Feb 29, 2016 - This work was supported by the American Heart Foundation (AHA. 13GRNT17290006). 1065-Pos Board B42. Regulation and Quality Control ...
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Monday, February 29, 2016
fighting the consequences of protein misfolding and aggregation or enhancing disaggregation of toxic aggregates by clearance mechanisms. In particular, recent studies have attributed to some chaperones the capability to directly interact with amyloid aggregation-prone species of peptide involved in Alzheimer Disease in solution and/or suppress the associated toxicity. Among them are the principal heat-shock proteins (HSPs), the small HSPs, but also molecules the exhibit marked chaperon like activity, like milk caseins or BRICHOS domains. There are several mechanisms by which chaperones exert their protective action, many of which involve protein disordered regions. However, in many cases, the scarcity of structural data has impeded an understanding of the recognition and antiaggregation mechanisms, that result crucially important when considering how to screen for and characterize potential inhibitors of amyloidosis. Here we show how a human chaperonin, Hsp60, and intrinsically disorder colloids with marked chaperon-like activity, the milk caseins, can specifically influence Ab40 fibrillogenesis. Inhibition studies are correlated with investigation on structural, self organization and stability properties of the chaperones under study performed by a battery of biophysical methods and aimed to understand what are molecular determinants responsible for their inhibitory action. 1064-Pos Board B41 The Unique Roles of UNC-45 Domains in Chaperoning Myosin Folding and Modulating Myosin Powerstroke Paul Nicholls1, Paul Bujalowski2, Andres Oberhauser1. 1 Physiology&Biophysics, UTMB, Galveston, TX, USA, 2BMB, UTMB, Galveston, TX, USA. Molecular chaperones increase de novo protein folding efficiency in the crowded environment of the cell. They typically function by shielding hydrophobic regions of unfolded and partially folded proteins, preventing misfolding and aggregation. Also, certain chaperones interact with proteins further along their folding process, including the native state, but the relevance of this role remains unclear. Investigation of the mechanisms used by the chaperones UNC-45 and Hsp90 to assist in folding the myosin motor domain offers an ideal opportunity to investigate these gaps in knowledge. The main driver of current research is our unexpected finding that the myosin chaperone UNC-45 inhibits myosin motor domain powerstroke until its partner chaperone Hsp90 reverses the inhibition. We have found that the distinct domains comprising UNC-45 participate in discretely different transactions during myosin folding and assembly: 1) the UCS domain shields misfolding of folding intermediates as they occur heat shock conditions (and hence is responsible for the ‘classic chaperone’ function); 2) Our binding studies indicate that the Central domain also interacts with myosin but does not prevent myosin aggregation thus it does not possess chaperone-like activities. We found that the Central domain alone acts as an inhibitor of myosin power stroke. This different behavior and the fact that the Central domain induces significant conformational changes within the myosin–UNC-45 complex through allosteric interactions, suggest that the Central domain performs a different function than the UCS domain; 3) the TPR domain recruits Hsp90 to allow the myosin powerstroke to resume actin translocation(‘unblocking’ function). Our studies should provide novel mechanistic insights into the pathogenesis of increased or reduced myosin activity (such as in certain hypertrophic and dilated cardiomyopathies) and its modulation by chaperones. This work was supported by the American Heart Foundation (AHA 13GRNT17290006). 1065-Pos Board B42 Regulation and Quality Control of Adiponectin Assembly by Endoplasmic Reticulum Chaperone ERp44 Lutz Hampe, Alok Kumar Mitra, Mazdak Radjainia. School of Biological Sciences, University of Auckland, Auckland, New Zealand. Adiponectin is a collagenous adipokine with anti-diabetic, anti-inflammatory and anti-tumor properties. Mediated by the conserved Cys39 located in the variable region of the N-terminus, the trimeric (low-molecular weight; LMW) adiponectin subunit assembles into different higher-order complexes e.g. hexamers (MMW) and 12-18mers (HMW) - the latter being mostly responsible for the insulin-sensitizing activity of adiponectin. In obese patients, adiponectin levels are greatly reduced with an accompanying loss of its profound protective effects. This reduction is partly due to obesity-induced dysregulation of endoplasmic reticulum (ER) chaperones such as ERp44, which mediate adiponectin assembly and secretion. In this study, we sought molecular-level understanding of the role of ER chaperone ERp44 in adiponectin assembly and secretion. Such investigations are mainly hampered by the complexity of adiponectin assemblies and its structural polymorphism. To overcome this problem, we developed multiple model peptides, which mimic the different oligomeric states of the N-terminal region of adiponectin containing the conserved Cys39.
Using these model peptides in in vitro assays and combined with cellular studies using full-length adiponectin we show that ERp44 specifically recognizes the LMW and MMW but not the HMW form. Our binding assays for short peptide mimetics of adiponectin suggest that ERp44 intercepts and converts the pool of fully oxidized LMW and MMW adiponectin, but not the HMW, into reduced trimeric precursors of the HMW form. Once the ERp44-precursorscomplex is formed in the acidic environment of the cisGolgi, the cargocomplex is transported back to the ER and the trimeric precursor is released triggered by the more alkaline pH there. Using such a process, we suggest that ERp44 enhances the population of adiponectin intermediates with appropriate oxidative state for HMW assembly, thereby underpinning the process of ERp44 quality control. 1066-Pos Board B43 Signature Networks Underlying Unfolded Intermediate of an Obligate Groel Substrate Lipi Thukral. IGIB, New Delhi, India. A properly folded protein is a prerequisite in nearly every cellular process. Several proteins fold spontaneously while others rely on molecular chaperones to reach their native states. In this work, we study DapA protein, which exhibits obligate requirement on GroEL chaperonin machinery for its folding. All-atom and replica-exchange molecular dynamics simulations were performed to reveal populated intermediate structures of DapA in atomic details. We found that surface exposed hydrophobic patches are significantly increased, primarily contributed from native and non-native b-sheet elements. We validate the structural properties of these conformers using experimental data, including circular dichroism (CD), 1-anilinonaphthalene-8-sulfonic acid (ANS) binding measurements and previously reported hydrogen-deutrium exchange coupled to mass spectrometry (HDX-MS). Further, we constructed network graphs to elucidate long-range intra-protein connectivity of native and intermediate topologies, demonstrating regions that serve as central ‘‘hubs’’. In summary, our work provides a molecular picture of an unfolded protein that is en route to chaperone binding, and these underlying structural properties might act as a molecular signal for their productive folding. Reference: Nagpal S, Tiwari S, Mapa K, Thukral L (2015). Decoding structural properties of a partially unfolded protein substrate: en route to chaperone binding. PLoS Computational Biol. 2015;11(9):e1004496. 1067-Pos Board B44 Studies on Domain Specific Aggregation Behavior of Huntingtin Exon1 Nitin K. Pandey, Jose Mario Isas, Ralf Langen. Biochemistry, Zilkha Neurogenetic Institute, University of southern california, Los Angeles, CA, USA. The pathogenesis of Huntington’s disease results from polyQ expansions (>36Q) in the Huntingtin (Htt) protein.1 These polyQ expansions make Htt less soluble and promote the formation of misfolded oligomers, fibrils and aggregates in Huntington’s disease.2 Efforts to prevent aggregation will greatly benefit from a detailed molecular understanding of the misfolding process. However, htt misfolding has been difficult to study as the protein is tough to handle and examine spectroscopically. Here we developed a novel purification scheme to generate recombinant, clean and monomeric Htt exon1, an aggregation prone fragment of htt that is highly expressed in disease. The misfolding of spin labeled derivatives of Htt exon1 were then monitored by EPR in order to obtain site-specific folding information for residues in different regions of the protein. Our data indicate that residues in the N-terminal N17, the middle polyQ region and the C-terminal, proline rich region all undergo structural changes on different time scales. Moreover, we have also found that the aggregation kinetics and mechanisms of aggregation significantly depend on Q length. Overall, the data indicate distinct phases in which different regions of the molecule undergo conformational changes. Each of these stages may afford the design of different compounds intended at altering the aggregation process. 1. Bates, GP.; Harper, PS.; Jones, L., editors. Huntington’s Disease. Oxford University Press; Oxford, U.K.: 2002. 2. Bates, GP.; Benn, C. The polyglutamine diseases. In: Bates, GP.; Harper, PS.; Jones, L., editors. Huntington’s Disease. Oxford University Press; Oxford, U.K.: 2002. p. 429-472. 1068-Pos Board B45 Contrasting Roles of Asparagine and Glutamine in the Aggregation of Prion-Like Proteins Yuan Zhang, Viet Hoang Man, Christopher Roland, Celeste Sagui. Physics, North Carolina State University, Raleigh, NC, USA. Sequences rich in glutamine (Q) and asparagine (N) are intrinsically disordered in monomeric form, but can aggregate into highly ordered amyloids, as seen in
Monday, February 29, 2016 Q/N-rich prion domains (PrDs). Amyloids are fibrillar protein aggregates rich in b-sheet structures that can self-propagate through protein-conformational chain reactions. It has been shown that tuning the amount of Ns and Qs in yeast PrDs results in very different effects: N-rich mutants lead to nonpathological self-seeding amyloids while Q-rich mutants lead to toxic nonamyloid structures. These structural preferences have been explained in terms of an enhanced b- hairpin turn propensity of Ns over Qs. Here, we consider a variety of N/Q-rich peptides, including sequences found in the yeast Sup35 PrD, in parallel and antiparallel b-sheet aggregates, and probe all their possible steric-zipper interfaces to determine their relative stability. Our results show that polyglutamine aggregates are more stable than polyasparagine aggregates. The observation that Q-rich PrD mutants lack amyloid structure can be attributed to three facts. First, although once formed polyglutamine aggregates are more stable, their entropic contribution to the free energy is less favorable: Q-rich sequences have a larger phase space to sample. Second, N-rich sequences favor parallel b sheets, for which the formation of hairpin turns is irrelevant: indeed polyasparagine b-hairpins are more unstable than polyglutamine hairpins. Third, when other amino acids are present, such as in the Sup35 PrD, their shorter side chains favor steric-zipper formation for N but not Q, as they preclude the in-register association of the long Q side chains. 1069-Pos Board B46 Structural Determinants of Polyqlutamine Protofibrils and Crystallites Viet H. Man, Christopher Roland, Celeste Sagui. Physics, North Carolina State University, Raleigh, NC, USA. Nine inherited neurodegenerative diseases are associated with the expansion of the CAG codon. Once the translated polyglutamine expansion becomes longer than ~36 residues, it triggers the formation of intraneural protein aggregates that often display the signature of cross-b amyloid fibrils. Here, we use fully atomistic molecular dynamics simulations with explicit solvent and state-ofthe-art force field to probe the structural stability and conformational dynamics of both previously proposed and new polyglutamine aggregate models, for a cumulative time of over 23 ms. We estimate the relative stability of parallel and antiparallel b sheets, and characterize possible steric interfaces between neighboring sheets and the effects of different alignments of the side-chain carboxamide dipoles. The results indicate that (i) different initial oligomer structures converge to crystals consistent with available diffraction data, after undergoing cooperative side-chain rotational transitions and quarter-stagger displacements on a microsecond time scale, (ii) structures previously deemed stable on a hundred nanosecond time scale are unstable over the microsecond time scale, and (iii) conversely, structures previously deemed unstable did not account for the correct side-chain packing and once the correct symmetry is considered the structures become stable for over a microsecond, due to tightly interdigitated side chains, which lock into highly regular polar zippers with inter-side-chain and backbone side-chain hydrogen bonds. With these insights, we built Q40 monomeric models with different combinations of arc and hairpin turns and tested them for stability. The stable monomers were further probed as a function of repeat length. Our results are consistent with the aggregation threshold. These results explain and reconcile previously reported experimental and model discrepancies about polyglutamine aggregate structures. 1070-Pos Board B47 Folding Pathways of Evolutionarily Related Proteins Probed by Hydrogen Exchange Mass Spectrometry Eric Bolin, Susan Marqusee, Shion Lim. UC Berkeley, Berkeley, CA, USA. The process by which proteins adopt their native structure has been an active field of study for decades. However, many basic questions about this protein folding process remain unanswered. For instance, the role of sequence in determining the pathway through which a protein folds has been difficult to characterize since most techniques to characterize pathways are slow, require large amounts of protein, or only probe folding in a small region of the protein. Hydrogen exchange measured by mass spectrometry (HX/MS) has proven to be a useful tool that can quickly determine folding pathway of a protein by measuring the buildup of hydrogen bonding along the protein backbone. In addition to its relative speed, HX/MS is able to measure to probe folding across the entire protein sequence allowing a pathway to be determined from a single experiment and uses nanomolar quantities of protein. Using HX/MS to determine the folding pathway for the RNase H from E. coli and T. thermophilus as well as the RNase H from the last common ancestor as determined through ancestral sequence reconstruction shows that despite folding to the same final structure these proteins fold through distinct pathways.
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Protein Assemblies I 1071-Pos Board B48 Determining Unitary Water Permeability of Membrane Proteins Reconstituted into Giant Unilamellar Vesicles Danila Boytsov, Christof Hannesschlaeger, Andreas Horner, Peter Pohl. Institute of Biophysics, Johannes Kepler University Linz, Linz, Austria. The high background water permeability of lipid bilayers may hamper determining unitary water permeability pf at low protein/lipid ratios. That is, reliable pf measurements require reconstitution of more than one water-channeling protein per large unilamellar vesicle (LUV)1. The copy number per LUV must be even higher for proteins that conduct water at slower rates than the most efficient aquaporins, which may prove impossible to achieve. Here we show that protein reconstitution into giant unilamellar vesicles (GUVs) provides a solution. Instead of assessing the volume change from the intensity of light scattering as performed with LUVs2, we implement the micropipette aspiration technique. It offers the possibility of measuring much smaller changes in relative volume. Additional advantages are that channel membrane abundancy and oligomeric state are directly determined by fluorescence correlation spectroscopy in the very same GUV from which the volume flow is extracted. In contrast, the channel number per LUV is extracted in retrospect: It is equal to the difference in particle number in the confocal volume of the correlation spectrometer before and subsequent to vesicle solubilization3. We test the new assay by reconstituting the purified and fluorescently-labeled glycerol facilitator from E.coli into GUVs. We found it mandatory to properly account for unstirred layer effects, i.e. for osmolyte and solute dilution adjacent to the external surface of the GUV and for solute concentration increases in the immediate vicinity of the internal leaflet. The pf value we determined with the novel assay agrees very well with recently published data2. 1. Hoomann et al. (2013). PNAS 110, 10842-10847. 2. Horner et al. (2015). Science Advances 1, e1400083. 3. Knyazev et al. (2013). JBC 288, 17941-17946. 1072-Pos Board B49 Regulation of ALIX during Exocytic Vesicle Release and Assembly of ESCRT Proteins on the Plasma Membrane Pei-I Ku, Saveez Saffarian. Physics and Astronomy, University of Utah, Salt Lake City, UT, USA. Endosomal sorting complexes required for transport (ESCRTs) are protein complexes that facilitate the release of budding vesicles into extracellular environment. ESCRTs are essential in most cellular processes requiring fission of budding membrane, including multi-vesicular body formation, envelope virus release, and exosome release. During HIV-1 budding, HIV-1 Gag assembles on the plasma membrane and drives the outward deformation of the membrane toward forming vesicles. HIV-1 virus like particles (VLPs) can be generated by expressing Gag alone in the cytosol, these VLPs are consistent of ~2000 copies of Gag and form vesicles with an average diameter of 120 nanometers and their assembly on the plasma membrane take ~20-30 minutes. HIV-1 utilizes ESCRTs extensively for the release of progeny virions into extracellular space and VLPs serve as a good model system for studying ESCRT recruitment on the plasma membrane. Gag has two late domain motifs which interact with TSG101 and ALIX, two of the early ESCRT components. Given the stoichiometry of Gag and its steady accumulation in the forming VLPs, it has been expected that ALIX would be recruited into the forming VLPs along with Gag during the assembly. Using live imaging to observe real time assembly of VLPs in the presence of fluorescently tagged ALIX proteins, we have observed that ALIX is recruited transiently at the end of Gag assembly and is mostly recycled after VLPs release. This observation implies high spatial and temporal regulation for recruitment of ALIX during budding. We have further dissected the essential interactions that govern ALIX recruitment, showed the interaction of ALIX Bro1 domain with CHMP4 is critical for recruitment of ALIX and proposed the emerging regulatory mechanism essential for recruitment of ALIX to the plasma membrane during vesicle budding. 1073-Pos Board B50 Functional Cooperativity among the Subunits of the Homotetrameric Aquaglyceroprotein GlpF Andreas Horner1, Danila Boytsov1, Christine Siligan1, Johannes Preiner2, Peter Pohl1. 1 Institute of Biophysics, Johannes Kepler University Linz, Linz, Austria, 2 Center for Advanced Bioanalysis GmbH, Linz, Austria. Aquaporins are conserved throughout all kingdoms of life. Each aquaporin monomer possesses a channel that facilitates the transport of water across the membrane, and yet the protein assembles into a tetramer. We investigated the physiological function of the tetrameric assembly by introducing
