Hydrogen bonding in aqueous solution and peptide helicity studied using model systems

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 1999.Includes bibliographical references.by Evan T. Powers.Ph.D

The proteostasis boundary in misfolding diseases of membrane traffic

AbstractProtein function is regulated by the proteostasis network (PN) [Balch, W.E., Morimoto, R.I., Dillin, A. and Kelly, J.W. (2008) Adapting proteostasis for disease intervention. Science 319, 916–919], an integrated biological system that generates and protects the protein fold. The composition of the PN is regulated by signaling pathways including the unfolded protein response (UPR), the heat-shock response (HSR), the ubiquitin proteasome system (UPS) and epigenetic programs. Mismanagement of protein folding and function during membrane trafficking through the exocytic and endocytic pathways of eukaryotic cells by the PN is responsible for a wide range of diseases that include, among others, lysosomal storage diseases, myelination diseases, cystic fibrosis, systemic amyloidoses such as light chain myeloma, and neurodegenerative diseases including Alzheimer’s. Toxicity from misfolding can be cell autonomous (affect the producing cell) or cell non-autonomous (affect a non-producing cell) or both, and have either a loss-of-function or gain-of-toxic function phenotype. Herein, we review the role of the PN and its regulatory transcriptional circuitry likely to be operational in managing the protein fold and function during membrane trafficking. We emphasize the enabling principle of a ‘proteostasis boundary (PB)’ [Powers, E.T., Morimoto, R.T., Dillin, A., Kelly, J.W., and Balch, W.E. (2009) Biochemical and chemical approaches to diseases of proteostasis deficiency. Annu. Rev. Biochem. 78, 959–991]. The PB is defined by the combined effects of the kinetics and thermodynamics of folding and the kinetics of misfolding, which are linked to the variable and adjustable PN capacity found different cell types. Differences in the PN account for the versatility of protein folding and function in health, and the cellular and tissue response to mutation and environmental challenges in disease. We discuss how manipulation of the folding energetics or the PB through metabolites and pharmacological intervention provides multiple routes for restoration of biological function in trafficking disease

Internally heated and fully compressible convection: flow morphology and scaling laws

In stars and planets natural processes heat convective flows in the bulk of a convective region rather than at hard boundaries. By characterizing how convective dynamics are determined by the strength of an internal heating source we can gain insight into the processes driving astrophysical convection. Internally heated convection has been studied extensively in incompressible fluids, but the effects of stratification and compressibility have not been examined in detail. In this work, we study fully compressible convection driven by a spatially uniform heating source in 2D and 3D Cartesian, hydrodynamic simulations. We use a fixed temperature upper boundary condition which results in a system that is internally heated in the bulk and cooled at the top. We find that the flow speed, as measured by the Mach number, and turbulence, as measured by the Reynolds number, can be independently controlled by separately varying the characteristic temperature gradient from internal heating and the diffusivities. 2D simulations at a fixed Mach number (flow speed) demonstrate consistent power at low wavenumber as diffusivities are decreased. We observe convection where the velocity distribution is skewed towards cold, fast downflows, and that the flow speed is related to the length scale and entropy gradient of the upper boundary where the downflows are driven. We additionally find a heat transport scaling law which is consistent with prior incompressible work.Comment: 22 pages, 12 figures, submitted to Phys. Rev. Fluid

Individual and Collective Contributions of Chaperoning and Degradation to Protein Homeostasis in E. coli

SummaryThe folding fate of a protein in vivo is determined by the interplay between a protein’s folding energy landscape and the actions of the proteostasis network, including molecular chaperones and degradation enzymes. The mechanisms of individual components of the E. coli proteostasis network have been studied extensively, but much less is known about how they function as a system. We used an integrated experimental and computational approach to quantitatively analyze the folding outcomes (native folding versus aggregation versus degradation) of three test proteins biosynthesized in E. coli under a variety of conditions. Overexpression of the entire proteostasis network benefited all three test proteins, but the effect of upregulating individual chaperones or the major degradation enzyme, Lon, varied for proteins with different biophysical properties. In sum, the impact of the E. coli proteostasis network is a consequence of concerted action by the Hsp70 system (DnaK/DnaJ/GrpE), the Hsp60 system (GroEL/GroES), and Lon

Semi-Quantitative Models for Identifying Potent and Selective Transthyretin Amyloidogenesis Inhibitors

Rate-limiting dissociation of the tetrameric protein transthyretin (TTR), followed by monomer misfolding and misassembly, appears to cause degenerative diseases in humans known as the transthyretin amyloidoses, based on human genetic, biochemical and pharmacologic evidence. Small molecules that bind to the generally unoccupied thyroxine binding pockets in the native TTR tetramer kinetically stabilize the tetramer, slowing subunit dissociation proportional to the extent that the molecules stabilize the native state over the dissociative transition state—thereby inhibiting amyloidogenesis. Herein, we use previously reported structure-activity relationship data to develop two semi-quantitative algorithms for identifying the structures of potent and selective transthyretin kinetic stabilizers/amyloidogenesis inhibitors. The viability of these prediction algorithms, in particular the more robust in silico docking model, is perhaps best validated by the clinical success of tafamidis, the first-in-class drug approved in Europe, Japan, South America, and elsewhere for treating transthyretin aggregation-associated familial amyloid polyneuropathy. Tafamidis is also being evaluated in a fully-enrolled placebo-controlled clinical trial for its efficacy against TTR cardiomyopathy. These prediction algorithms will be useful for identifying second generation TTR kinetic stabilizers, should these be needed to ameliorate the central nervous system or ophthalmologic pathology caused by TTR aggregation in organs not accessed by oral tafamidis administration

Enhanced Aromatic Sequons Increase Oligosaccharyltransferase Glycosylation Efficiency and Glycan Homogeneity

SummaryN-Glycosylation plays an important role in protein folding and function. Previous studies demonstrate that a phenylalanine residue introduced at the n-2 position relative to an Asn-Xxx-Thr/Ser N-glycosylation sequon increases the glycan occupancy of the sequon in insect cells. Here, we show that any aromatic residue at n-2 increases glycan occupancy in human cells and that this effect is dependent upon oligosaccharyltransferase substrate preferences rather than differences in other cellular processing events such as degradation or trafficking. Moreover, aromatic residues at n-2 alter glycan processing in the Golgi, producing proteins with less complex N-glycan structures. These results demonstrate that manipulating the sequence space surrounding N-glycosylation sequons is useful both for controlling glycosylation efficiency, thus enhancing glycan occupancy, and for influencing the N-glycan structures produced

A human, compact, fully functional anti-ErbB2 antibody as a novel antitumour agent

A new human, compact antibody was engineered by fusion of a human, antitumour ErbB2-directed scFv with a human IgG1 Fc domain. Overexpression of the ErbB2 receptor is related to tumour aggressiveness and poor prognosis. This new immunoagent meets all criteria for a potential anticancer drug: it is human, hence poorly or not immunogenic; it binds selectively and with high affinity to target cells, on which it exerts an effective and selective antiproliferative action, including both antibody-dependent and complement-dependent cytotoxicity; it effectively inhibits tumour growth in vivo. Its compact molecular size should provide for an efficient tissue penetration, yet suitable to a prolonged serum half-life

Using decision analysis to support proactive management of emerging infectious wildlife diseases

Despite calls for improved responses to emerging infectious diseases in wildlife, management is seldom considered until a disease has been detected in affected populations. Reactive approaches may limit the potential for control and increase total response costs. An alternative, proactive management framework can identify immediate actions that reduce future impacts even before a disease is detected, and plan subsequent actions that are conditional on disease emergence. We identify four main obstacles to developing proactive management strategies for the newly discovered salamander pathogen Batrachochytrium salamandrivorans (Bsal). Given that uncertainty is a hallmark of wildlife disease management and that associated decisions are often complicated by multiple competing objectives, we advocate using decision analysis to create and evaluate trade-offs between proactive (pre-emergence) and reactive (post-emergence) management options. Policy makers and natural resource agency personnel can apply principles from decision analysis to improve strategies for countering emerging infectious diseases

Individual Differences in Sound-in-Noise Perception Are Related to the Strength of Short-Latency Neural Responses to Noise

Important sounds can be easily missed or misidentified in the presence of extraneous noise. We describe an auditory illusion in which a continuous ongoing tone becomes inaudible during a brief, non-masking noise burst more than one octave away, which is unexpected given the frequency resolution of human hearing. Participants strongly susceptible to this illusory discontinuity did not perceive illusory auditory continuity (in which a sound subjectively continues during a burst of masking noise) when the noises were short, yet did so at longer noise durations. Participants who were not prone to illusory discontinuity showed robust early electroencephalographic responses at 40–66 ms after noise burst onset, whereas those prone to the illusion lacked these early responses. These data suggest that short-latency neural responses to auditory scene components reflect subsequent individual differences in the parsing of auditory scenes

Sensing and Integration of Erk and PI3K Signals by Myc

The transcription factor Myc plays a central role in regulating cell-fate decisions, including proliferation, growth, and apoptosis. To maintain a normal cell physiology, it is critical that the control of Myc dynamics is precisely orchestrated. Recent studies suggest that such control of Myc can be achieved at the post-translational level via protein stability modulation. Myc is regulated by two Ras effector pathways: the extracellular signal-regulated kinase (Erk) and phosphatidylinositol 3-kinase (PI3K) pathways. To gain quantitative insight into Myc dynamics, we have developed a mathematical model to analyze post-translational regulation of Myc via sequential phosphorylation by Erk and PI3K. Our results suggest that Myc integrates Erk and PI3K signals to result in various cellular responses by differential stability control of Myc protein isoforms. Such signal integration confers a flexible dynamic range for the system output, governed by stability change. In addition, signal integration may require saturation of the input signals, leading to sensitive signal integration to the temporal features of the input signals, insensitive response to their amplitudes, and resistance to input fluctuations. We further propose that these characteristics of the protein stability control module in Myc may be commonly utilized in various cell types and classes of proteins