Control of the orientational order and nonlinear optical response of the "push-pull" chromophore RuPZn via specific incorporation into densely packed monolayer ensembles of an amphiphilic four-helix bundle peptide: characterization of the peptide-chromophore complexes.

"Push-pull" chromophores based on extended pi-electron systems have been designed to exhibit exceptionally large molecular hyperpolarizabilities. We have engineered an amphiphilic four-helix bundle peptide to vectorially incorporate such hyperpolarizable chromophores having a metalloporphyrin moiety, with high specificity into the interior core of the bundle. The amphiphilic exterior of the bundle facilitates the formation of densely packed monolayer ensembles of the vectorially oriented peptide-chromophore complexes at the liquid-gas interface. Chemical specificity designed into the ends of the bundle facilitates the subsequent covalent attachment of these monolayer ensembles onto the surface of an inorganic substrate. In this article, we describe the structural characterization of these monolayer ensembles at each stage of their fabrication for one such peptide-chromophore complex designated as AP0-RuPZn. In the accompanying article, we describe the characterization of their macroscopic nonlinear optical properties.

Using Helix-coil Models to Study Protein Unfolded States

An abstract of a thesis devoted to using helix-coil models to study unfolded states.\\

Research on polypeptide unfolded states has received much more attention in the last decade or so than it has in the past. Unfolded states are thought to be implicated in various

misfolding diseases and likely play crucial roles in protein folding equilibria and folding rates. Structural characterization of unfolded states has proven to be

much more difficult than the now well established practice of determining the structures of folded proteins. This is largely because many core assumptions underlying

folded structure determination methods are invalid for unfolded states. This has led to a dearth of knowledge concerning the nature of unfolded state conformational

distributions. While many aspects of unfolded state structure are not well known, there does exist a significant body of work stretching back half a century that

has been focused on structural characterization of marginally stable polypeptide systems. This body of work represents an extensive collection of experimental

data and biophysical models associated with describing helix-coil equilibria in polypeptide systems. Much of the work on unfolded states in the last decade has not been devoted

specifically to the improvement of our understanding of helix-coil equilibria, which arguably is the most well characterized of the various conformational equilibria

that likely contribute to unfolded state conformational distributions. This thesis seeks to provide a deeper investigation of helix-coil equilibria using modern

statistical data analysis and biophysical modeling techniques. The studies contained within seek to provide deeper insights and new perspectives on what we presumably

know very well about protein unfolded states. \\

Chapter 1 gives an overview of recent and historical work on studying protein unfolded states. The study of helix-coil equilibria is placed in the context

of the general field of unfolded state research and the basics of helix-coil models are introduced.\\

Chapter 2 introduces the newest incarnation of a sophisticated helix-coil model. State of the art modern statistical techniques are employed to estimate the energies

of various physical interactions that serve to influence helix-coil equilibria. A new Bayesian model selection approach is utilized to test many long-standing

hypotheses concerning the physical nature of the helix-coil transition. Some assumptions made in previous models are shown to be invalid and the new model

exhibits greatly improved predictive performance relative to its predecessor. \\

Chapter 3 introduces a new statistical model that can be used to interpret amide exchange measurements. As amide exchange can serve as a probe for residue-specific

properties of helix-coil ensembles, the new model provides a novel and robust method to use these types of measurements to characterize helix-coil ensembles experimentally

and test the position-specific predictions of helix-coil models. The statistical model is shown to perform exceedingly better than the most commonly used

method for interpreting amide exchange data. The estimates of the model obtained from amide exchange measurements on an example helical peptide

also show a remarkable consistency with the predictions of the helix-coil model. \\

Chapter 4 involves a study of helix-coil ensembles through the enumeration of helix-coil configurations. Aside from providing new insights into helix-coil ensembles,

this chapter also introduces a new method by which helix-coil models can be extended to calculate new types of observables. Future work on this approach could potentially

allow helix-coil models to move into use domains that were previously inaccessible and reserved for other types of unfolded state models that were introduced in chapter 1.

Short-lived alpha-helical intermediates in the folding of beta-sheet proteins.

Several lines of evidence point strongly toward the importance of highly alpha-helical intermediates in the folding of all globular proteins, regardless of their native structure. However, experimental refolding studies demonstrate no observable alpha-helical intermediate during refolding of some beta-sheet proteins and have dampened enthusiasm for this model of protein folding. In this study, beta-sheet proteins were hypothesized to have potential to form amphiphilic helices at a period of <3.6 residues/turn that matches or exceeds the potential at 3.6 residues/turn. Hypothetically, such potential is the basis for an effective and unidirectional mechanism by which highly alpha-helical intermediates might be rapidly disassembled during folding and potentially accounts for the difficulty in detecting highly alpha-helical intermediates during the folding of some proteins. The presence of this potential was confirmed, indicating that a model entailing ubiquitous formation of alpha-helical intermediates during the folding of globular proteins predicts previously unrecognized features of primary structure. Further, the folding of fatty acid binding protein, a predominantly beta-sheet protein that exhibits no apparent highly alpha-helical intermediate during folding, was dramatically accelerated by 2,2,2-trifluoroethanol, a solvent that stabilizes alpha-helical structure. This observation suggests that formation of an alpha-helix can be a rate-limiting step during folding of a predominantly beta-sheet protein and further supports the role of highly alpha-helical intermediates in the folding of all globular proteins.

Lactic acidosis triggers starvation response with paradoxical induction of TXNIP through MondoA.

Although lactic acidosis is a prominent feature of solid tumors, we still have limited understanding of the mechanisms by which lactic acidosis influences metabolic phenotypes of cancer cells. We compared global transcriptional responses of breast cancer cells in response to three distinct tumor microenvironmental stresses: lactic acidosis, glucose deprivation, and hypoxia. We found that lactic acidosis and glucose deprivation trigger highly similar transcriptional responses, each inducing features of starvation response. In contrast to their comparable effects on gene expression, lactic acidosis and glucose deprivation have opposing effects on glucose uptake. This divergence of metabolic responses in the context of highly similar transcriptional responses allows the identification of a small subset of genes that are regulated in opposite directions by these two conditions. Among these selected genes, TXNIP and its paralogue ARRDC4 are both induced under lactic acidosis and repressed with glucose deprivation. This induction of TXNIP under lactic acidosis is caused by the activation of the glucose-sensing helix-loop-helix transcriptional complex MondoA:Mlx, which is usually triggered upon glucose exposure. Therefore, the upregulation of TXNIP significantly contributes to inhibition of tumor glycolytic phenotypes under lactic acidosis. Expression levels of TXNIP and ARRDC4 in human cancers are also highly correlated with predicted lactic acidosis pathway activities and associated with favorable clinical outcomes. Lactic acidosis triggers features of starvation response while activating the glucose-sensing MondoA-TXNIP pathways and contributing to the "anti-Warburg" metabolic effects and anti-tumor properties of cancer cells. These results stem from integrative analysis of transcriptome and metabolic response data under various tumor microenvironmental stresses and open new paths to explore how these stresses influence phenotypic and metabolic adaptations in human cancers.

Transcription factors MYOCD, SRF, Mesp1 and SMARCD3 enhance the cardio-inducing effect of GATA4, TBX5, and MEF2C during direct cellular reprogramming.

Transient overexpression of defined combinations of master regulator genes can effectively induce cellular reprogramming: the acquisition of an alternative predicted phenotype from a differentiated cell lineage. This can be of particular importance in cardiac regenerative medicine wherein the heart lacks the capacity to heal itself, but simultaneously contains a large pool of fibroblasts. In this study we determined the cardio-inducing capacity of ten transcription factors to actuate cellular reprogramming of mouse embryonic fibroblasts into cardiomyocyte-like cells. Overexpression of transcription factors MYOCD and SRF alone or in conjunction with Mesp1 and SMARCD3 enhanced the basal but necessary cardio-inducing effect of the previously reported GATA4, TBX5, and MEF2C. In particular, combinations of five or seven transcription factors enhanced the activation of cardiac reporter vectors, and induced an upregulation of cardiac-specific genes. Global gene expression analysis also demonstrated a significantly greater cardio-inducing effect when the transcription factors MYOCD and SRF were used. Detection of cross-striated cells was highly dependent on the cell culture conditions and was enhanced by the addition of valproic acid and JAK inhibitor. Although we detected Ca(2+) transient oscillations in the reprogrammed cells, we did not detect significant changes in resting membrane potential or spontaneously contracting cells. This study further elucidates the cardio-inducing effect of the transcriptional networks involved in cardiac cellular reprogramming, contributing to the ongoing rational design of a robust protocol required for cardiac regenerative therapies.

Later Life Consequences of Developmental Mitochondrial DNA Damage in C. elegans

Mitochondria are responsible for producing the vast majority of cellular ATP, and are therefore critical to organismal health [1]. They contain thir own genomes (mtDNA) which encode 13 proteins that are all subunits of the mitochondrial respiratory chain (MRC) and are essential for oxidative phosphorylation [2]. mtDNA is present in multiple copies per cell, usually between 103 and 104 , though this number is reduced during certain developmental stages [3, 4]. The health of the mitochondrial genome is also important to the health of the organism, as mutations in mtDNA lead to human diseases that collectively affect approximately 1 in 4000 people [5, 6]. mtDNA is more susceptible than nuclear DNA (nucDNA) to damage by many environmental pollutants, for reasons including the absence of Nucleotide Excision Repair (NER) in the mitochondria [7]. NER is a highly functionally conserved DNA repair pathway that removes bulky, helix distorting lesions such as those caused by ultraviolet C (UVC) radiation and also many environmental toxicants, including benzo[a]pyrene (BaP) [8]. While these lesions cannot be repaired, they are slowly removed through a process that involves mitochondrial dynamics and autophagy [9, 10]. However, when present during development in C. elegans, this damage reduces mtDNA copy number and ATP levels [11]. We hypothesize that this damage, when present during development, will result in mitochondrial dysfunction and increase the potential for adverse outcomes later in life.

To test this hypothesis, 1st larval stage (L1) C. elegans are exposed to 3 doses of 7.5J/m2 ultraviolet C radiation 24 hours apart, leading to the accumulation of mtDNA damage [9, 11]. After exposure, many mitochondrial endpoints are assessed at multiple time points later in life. mtDNA and nucDNA damage levels and genome copy numbers are measured via QPCR and real-time PCR , respectively, every 2 day for 10 days. Steady state ATP levels are measured via luciferase expressing reporter strains and traditional ATP extraction methods. Oxygen consumption is measured using a Seahorse XFe24 extra cellular flux analyzer. Gene expression changes are measured via real time PCR and targeted metabolomics via LC-MS are used to investigate changes in organic acid, amino acid and acyl-carnitine levels. Lastly, nematode developmental delay is assessed as growth, and measured via imaging and COPAS biosort.

I have found that despite being removed, UVC induced mtDNA damage during development leads to persistent deficits in energy production later in life. mtDNA copy number is permanently reduced, as are ATP levels, though oxygen consumption is increased, indicating inefficient or uncoupled respiration. Metabolomic data and mutant sensitivity indicate a role for NADPH and oxidative stress in these results, and exposed nematodes are more sensitive to the mitochondrial poison rotenone later in life. These results fit with the developmental origin of health and disease hypothesis, and show the potential for environmental exposures to have lasting effects on mitochondrial function.

Lastly, we are currently working to investigate the potential for irreparable mtDNA lesions to drive mutagenesis in mtDNA. Mutations in mtDNA lead to a wide range of diseases, yet we currently do not understand the environmental component of what causes them. In vitro evidence suggests that UVC induced thymine dimers can be mutagenic [12]. We are using duplex sequencing of C. elegans mtDNA to determine mutation rates in nematodes exposed to our serial UVC protocol. Furthermore, by including mutant strains deficient in mitochondrial fission and mitophagy, we hope to determine if deficiencies in these processes will further increase mtDNA mutation rates, as they are implicated in human diseases.

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.

Inactivation of the von Hippel-Lindau tumor suppressor leads to selective expression of a human endogenous retrovirus in kidney cancer.

A human endogenous retrovirus type E (HERV-E) was recently found to be selectively expressed in most renal cell carcinomas (RCCs). Importantly, antigens derived from this provirus are immunogenic, stimulating cytotoxic T cells that kill RCC cells in vitro and in vivo. Here, we show HERV-E expression is restricted to the clear cell subtype of RCC (ccRCC) characterized by an inactivation of the von Hippel-Lindau (VHL) tumor-suppressor gene with subsequent stabilization of hypoxia-inducible transcription factors (HIFs)-1α and -2α. HERV-E expression in ccRCC linearly correlated with HIF-2α levels and could be silenced in tumor cells by either transfection of normal VHL or small interfering RNA inhibition of HIF-2α. Using chromatin immunoprecipitation, we demonstrated that HIF-2α can serve as transcriptional factor for HERV-E by binding with HIF response element (HRE) localized in the proviral 5' long terminal repeat (LTR). Remarkably, the LTR was found to be hypomethylated only in HERV-E-expressing ccRCC while other tumors and normal tissues possessed a hypermethylated LTR preventing proviral expression. Taken altogether, these findings provide the first evidence that inactivation of a tumor suppressor gene can result in aberrant proviral expression in a human tumor and give insights needed for translational research aimed at boosting human immunity against antigenic components of this HERV-E.