX (stability of unpurified proteins from rates of H-D exchange), an H-D exchange and MALDI-based technique, in studying the binding interaction between the molecular chaperone Hsp33 and four different unfolded protein substrates, including citrate synthase, lactate dehydrogenase, malate dehydrogenase, and aldolase. The results of our studies suggest that the cooperativity of the Hsp33 folding-unfolding reaction increases upon binding with denatured protein substrates. This is consistent with the burial of significant hydrophobic surface area in Hsp33 when it interacts with its substrate proteins. The SUPREX-derived K(d) values for Hsp33 complexes with four different substrates were all found to be within the range of 3-300 nM.

Identification of the endophilins (SH3p4/p8/p13) as novel binding partners for the beta1-adrenergic receptor.

Several G-protein coupled receptors, such as the beta1-adrenergic receptor (beta1-AR), contain polyproline motifs within their intracellular domains. Such motifs in other proteins are known to mediate protein-protein interactions such as with Src homology (SH)3 domains. Accordingly, we used the proline-rich third intracellular loop of the beta1-AR either as a glutathione S-transferase fusion protein in biochemical "pull-down" assays or as bait in the yeast two-hybrid system to search for interacting proteins. Both approaches identified SH3p4/p8/p13 (also referred to as endophilin 1/2/3), a SH3 domain-containing protein family, as binding partners for the beta1-AR. In vitro and in human embryonic kidney (HEK) 293 cells, SH3p4 specifically binds to the third intracellular loop of the beta1-AR but not to that of the beta2-AR. Moreover, this interaction is mediated by the C-terminal SH3 domain of SH3p4. Functionally, overexpression of SH3p4 promotes agonist-induced internalization and modestly decreases the Gs coupling efficacy of beta1-ARs in HEK293 cells while having no effect on beta2-ARs. Thus, our studies demonstrate a role of the SH3p4/p8/p13 protein family in beta1-AR signaling and suggest that interaction between proline-rich motifs and SH3-containing proteins may represent a previously underappreciated aspect of G-protein coupled receptor signaling.

Overexpression of the cardiac beta(2)-adrenergic receptor and expression of a beta-adrenergic receptor kinase-1 (betaARK1) inhibitor both increase myocardial contractility but have differential effects on susceptibility to ischemic injury.

Cardiac beta(2)-adrenergic receptor (beta(2)AR) overexpression is a potential contractile therapy for heart failure. Cardiac contractility was elevated in mice overexpressing beta(2)ARs (TG4s) with no adverse effects under normal conditions. To assess the consequences of beta(2)AR overexpression during ischemia, perfused hearts from TG4 and wild-type mice were subjected to 20-minute ischemia and 40-minute reperfusion. During ischemia, ATP and pH fell lower in TG4 hearts than wild type. Ischemic injury was greater in TG4 hearts, as indicated by lower postischemic recoveries of contractile function, ATP, and phosphocreatine. Because beta(2)ARs, unlike beta(1)ARs, couple to G(i) as well as G(s), we pretreated mice with the G(i) inhibitor pertussis toxin (PTX). PTX treatment increased basal contractility in TG4 hearts and abolished the contractile resistance to isoproterenol. During ischemia, ATP fell lower in TG4+PTX than in TG4 hearts. Recoveries of contractile function and ATP were lower in TG4+PTX than in TG4 hearts. We also studied mice that overexpressed either betaARK1 (TGbetaARK1) or a betaARK1 inhibitor (TGbetaARKct). Recoveries of function, ATP, and phosphocreatine were higher in TGbetaARK1 hearts than in wild-type hearts. Despite basal contractility being elevated in TGbetaARKct hearts to the same level as that of TG4s, ischemic injury was not increased. In summary, beta(2)AR overexpression increased ischemic injury, whereas betaARK1 overexpression was protective. Ischemic injury in the beta(2)AR overexpressors was exacerbated by PTX treatment, implying that it was G(s) not G(i) activity that enhanced injury. Unlike beta(2)AR overexpression, basal contractility was increased by betaARK1 inhibitor expression without increasing ischemic injury, thus implicating a safer potential therapy for heart failure.

A constitutively active mutant beta 2-adrenergic receptor is constitutively desensitized and phosphorylated.

The beta 2-adrenergic receptor (beta 2AR) can be constitutively activated by mutations in the third intracellular loop. Whereas the wild-type receptor exists predominantly in an inactive conformation (R) in the absence of agonist, the mutant receptor appears to spontaneously adopt an active conformation (R*). We now demonstrate that not only is the mutant beta 2AR constitutively active, it is also constitutively desensitized and down-regulated. To assess whether the mutant receptor can constitutively engage a known element of the cellular desensitization machinery, the receptor was purified and reconstituted into phospholipid vesicles. These preparations retained the essential properties of the constitutively active mutant receptor: agonist-independent activity [to stimulate guanine nucleotide-binding protein (Gs)-GTPase] and agonist-specific increase in binding affinity. Moreover, the purified mutant receptor, in the absence of agonist, was phosphorylated by recombinant beta AR-specific kinase (beta ARK) in a fashion comparable to the agonist-occupied wild-type receptor. Thus, the conformation of the mutated receptor is equivalent to the active conformation (R*), which stimulates Gs protein and is identical to the beta ARK substrate.

Expansion of the alpha 2-adrenergic receptor family: cloning and characterization of a human alpha 2-adrenergic receptor subtype, the gene for which is located on chromosome 2.

Pharmacologic, biochemical, and genetic analyses have demonstrated the existence of multiple alpha 2-adrenergic receptor (alpha 2AR) subtypes. We have cloned a human alpha 2AR by using the polymerase chain reaction with oligonucleotide primers homologous to conserved regions of the previously cloned alpha 2ARs, the genes for which are located on human chromosomes 4 (C4) and 10 (C10). The deduced amino acid sequence encodes a protein of 450 amino acids whose putative topology is similar to that of the family of guanine nucleotide-binding protein-coupled receptors, but whose structure most closely resembles that of the alpha 2ARs. Competition curve analysis of the binding properties of the receptor expressed in COS-7 cells with a variety of adrenergic ligands demonstrates a unique alpha 2AR pharmacology. Hybridization with somatic cell hybrids shows that the gene for this receptor is located on chromosome 2. Northern blot analysis of various rat tissues shows expression in liver and kidney. The unique pharmacology and tissue localization of this receptor suggest that this is an alpha 2AR subtype not previously identified by classical pharmacological or ligand binding approaches.

Cloning and expression of a human kidney cDNA for an alpha 2-adrenergic receptor subtype.

An alpha 2-adrenergic receptor subtype has been cloned from a human kidney cDNA library using the gene for the human platelet alpha 2-adrenergic receptor as a probe. The deduced amino acid sequence resembles the human platelet alpha 2-adrenergic receptor and is consistent with the structure of other members of the family of guanine nucleotide-binding protein-coupled receptors. The cDNA was expressed in a mammalian cell line (COS-7), and the alpha 2-adrenergic ligand [3H]rauwolscine was bound. Competition curve analysis with a variety of adrenergic ligands suggests that this cDNA clone represents the alpha 2B-adrenergic receptor. The gene for this receptor is on human chromosome 4, whereas the gene for the human platelet alpha 2-adrenergic receptor (alpha 2A) lies on chromosome 10. This ability to express the receptor in mammalian cells, free of other adrenergic receptor subtypes, should help in developing more selective alpha-adrenergic ligands.

Wee1-regulated apoptosis mediated by the crk adaptor protein in Xenopus egg extracts.

Many of the biochemical reactions of apoptotic cell death, including mitochondrial cytochrome c release and caspase activation, can be reconstituted in cell-free extracts derived from Xenopus eggs. In addition, because caspase activation does not occur until the egg extract has been incubated for several hours on the bench, upstream signaling processes occurring before full apoptosis are rendered accessible to biochemical manipulation. We reported previously that the adaptor protein Crk is required for apoptotic signaling in egg extracts (Evans, E.K., W. Lu, S.L. Strum, B.J. Mayer, and S. Kornbluth. 1997. EMBO (Eur. Mol. Biol. Organ.) J. 16:230-241). Moreover, we demonstrated that removal of Crk Src homology (SH)2 or SH3 interactors from the extracts prevented apoptosis. We now report the finding that the relevant Crk SH2-interacting protein, important for apoptotic signaling in the extract, is the well-known cell cycle regulator, Wee1. We have demonstrated a specific interaction between tyrosine-phosphorylated Wee1 and the Crk SH2 domain and have shown that recombinant Wee1 can restore apoptosis to an extract depleted of SH2 interactors. Moreover, exogenous Wee1 accelerated apoptosis in egg extracts, and this acceleration was largely dependent on the presence of endogenous Crk protein. As other Cdk inhibitors, such as roscovitine and Myt1, did not act like Wee1 to accelerate apoptosis, we propose that Wee1-Crk complexes signal in a novel apoptotic pathway, which may be unrelated to Wee1's role as a cell cycle regulator.

Suppression of conformational heterogeneity at a protein-protein interface.

Staphylococcal protein A (SpA) is an important virulence factor from Staphylococcus aureus responsible for the bacterium's evasion of the host immune system. SpA includes five small three-helix-bundle domains that can each bind with high affinity to many host proteins such as antibodies. The interaction between a SpA domain and the Fc fragment of IgG was partially elucidated previously in the crystal structure 1FC2. Although informative, the previous structure was not properly folded and left many substantial questions unanswered, such as a detailed description of the tertiary structure of SpA domains in complex with Fc and the structural changes that take place upon binding. Here we report the 2.3-Å structure of a fully folded SpA domain in complex with Fc. Our structure indicates that there are extensive structural rearrangements necessary for binding Fc, including a general reduction in SpA conformational heterogeneity, freezing out of polyrotameric interfacial residues, and displacement of a SpA side chain by an Fc side chain in a molecular-recognition pocket. Such a loss of conformational heterogeneity upon formation of the protein-protein interface may occur when SpA binds its multiple binding partners. Suppression of conformational heterogeneity may be an important structural paradigm in functionally plastic proteins.

Dual modulation of cell survival and cell death by beta(2)-adrenergic signaling in adult mouse cardiac myocytes.

The goal of this study was to determine whether beta(1)-adrenergic receptor (AR) and beta(2)-AR differ in regulating cardiomyocyte survival and apoptosis and, if so, to explore underlying mechanisms. One potential mechanism is that cardiac beta(2)-AR can activate both G(s) and G(i) proteins, whereas cardiac beta(1)-AR couples only to G(s). To avoid complicated crosstalk between beta-AR subtypes, we expressed beta(1)-AR or beta(2)-AR individually in adult beta(1)/beta(2)-AR double knockout mouse cardiac myocytes by using adenoviral gene transfer. Stimulation of beta(1)-AR, but not beta(2)-AR, markedly induced myocyte apoptosis, as indicated by increased terminal deoxynucleotidyltransferase-mediated UTP end labeling or Hoechst staining positive cells and DNA fragmentation. In contrast, beta(2)-AR (but not beta(1)-AR) stimulation elevated the activity of Akt, a powerful survival signal; this effect was fully abolished by inhibiting G(i), G(beta gamma), or phosphoinositide 3 kinase (PI3K) with pertussis toxin, beta ARK-ct (a peptide inhibitor of G(beta gamma)), or LY294002, respectively. This indicates that beta(2)-AR activates Akt via a G(i)-G(beta gamma)-PI3K pathway. More importantly, inhibition of the G(i)-G(beta gamma)-PI3K-Akt pathway converts beta(2)-AR signaling from survival to apoptotic. Thus, stimulation of a single class of receptors, beta(2)-ARs, elicits concurrent apoptotic and survival signals in cardiac myocytes. The survival effect appears to predominate and is mediated by the G(i)-G(beta gamma)-PI3K-Akt signaling pathway.