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Journal of molecular biology [journal]
- Gly25-Ser26 amyloid β-protein structural isomorphs produce distinct Aβ42 conformational dynamics and assembly characteristics. [JOURNAL ARTICLE]
- J Mol Biol 2014 Apr 12.
One of the earliest events in amyloid β-protein (Aβ) self-association is nucleation of Aβ monomer folding through formation of a turn at Gly25-Lys28. We report here the effects of structural changes at the center of the turn, Gly25-Ser26, on Aβ42 conformational dynamics and assembly. We used "click peptide" chemistry to quasi-synchronously create Aβ42 from 26-O-acyliso-Aβ42 (iAβ42) through a pH jump from 3→7.4. We also synthesized Nα-acetyl-Ser26iAβ42 (Ac-iAβ42), which cannot undergo O→N acyl chemistry, to study the behavior of this ester form of Aβ42 itself at neutral pH. Data from experiments monitoring increases in β-sheet formation (ThT, CD), hydrodynamic radius (RH), scattering intensity (QLS), and extent of oligomerization (IMS-MS), were quite consistent. A rank order of Ac-iAβ42>iAβ42>Aβ42 was observed. Photochemically cross-linked iAβ42 displayed an oligomer distribution with a prominent dimer band that was not present with Aβ42. These dimers also were observed selectively in iAβ42 in ion mobility spectrometry experiments. The distinct biophysical behaviors of iAβ42 and Aβ42 appear to be due to the conversion of iAβ42 into "pure" Aβ42 monomer, a nascent form of Aβ42 that does not comprise the variety of oligomeric and aggregated states present in pre-existent Aβ42. These results emphasize the importance of the Gly25-Ser26 dipeptide in organizing Aβ42 monomer structure and thus suggest that drugs altering the interactions of this dipeptide with neighboring side-chain atoms or with the peptide backbone could be useful in therapeutic strategies targeting formation of Aβ oligomers and higher-order assemblies.
- The Nucleolar PICT-1/GLTSCR2 Protein Forms Homo-Oligomers. [JOURNAL ARTICLE]
- J Mol Biol 2014 Apr 12.
The human 'protein interacting with carboxyl terminus 1' (PICT-1), also designated as the 'glioma tumor suppressor candidate region 2 gene product', GLTSCR2, is a nucleolar protein whose activity is, as yet, unknown. Contradictory results regarding the role of PICT-1 in cancer have been reported, and PICT-1 has been suggested to function either as a tumor suppressor protein or as an oncogene. In this study, we demonstrate self-association of PICT-1. Through yeast two-hybrid assay, we identified PICT-1 as its own interaction partner. We confirmed the interaction of PICT-1 with itself by direct yeast two-hybrid assay and also showed self-association of PICT-1 in mammalian cells by co-immunoprecipitation and FRET assays. Furthermore, we confirmed direct self-association of PICT-1 by using in vitro microfluidic affinity binding assays. The later assay also identified the carboxy-terminus domain as mediating self-interaction of PICT-1. Glutaraldehyde cross-linking and gel filtration assays suggest that PICT-1 forms dimers, though it may form higher order complexes as well. Our findings add another layer of complexity to understanding the different functions of PICT-1 and may help provide insights regarding the activities of this protein.
- The molecular basis of bacterial-insect symbiosis. [REVIEW]
- J Mol Biol 2014 Apr 12.
Insects provide experimentally tractable and cost-effective model systems to investigate the molecular basis of animal-bacterial interactions. Recent research is revealing the central role of the insect innate immune system, especially anti-microbial peptides and reactive oxygen species, in regulating the abundance and composition of the microbiota in various insects, including Drosophila and the mosquitoes Aedes and Anopheles. Interactions between the immune system and microbiota are, however, bidirectional with evidence that members of the resident microbiota can promote immune function, conferring resistance to pathogens and parasites by both activation of immune effectors and production of toxins. Antagonistic and mutualistic interactions among bacteria have also been implicated as determinants of the microbiota composition, including exclusion of pathogens, but the molecular mechanisms are largely unknown. Some bacteria are crucial for insect nutrition, through provisioning of specific nutrients (e.g. B vitamins, essential amino acids) and modulation of the insect nutritional sensing and signaling pathways (e.g. insulin signaling) that regulate nutrient allocation, especially to lipid and other energy reserves. A key challenge for future research is to identify the molecular interaction between specific bacterial effectors and animal receptors, and to determine how these interactions translate into microbiota-dependent signaling, metabolism and immune function in the host.
- A genome-wide sequence-structure analysis suggests aggregation gatekeepers constitute an evolutionary constrained functional class. [JOURNAL ARTICLE]
- J Mol Biol 2014 Apr 12.
Protein aggregation is geared by aggregation prone sequences (APRs) that self-associate by β-strand interactions. Charged residues and prolines are enriched at the flanks of APRs resulting in decreased aggregation. It is still unclear what drives the overrepresentation of these 'aggregation gatekeepers', i.e. whether their presence results from structural constraints determining protein stability or whether they constitute a bona fide functional class selectively maintained to control protein aggregation. As functional residues are typically conserved regardless of their cost to protein stability we compared sequence conservation and thermodynamic cost of these residues in 2659 protein families in E. coli. Across protein families we find gatekeepers to be under strong selective conservation while at the same time representing a significant thermodynamic cost to protein structure. This finding supports the notion that aggregation gatekeepers are not structurally determined but evolutionary selected to control protein aggregation.
- The crystal structure of the anti-σ factor CnrY in complex with the σ factor CnrH shows a new structural class of anti-σ factors targeting ExtraCytoplasmic-Function σ factors. [JOURNAL ARTICLE]
- J Mol Biol 2014 Apr 9.
Gene expression in bacteria is regulated at the level of transcription initiation, a process driven by σ factors. The regulation of σ factor activity proceeds from the regulation of their cytoplasmic availability, which relies on specific inhibitory proteins called anti-σ factors. With anti-σ factors regulating their availability according to diverse cues, ExtraCytoplasmic Function σ factors (σ(ECF)) form a major signal transduction system in bacteria. Here, structure:function relationships have been characterized in an emerging class of minimal-size, transmembrane anti-σ factors, using CnrY from Cupriavidus metallidurans CH34 as a model. This study reports the 1.75 Å resolution structure of CnrY cytosolic domain in complex with CnrH, its cognate σ(ECF) and identifies a small hydrophobic knob in CnrY as the major determinant of this interaction in vivo. Unsuspected structural similarity with the molecular switch regulating the general stress response in α-proteobacteria unravels a new class of anti-σ factors targeting σ(ECF). Members of this class carry out their function via a 30-residue stretch that displays helical propensity but no canonical structure on its own.
- Towards predictive models of the human gut microbiome. [REVIEW]
- J Mol Biol 2014 Apr 9.
The intestinal microbiota is an ecosystem susceptible to external perturbations such as dietary changes and antibiotic therapies. Mathematical models of microbial communities could be of great value in the rational design of microbiota-tailoring diets and therapies. Here, we discuss how advances in another field, engineering of microbial communities for wastewater treatment bioreactors, could inspire development of mechanistic mathematical models of the gut microbiota. We review the current state-of-the-art in bioreactor modeling and current efforts in modeling the intestinal microbiota. Mathematical modeling could benefit greatly from the deluge of data emerging from metagenomic studies, but data-driven approaches such as network inference that aim to predict microbiome dynamics without explicit mechanistic knowledge seem better suited to model these data. Finally, we discuss how the integration of microbiome shotgun sequencing and metabolic modeling approaches such as flux balance analysis may fulfill the promise of a mechanistic model of the intestinal microbiota.
- Elucidating the mechanism of substrate recognition by the bacterial Hsp90 molecular chaperone. [JOURNAL ARTICLE]
- J Mol Biol 2014 Apr 8.
Hsp90 is a conformationally dynamic molecular chaperone known to promote the folding and activation of a broad array of protein substrates ("clients"). Hsp90 is believed to preferentially interact with partially folded substrates, and it has been hypothesized that the chaperone can significantly alter substrate structure as a mechanism to alter the substrate functional state. However, critically testing the mechanism of substrate recognition and remodeling by Hsp90 has been challenging. Using a partially folded protein as a model system, we find that the bacterial Hsp90 adapts its conformation to the substrate, forming a binding site that spans the middle and C-terminal domains of the chaperone. Crosslinking and NMR measurements indicate that Hsp90 binds to a large partially-folded region of the substrate and significantly alters both its local and long-range structure. These findings implicate Hsp90's conformational dynamics in its ability to bind and remodel partially folded proteins. Moreover, native-state hydrogen exchange indicates that Hsp90 can also interact with partially folded states only transiently populated from within a thermodynamically stable native state ensemble. These results suggest a general mechanism by which Hsp90 can recognize and remodel native proteins by binding and remodeling partially folded states that are transiently sampled from within the native ensemble.
- A mutation in the catalytic loop of Hsp90 specifically impairs ATPase stimulation by Aha1p, but not Hch1p. [JOURNAL ARTICLE]
- J Mol Biol 2014 Apr 8.
Hsp90 is a molecular chaperone that plays a central role in maintaining cellular homeostasis by facilitating activation of a large number of client proteins. ATP-dependent client activation by Hsp90 is tightly regulated by a host of co-chaperone proteins that control progression through the activation cycle. ATPase stimulation of Hsp90 by Aha1p requires a conserved RKxK motif that interacts with the catalytic loop of Hsp90. In this study, we explore the role of this RKxK motif in the biological and biochemical properties of Hch1p. We found that this motif is required for Hch1p-mediated ATPase stimulation in vitro, but mutations that block stimulation do not impair the action of Hch1p in vivo. This suggests that the biological function of Hch1p is not directly linked to ATPase stimulation. Moreover, a mutation in the catalytic loop of Hsp90 specifically impairs ATPase stimulation by Aha1p but not by Hch1p. Our work here suggests that both Hch1p and Aha1p regulate Hsp90 function through interaction with the catalytic loop but do so in different ways.
- Epigenetic Signaling in Psychiatric Disorders. [REVIEW]
- J Mol Biol 2014 Apr 4.
Psychiatric disorders are complex multifactorial illnesses involving chronic alterations in neural circuit structure and function. While genetic factors are important in the etiology of disorders such as depression and addiction, relatively high rates of discordance among identical twins clearly indicate the importance of additional mechanisms. Environmental factors such as stress or prior drug exposure are known to play a role in the onset of these illnesses. Such exposure to environmental insults induces stable changes in gene expression, neural circuit function, and ultimately behavior, and these maladaptations appear distinct between developmental and adult exposures. Increasing evidence indicates that these sustained abnormalities are maintained by epigenetic modifications in specific brain regions. Indeed, transcriptional dysregulation and associated aberrant epigenetic regulation is a unifying theme in psychiatric disorders. Aspects of depression and addiction can be modeled in animals by inducing disease-like states through environmental manipulations (e.g., chronic-stress, drug administration). Understanding how environmental factors recruit the epigenetic machinery in animal models is revealing new insight into disease mechanisms in humans.
- Architectures of Whole-Module and Bimodular Proteins from the 6-Deoxyerythronolide B Synthase. [JOURNAL ARTICLE]
- J Mol Biol 2014 Apr 2.
The 6-deoxyerythronolide B synthase (DEBS) is a prototypical assembly line polyketide synthase (PKS) produced by the actinomycete Saccharopolyspora erythraea that synthesizes the macrocyclic core of the antibiotic erythromycin, 6-deoxyerythronolide B (6-dEB). The megasynthase is a 2 MDa trimeric complex comprised of three unique homodimers assembled from the gene products DEBS1, DEBS2, and DEBS3, which are housed within the erythromycin biosynthetic gene cluster. Each homodimer contains two clusters of catalytically independent enzymatic domains, each referred to as a module, which catalyzes one round of polyketide chain extension and modification. Modules are named sequentially to indicate the order in which they are utilized during synthesis of 6-dEB. We report small angle X-ray scattering (SAXS) analyses of a whole module and bimodule from DEBS as well as a set of domains for which high-resolution structures are available. In all cases, the solution state was probed under previously established conditions that ensure each protein is catalytically active. SAXS data are consistent with atomic-resolution structures of DEBS fragments. Therefore, we used the available high-resolution structures of DEBS domains to model the architectures of the larger protein assemblies using rigid body refinement. Our data supports a model in which, the third module of DEBS forms a disc-shaped structure capable of caging the acyl carrier protein domain proximal to each active site. The molecular envelope of DEBS3 is a thin, elongated ellipsoid, and the results of rigid body modeling suggest that modules 5 and 6 stack colinearly along the 2-fold axis of symmetry.