Alison M. Maxwell’s research while affiliated with UCSF University of California, San Francisco and other places

Down syndrome (DS) is caused by the triplication of chromosome 21 and is the most common chromosomal disorder in humans. Those individuals with DS who live beyond age 40 y develop a progressive dementia that is similar to Alzheimer's disease (AD). Both DS and AD brains exhibit numerous extracellular amyloid plaques composed of Aβ and intracellular neurofibrillary tangles composed of tau. Since AD is a double-prion disorder, we asked if both Aβ and tau prions feature in DS. Frozen brains from people with DS, familial AD (fAD), sporadic AD (sAD), and age-matched controls were procured from brain biorepositories. We selectively precipitated Aβ and tau prions from DS brain homogenates and measured the number of prions using cellular bioassays. In brain extracts from 28 deceased donors with DS, ranging in age from 19 to 65 y, we found nearly all DS brains had readily measurable levels of Aβ and tau prions. In a cross-sectional analysis of DS donor age at death, we found that the levels of Aβ and tau prions increased with age. In contrast to DS brains, the levels of Aβ and tau prions in the brains of 37 fAD and sAD donors decreased as a function of age at death. Whether DS is an ideal model for assessing the efficacy of putative AD therapeutics remains to be determined.

Amyloid beta (Aβ) is thought to play a critical role in the pathogenesis of Alzheimer’s disease (AD). Prion-like Aβ polymorphs, or “strains”, can have varying pathogenicity and may underlie the phenotypic heterogeneity of the disease. In order to develop effective AD therapies, it is critical to identify the strains of Aβ that might arise prior to the onset of clinical symptoms and understand how they may change with progressing disease. Down syndrome (DS), as the most common genetic cause of AD, presents promising opportunities to compare such features between early and advanced AD. In this work, we evaluate the neuropathology and Aβ strain profile in the post-mortem brain tissues of 210 DS, AD, and control individuals. We assayed the levels of various Aβ and tau species and used conformation-sensitive fluorescent probes to detect differences in Aβ strains among individuals and populations. We found that these cohorts have some common but also some distinct strains from one another, with the most heterogeneous populations of Aβ emerging in subjects with high levels of AD pathology. The emergence of distinct strains in DS at these later stages of disease suggests that the confluence of aging, pathology, and other DS-linked factors may favor conditions that generate strains that are unique from sporadic AD.

Amyloid beta (Aβ) is thought to play a critical role in the pathogenesis of Alzheimer’s disease (AD). Prion-like Aβ polymorphs, or “strains”, can have varying pathogenicity and may underlie the phenotypic heterogeneity of the disease. In order to develop effective AD therapies, it is critical to identify the strains of Aβ that might arise prior to the onset of clinical symptoms and understand how they may change with progressing disease. Down syndrome (DS), as the most common genetic cause of AD, presents promising opportunities to compare such features between early and advanced AD. In this work, we evaluate the neuropathology and Aβ strain profile in the post-mortem brain tissues of 210 DS, AD, and control individuals. We assayed the levels of various Aβ and tau species and used conformation-sensitive fluorescent probes to detect differences in Aβ strains among individuals and populations. We found that these cohorts have some common but also some distinct strains from one another, with the most heterogeneous populations of Aβ emerging in subjects with high levels of AD pathology. The emergence of distinct strains in DS at these later stages of disease suggests that the confluence of aging, pathology, and other DS-linked factors may favor conditions that generate strains that are unique from sAD.

Protein conformations are shaped by cellular environments, but how environmental changes alter the conformational landscapes of specific proteins in vivo remains largely uncharacterized, in part due to the challenge of probing protein structures in living cells. Here, we use deep mutational scanning to investigate how a toxic conformation of α-synuclein, a dynamic protein linked to Parkinson’s disease, responds to perturbations of cellular proteostasis. In the context of a course for graduate students in the UCSF Integrative Program in Quantitative Biology, we screened a comprehensive library of α-synuclein missense mutants in yeast cells treated with a variety of small molecules that perturb different cellular processes linked to α-synuclein biology and pathobiology. We found that the conformation of α-synuclein previously shown to drive yeast toxicity—an extended, membrane-bound helix—is largely unaffected by these chemical perturbations, underscoring the importance of this conformational state as a driver of cellular toxicity. On the other hand, the chemical perturbations have a significant effect on the ability of mutants to suppress α-synuclein toxicity. Moreover, we find that sequence determinants of α-synuclein toxicity are well described by a simple structural model of the membrane-bound helix. This model predicts that α-synuclein penetrates the membrane to constant depth across its length but that membrane affinity decreases toward the C terminus, which is consistent with orthogonal biophysical measurements. Finally, we discuss how parallelized chemical genetics experiments can provide a robust framework for inquiry-based graduate coursework.

Protein conformations are shaped by cellular environments, but how environmental changes alter the conformational landscapes of specific proteins in vivo remains largely uncharacterized, in part due to the challenge of probing protein structures in living cells. Here, we use deep mutational scanning to investigate how a toxic conformation of α-synuclein, a dynamic protein linked to Parkinson's disease, responds to perturbations of cellular proteostasis. In the context of a course for graduate students in the UCSF Integrative Program in Quantitative Biology, we screened a comprehensive library of α-synuclein point mutants in yeast cells treated with a variety of small molecules that perturb cellular processes linked to α-synuclein biology and pathobiology. We found that the conformation of α-synuclein previously shown to drive yeast toxicity - an extended, membrane-bound helix - is largely unaffected by these chemical perturbations, underscoring the importance of this conformational state as a driver of cellular toxicity. On the other hand, the chemical perturbations have a significant effect on the ability of mutations to suppress α-synuclein toxicity. Moreover, we find that sequence determinants of α-synuclein toxicity are well described by a simple structural model of the membrane-bound helix. This model predicts that α-synuclein penetrates the membrane to constant depth across its length but that membrane affinity decreases toward the C terminus, which is consistent with orthogonal biophysical measurements. Finally, we discuss how parallelized chemical genetics experiments can provide a robust framework for inquiry-based graduate coursework.

Although the primary protein sequence of ubiquitin (Ub) is extremely stable over evolutionary time, it is highly tolerant to mutation during selection experiments performed in the laboratory. We have proposed that this discrepancy results from the difference between fitness under laboratory culture conditions and the selective pressures in changing environments over evolutionary timescales. Building on our previous work (Mavor et al., 2016), we used deep mutational scanning to determine how twelve new chemicals (3-Amino-1,2,4-triazole, 5-fluorocytosine, Amphotericin B, CaCl2, Cerulenin, Cobalt Acetate, Menadione, Nickel Chloride, p-Fluorophenylalanine, Rapamycin, Tamoxifen, and Tunicamycin) reveal novel mutational sensitivities of ubiquitin residues. Collectively, our experiments have identified eight new sensitizing conditions for Lys63 and uncovered a sensitizing condition for every position in Ub except Ser57 and Gln62. By determining the ubiquitin fitness landscape under different chemical constraints, our work helps to resolve the inconsistencies between deep mutational scanning experiments and sequence conservation over evolutionary timescales.

Significance An expanding body of evidence argues that the Aβ and tau proteins share important characteristics of prion propagation to cause pathogenesis in Alzheimer’s disease (AD). Aβ and tau form a number of amyloids (β-sheet–rich structures) with distinct conformations (“strains”), some of which give rise to different diseases and associated pathologies. We develop new probes of amyloid structure and use these to identify conformational strains of Aβ in heritable and sporadic forms of AD patient samples. We demonstrate that distinct strains of Aβ can be discerned in different disease types, or in different brain compartments within a given patient. Our findings may potentially explain the spectrum of clinical and pathologic features observed in AD.

Protein catalysis requires the atomic-level orchestration of side chains, substrates and cofactors, and yet the ability to design a small-molecule-binding protein entirely from first principles with a precisely predetermined structure has not been demonstrated. Here we report the design of a novel protein, PS1, that binds a highly electron-deficient non-natural porphyrin at temperatures up to 100 °C. The high-resolution structure of holo-PS1 is in sub-Å agreement with the design. The structure of apo-PS1 retains the remote core packing of the holoprotein, with a flexible binding region that is predisposed to ligand binding with the desired geometry. Our results illustrate the unification of core packing and binding-site definition as a central principle of ligand-binding protein design.

Although the primary protein sequence of ubiquitin (Ub) is extremely stable over evolutionary time, it is highly tolerant to mutation during selection experiments performed in the laboratory. We have proposed that this discrepancy results from the difference between fitness under laboratory culture conditions and the selective pressures in changing environments over evolutionary time scales. Building on our previous work (Mavor et al 2016), we used deep mutational scanning to determine how twelve new chemicals (3-Amino-1,2,4-triazole, 5-fluorocytosine, Amphotericin B, CaCl 2 , Cerulenin, Cobalt Acetate, Menadione, Nickel Chloride, p-fluorophenylalanine, Rapamycin, Tamoxifen, and Tunicamycin) reveal novel mutational sensitivities of ubiquitin residues. We found sensitization of Lys63 in eight new conditions. In total, our experiments have uncovered a sensitizing condition for every position in Ub except Ser57 and Gln62. By determining the Ubiquitin fitness landscape under different chemical constraints, our work helps to resolve the inconsistencies between deep mutational scanning experiments and sequence conservation over evolutionary timescales. Builds on Mavor D, Barlow KA, Thompson S, Barad BA, Bonny AR, Cario CL, Gaskins G, Liu Z, Deming L, Axen SD, Caceres E, Chen W, Cuesta A, Gate R, Green EM, Hulce KR, Ji W, Kenner LR, Mensa B, Morinishi LS, Moss SM, Mravic M, Muir RK, Niekamp S, Nnadi CI, Palovcak E, Poss EM, Ross TD, Salcedo E, See S, Subramaniam M, Wong AW, Li J, Thorn KS, Conchúir SÓ, Roscoe BP, Chow ED, DeRisi JL, Kortemme T, Bolon DN, Fraser JS. Determination of Ubiquitin Fitness Landscapes Under Different Chemical Stresses in a Classroom Setting. eLife . 2016. Impact Statement We organized a project-based course that used deep mutational scanning in multiple chemical conditions to resolve the inconsistencies between tolerance to mutations in laboratory conditions and sequence conservation over evolutionary timescales.