163 research outputs found
Using Mutant Cycle Analysis to Elucidate Long-Range Functional Coupling in Allosteric Receptors
No full textFunctional coupling of residues that are far apart in space is the quintessential property of allosteric
receptors. Data from functional studies of allosteric receptors, such as whole-cell dose–response relations,
can be used to determine if mutation to a receptor significantly impacts agonist potency. However, the
classification of perturbations as primarily impacting binding or allosteric function is more challenging,
often requiring detailed kinetic studies. This protocol describes a simple strategy, derived from mutant
cycle analysis, for elucidating long-range functional coupling in allosteric receptors (ELFCAR). Introduction
of a gain-of-function reporter mutation, followed by a mutant cycle analysis of the readily measured
macroscopic EC_(50) values can provide insight into the role of many physically distant targets. This new
method should find broad application in determining the functional roles of residues in allosteric receptors
Joseph and Evelyn Lowery With Others, circa 1987
No full textJoseph and Evelyn Lowery stand with Sunanda Gandhi and her husband, Aron Gandhi, outside the headquarters building for the Southern Christian Leadership Conference in Atlanta, Georgia. Aron Gandhi is the grandson of Mahatma Gandhi.The Atlanta University Center Robert W. Woodruff Library acknowledges the generous support of the Joseph & Evelyn Lowery Institute for Justice and Human Rights, the Joseph Echols Lowery Irrevocable Trust, and other donors in supporting the processing and digitization of Morehouse College's Joseph Echols and Evelyn Gibson Lowery Collection
Attraction at first fright? What Datton & Aron really demonstrated almost 40 years ago
No full text
Almost four decades have passed since Dutton and Aron (1974) published their classic article in JPSP in which they
present the results of three studies. According to interpretations of the results done by the authors, the suffi cient condition
of obtaining the effect of increased sexual attraction toward the object (an attractive woman) - which must be present
shortly after or while waiting to become an aversive stimulus - is the induction in the subjects of a strong autonomic
arousal. This can be done via crossing a high suspended bridge or anticipating the receipt of strong electric shocks.
However, the results of reanalysis do not allow such a conclusion. In the article the author presents the results of secondary
analysis and lists methodological, theoretical and interpretative incoherences.
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STRUCTURAL AND FUNCTIONAL ANALYSIS ON HUMAN INO80 CHROMATIN REMODELING COMPLEXES
No full textThe Conaway lab previously identified and purified a human ATP-dependent chromatin remodeling complex with similarity to the Saccharomyces cerevisiae INO80 complex (65) and demonstrated that it is composed of (i) a Snf2 family ATPase (hIno80) related in sequence to the S. cerevisiae Ino80 ATPase, (ii) 7 additional evolutionarily conserved subunits orthologous to yeast INO80 complex subunits, and (iii) 6 apparently metazoan-specific subunits. In the first part of my thesis, we present evidence that the human INO80 complex is composed of three modules that assemble with three distinct domains of the hIno80 ATPase. These modules include (i) one that is composed of the N-terminus of the hIno80 protein and all of the metazoan-specific subunits and is not required for ATP-dependent nucleosome remodeling, (ii) a second that is composed of the hIno80 HSA/PTH domain, the actin-related proteins Arp4 and Arp8, and the GLI-Kruppel family transcription factor YY1, and (iii) a third that is composed of the hIno80 Snf2 ATPase domain, the Ies2 and Ies6 proteins, the AAA+ ATPases Tip49a and Tip49b, and the actin-related protein Arp5. Through purification and characterization of hINO80 complex subassemblies, we demonstrate that ATP-dependent nucleosome remodeling by the hINO80 complex is catalyzed by a core complex comprised of the hIno80 protein HSA/PTH and Snf2 ATPase domains acting in concert with YY1 and the complete set of its evolutionarily conserved subunits. In the follow-up chapter, we seek to define the requirement for assembling core subunits Ies2, Ies6, Arp5, Tip49a and Tip49b, and distinguish their functional contribution to INO80 chromatin remodeling process. We obtained evidence that the ATPase insertion regions of INO80 family ATPases are necessary and sufficient for assembling all of the five ATPase-associating subunits Ies2, Ies6, Arp5, Tip49a and Tip49b. The missing or inclusion of this insertion module correlates with loss or gain of nucleosome binding capacity of the INO80 subcomplexes, suggesting they contribute to nucleosome binding. Consistent with this hypothesis, the subcomplexes missing the insertion module were not able to bind to nucleosome, thus they were deficient in nucleosome-stimulated ATPase and ATP dependent nucleosome remodeling activities. Within the insertion module, Ies6 and Arp5 form a heterodimer, and are mutually dependent for assembly into INO80. The heterodimer is dispensable for INO80's ATPase activity, but is required for the optimal nucleosome remodeling, presumably via its contribution in nucleosome binding. On the contrary, Ies2 assembles independently of the Arp5-Ies6 dimer, and is absolutely required for the catalytic activities of the INO80 complex, while dispensable for the binding affinity to nucleosomes. Our studies described in this thesis shed light on the structure and function of the human INO80 chromatin remodeling complex
Are all regions of folded proteins that undergo ligand‐dependent order–disorder transitions targets for allosteric peptide mimetics?
No full textRegulation of telomerase reverse transcriptase expression in Schizosaccharomyces pombe
No full textEukaryotic cells undergo chromosome shortening during each cell division due to the inability of DNA polymerase to replicate chromosome ends. This end replication problem is counteracted by the ribonucleoprotein telomerase, which functions as a reverse transcriptase to add DNA repeats to chromosome ends. These DNA repeats, called telomeres, delay cellular senescence and recruit DNA binding proteins which protect chromosome ends and distinguish them from double-strand breaks. Telomere attrition is thought to function as a means of tumor suppression as cells can only undergo a limited number of divisions in the absence of telomerase or an alternative mechanism for replenishing DNA at chromosome ends. In multicellular organisms, telomerase is present and active during the early stages of development, but is later downregulated, resulting in little or no activity in most somatic cell types. In contrast, approximately 90% of cancer cells do have detectable telomerase activity. This discovery has identified telomerase components, and other molecules regulating telomerase function, as potential targets for cancer treatment. Unlike multicellular organisms, single-celled organisms, such as the fission yeast Schizosaccharomyces pombe, are widely believed to constitutively express telomerase, despite insufficient studies on expression or activity in this species outside of typical laboratory conditions. Surprisingly, we have observed that the level of telomerase reverse transcriptase (Trt1) protein in this organism decreases in cells which have been arrested by entry into stationary phase, which corresponds with a decrease in in vitro telomerase activity from these cells. This decrease in Trt1 level is similarly observed in cells which have been starved of nitrogen or glucose, and the rate of turnover appears to be quite rapid. The downregulation of Trt1 protein is independent of trt1 mRNA transcript level, and regions within the protein coding sequence appear to be sufficient for causing the observed decrease in expression. These results indicate that telomerase reverse transcriptase expression in the fission yeast S. pombe is regulated in nutrient-starved cells, likely through degradation of the protein, as triggered by multiple protein sequence or structural elements. Further studies in this model organism will likely reveal great insights into mechanisms and functions of telomerase regulation
REGULATION OF ALC1'S SNF2 ATPASE: INTERPLAY BETWEEN THE SNF2 DOMAIN, THE MACRODOMAIN AND OTHER CONSERVED ELEMENTS
No full textALC1, also known as CHD1L, was originally identified as a gene present on a human chromosome 1q21 region amplified in ~50% of human hepatocellular carcinomas (HCC). ALC1 overexpressing cells form tumors in nude mice, and transgenic mice overexpressing ALC1 develop several types of spontaneous tumors. ALC1 has also been proposed as a novel candidate gene for congenital anomalies of the kidneys and urinary tract (CAKUT). ALC1, a member of the SNF2 family of ATPases, has an N-terminal SNF2-like ATPase that is most closely related to that of ISWI, and a C-terminal macrodomain that binds selectively to poly(ADP-ribose) (PAR). Between the ATPase and the macrodomain is an evolutionarily conserved region with no clear homology to any known domains. This "linker" region can be further divided into three sub-regions of greatest conservation. It was previously shown that wild type ALC1 possesses DNA-dependent ATPase and ATP-dependent nucleosome remodeling activities that are strongly dependent on the presence of poly(ADP-ribose) polymerase PARP1 (or the closely related PARP2) and its substrate NAD+. Importantly, a point mutation in the ALC1 macrodomain that interferes with PAR binding prevents PARP1- and NAD-dependent ALC1 activation. In this work, we dissected the mechanism by which PARP1 and NAD+ activate ALC1 nucleosome remodeling. We demonstrate that ALC1 activation depends on the formation of a stable ALC1·PARylated PARP1·nucleosome intermediate. In addition, by exploiting a novel PAR footprinting assay, we obtained evidence that the ALC1 macrodomain remains stably associated with PAR on autoPARylated PARP1 during the course of nucleosome remodeling reactions. Results of biochemical experiments described here argue (i) that stable binding of the ALC1 macrodomain to autoPARylated PARP1 is critical for ALC1 activation and (ii) that activation of ALC1 depends on formation of a stable ALC1-autoPARylated PARP1-nucleosome intermediate. In the course of this study, we also find that apart from being regulated by PARP and NAD+, ALC1 possesses and additional mode of intradomain control via conserved domains in the linker region and the macrodomain. First, we identified a region, NMAC (N-terminal to Macrodomain ATPase Coupling) domain needed to couple ATP hydrolysis to nucleosome remodeling. Deleting NMAC led to a robust PARP and NAD+-dependent ATPase, which lacked appreciable remodeling activity. In addition, we identified an additional mode of control via the macrodomain when we replaced ALC1's PAR binding macrodomain with another macrodomain from the variant histone macroH2A1.1 and discovered this chimeric protein was constitutively active independent of PARP and NAD+. Taken together our findings suggest a model of positive control of the SNF2 ATPase via the macrodomain and an additional level of control over its remodeling activities via the NMAC and other conserved linker elements
Modulation of Protein-Membrane Interactions by Lipids and Divalent Cations
No full textThe post-translational insertion of proteins into membranes is common in a number of biologically important processes, such as the cellular entry of bacterial toxins or regulation of apoptosis by the Bcl-2 family of proteins, as well as in targeted drug delivery. While our ability to target or manipulate these processes can be beneficial for human health, the basic knowledge of the mechanism of membrane modulation of conformational switching is lacking. To provide a more complete picture of protein-membrane interactions in the cell, this manuscript explores the effects of lipid composition and physiological [Ca2+/Mg2+] on the membrane insertion and refolding of peptides and proteins. The main focus is on the peptide pHLIP (pH-Low Insertion Peptide), a promising cancer targeting system, and the apoptotic inhibitor Bcl-xL. The results presented here demonstrate that physiological concentrations of Ca2+ and Mg2+ induce the membrane insertion and refolding of both pHLIP and Bcl-xL. In both cases, the divalent cation-dependent effects are further modulated by lipid composition. These results highlight the importance of physiological conditions on the study of protein membrane interactions and shows that their omission can lead to false assumptions of unfavorable interactions
Molecular and Structural Basis of Anthrax Lethal Toxin Pore Formation and Translocation
No full textThe morbidity and mortality of anthrax disease are associated with the anthrax toxin, which is generated by the gram-positive bacterium Bacillus anthracis. The anthrax toxin is an AB toxin composed of two components: an active (A) moiety named lethal factor (LF) and a binding (B) moiety termed protective antigen (PA). In order for the anthrax toxin to elicit its cytotoxic effect, the LF component must first enter the cell. To accomplish this, the PA component binds to a target host cell receptor and forms a pore (PApore) that translocates LF into the cytosol. In this work, we explore PA binding the host cell receptor, the interactions between PA and LF during pore formation, and the translocation of LF through the PApore. We hypothesize PA dissociates from its cellular receptor to facilitate pore formation. To test this, in Chapter 2, we investigated the anthrax toxin receptor CMG2 binding capabilities to the PApore under endosomal conditions. Our results provide evidence for receptor release prior to pore formation. In Chapter 3, we hypothesized the LF N-terminal tail travels down the pore lumen and interacts with the narrowest part of the pore. To test this, we characterized the structure of three LFN bound to PApore at neutral pH. Our results indicate the N-terminal tail of LF remains flexible in the translocation incompetent neutral pH environment and underscores the necessity of using physiologically relevant conditions. In Chapter 4, we hypothesize LF begins to refold inside the PApore during translocation. To test this, we captured intermediates of LF translocation through PApore using cryoEM. Our results support the hypothesis that initial refolding of LF structural elements occurs in the PApore beta barrel during translocation. Cumulatively, we have made significant contributions to our understanding of the anthrax intoxication mechanism at multiple biologically relevant steps
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