Jessica L. Goodman

Notch and Kras reprogram pancreatic acinar cells to ductal intraepithelial neoplasia

Efforts to model pancreatic cancer in mice have focused on mimicking genetic changes found in the human disease, particularly the activating KRAS mutations that occur in pancreatic tumors and their putative precursors, pancreatic intraepithelial neoplasia (PanIN). Although activated mouse Kras mutations induce PanIN lesions similar to those of human, only a small minority of cells that express mutant Kras go on to form PanINs. The basis for this selective response is unknown, and it is similarly unknown what cell types in the mature pancreas actually contribute to PanINs. One clue comes from the fact that PanINs, unlike most cells in the adult pancreas, exhibit active Notch signaling. We hypothesize that Notch, which inhibits differentiation in the embryonic pancreas, contributes to PanIN formation by abrogating the normal differentiation program of tumor-initiating cells. Through conditional expression in the mouse pancreas, we find dramatic synergy between activated Notch and Kras in inducing PanIN formation. Furthermore, we find that Kras activation in mature acinar cells induces PanIN lesions identical to those seen upon ubiquitous Kras activation, and that Notch promotes both initiation and dysplastic progression of these acinar-derived PanINs, albeit short of invasive adenocarcinoma. At the cellular level, Notch/Kras coactivation promotes rapid reprogramming of acinar cells to a duct-like phenotype, providing an explanation for how a characteristically ductal tumor can arise from nonductal acinar cells.

Functional Links Between Aβ Toxicity, Endocytic Trafficking, and Alzheimer’s Disease Risk Factors in Yeast

The use of yeast as a model organism reveals cellular factors involved in beta-amyloid toxicity. Aβ (beta-amyloid peptide) is an important contributor to Alzheimer’s disease (AD). We modeled Aβ toxicity in yeast by directing the peptide to the secretory pathway. A genome-wide screen for toxicity modifiers identified the yeast homolog of phosphatidylinositol binding clathrin assembly protein (PICALM) and other endocytic factors connected to AD whose relationship to Aβ was previously unknown. The factors identified in yeast modified Aβ toxicity in glutamatergic neurons of Caenorhabditis elegans and in primary rat cortical neurons. In yeast, Aβ impaired the endocytic trafficking of a plasma membrane receptor, which was ameliorated by endocytic pathway factors identified in the yeast screen. Thus, links between Aβ, endocytosis, and human AD risk factors can be ascertained with yeast as a model system.

The cellular prion protein mediates neurotoxic signalling of β‐sheet‐rich conformers independent of prion replication

Formation of aberrant protein conformers is a common pathological denominator of different neurodegenerative disorders, such as Alzheimers disease or prion diseases. Moreover, increasing evidence indicates that soluble oligomers are associated with early pathological alterations and that oligomeric assemblies of different disease‐associated proteins may share common structural features. Previous studies revealed that toxic effects of the scrapie prion protein (PrPSc), a β‐sheet‐rich isoform of the cellular PrP (PrPC), are dependent on neuronal expression of PrPC. In this study, we demonstrate that PrPC has a more general effect in mediating neurotoxic signalling by sensitizing cells to toxic effects of various β‐sheet‐rich (β) conformers of completely different origins, formed by (i) heterologous PrP, (ii) amyloid β‐peptide, (iii) yeast prion proteins or (iv) designed β‐peptides. Toxic signalling via PrPC requires the intrinsically disordered N‐terminal domain (N‐PrP) and the GPI anchor of PrP. We found that the N‐terminal domain is important for mediating the interaction of PrPC with β‐conformers. Interestingly, a secreted version of N‐PrP associated with β‐conformers and antagonized their toxic signalling via PrPC. Moreover, PrPC‐mediated toxic signalling could be blocked by an NMDA receptor antagonist or an oligomer‐specific antibody. Our study indicates that PrPC can mediate toxic signalling of various β‐sheet‐rich conformers independent of infectious prion propagation, suggesting a pathophysiological role of the prion protein beyond of prion diseases.

Structures of phi29 DNA polymerase complexed with substrate: the mechanism of translocation in B-family polymerases

Replicative DNA polymerases (DNAPs) move along template DNA in a processive manner. The structural basis of the mechanism of translocation has been better studied in the A‐family of polymerases than in the B‐family of replicative polymerases. To address this issue, we have determined the X‐ray crystal structures of phi29 DNAP, a member of the protein‐primed subgroup of the B‐family of polymerases, complexed with primer‐template DNA in the presence or absence of the incoming nucleoside triphosphate, the pre‐ and post‐translocated states, respectively. Comparison of these structures reveals a mechanism of translocation that appears to be facilitated by the coordinated movement of two conserved tyrosine residues into the insertion site. This differs from the mechanism employed by the A‐family polymerases, in which a conserved tyrosine moves into the templating and insertion sites during the translocation step. Polymerases from the two families also interact with downstream single‐stranded template DNA in very different ways.

Conserved features of intermediates in amyloid assembly determine their benign or toxic states

Some amyloid-forming polypeptides are associated with devastating human diseases and others provide important biological functions. For both, oligomeric intermediates appear during amyloid assembly. Currently we have few tools for characterizing these conformationally labile intermediates and discerning what governs their benign versus toxic states. Here, we examine intermediates in the assembly of a normal, functional amyloid, the prion-determining region of yeast Sup35 (NM). During assembly, NM formed a variety of oligomers with different sizes and conformation-specific antibody reactivities. Earlier oligomers were less compact and reacted with the conformational antibody A11. More mature oligomers were more compact and reacted with conformational antibody OC. We found we could arrest NM in either of these two distinct oligomeric states with small molecules or crosslinking. The A11-reactive oligomers were more hydrophobic (as measured by Nile Red binding) and were highly toxic to neuronal cells, while OC-reactive oligomers were less hydrophobic and were not toxic. The A11 and OC antibodies were originally raised against oligomers of Aβ, an amyloidogenic peptide implicated in Alzheimer’s disease (AD) that is completely unrelated to NM in sequence. Thus, this natural yeast prion samples two conformational states similar to those sampled by Aβ, and when assembly stalls at one of these two states, but not the other, it becomes extremely toxic. Our results have implications for selective pressures operating on the evolution of amyloid folds across a billion years of evolution. Understanding the features that govern such conformational transitions will shed light on human disease and evolution alike.

β-Peptide Bundles with Fluorous Cores

We reported recently that certain beta-peptides self-assemble spontaneously into cooperatively folded bundles whose kinetic and thermodynamic metrics mirror those of natural helix bundle proteins. The structures of four such beta-peptide bundles are known in atomic detail. These structures reveal a solvent-sequestered, hydrophobic core stabilized by a unique arrangement of leucine side chains and backbone methylene groups. Here we report that this hydrophobic core can be re-engineered to contain a fluorous subdomain while maintaining the characteristic beta-peptide bundle fold. Like alpha-helical bundles possessing fluorous cores, fluorous beta-peptide bundles are stabilized relative to hydrocarbon analogues and undergo cold denaturation. Beta-peptide bundles with fluorous cores represent the essential first step in the synthesis of orthogonal protein assemblies that can sequester selectively in an interstitial membrane environment.

Bipartite Tetracysteine Display Requires Site Flexibility for ReAsH Coordination

Flexibility required: We designed intramolecular bipartite tetracysteine sites in loops of p53 and the β‐sheets of EmGFP. We found that ReAsH binding preferentially favors tetracysteine sites with flexible geometries such as loops; flexibility was assessed by comparing Cα B‐factor values. This information is important for directing successful bipartite tetracysteine site designs.

Tetrameric β3‐Peptide Bundles

There is considerable current interest in the design of non-proteinaceous quaternary structures with defined oligomeric states, as these materials have potential as nanomaterial scaffolds, drug delivery tools and enzymatic platforms.[1, 2] We recently described a series of β3-peptides, exemplified by the sequences of Zwit-1F and Acid-1Y (Figure 1A), that assemble into octameric β-peptide bundles of known structure and high stability.[3–6] The 314 helices comprising these octamers form three distinct faces: a leucine face whose side chains form the bundle core, a salt bridge face of alternating β3-ornithine and β3-aspartate resides, and an aromatic face containing two β3-tyrosine or β3-phenylalanine residues.[7–9] Figure 1 (A) Helical net representation of Zwit-1F, Acid-1Y, Zwit-VY and Acid-VY. (B, C) Zwit-VY and Acid-VY self-association monitored by analytical ultracentrifugation and fit to monomer-n-mer equilibria. Samples were prepared in 10 mM NaH2PO4, 200 mM NaCl (pH ... It is well established that natural coiled coil bundle stoichiometries are controlled by the identity and conformation of side chains buried at the bundle interface.[10] For example, substitution of valine or isoleucine for leucine at the GCN4 dimer interface leads to trimeric and tetrameric bundles.[10] Here we show that β3-peptide bundle stoichiometry is also controlled by side chain identity within the bundle core. Specifically, substitution of valine for leucine at the octamer interfaces of Zwit-1F and Acid-1Y generates valine derivatives, Zwit-VY and Acid-VY. These second generation β-peptides assemble into discrete and stable tetrameric bundles that were characterized by analytical ultracentrifugation (AU), circular dichorism (CD), 1-anilino-8-naphthalenesulfonate (ANS) binding, and deuterium exchange NMR.[4, 6, 11] First we used analytical ultracentrifugation (AU) to determine whether Zwit-VY and Acid-VY formed bundles possessing a discrete stoichiometry in solution (Figure 1). The Zwit-VY AU data fit best to a monomer-n-mer equilibrium where n = 4.07 with an RMSD of 0.00670 (Figure 1B). A comparable fit (RMSD = 0.00672) resulted when n was set to equal 4 (Figure S2A). The ln Ka value calculated from these two fits are 37.70 ± 0.07 and 38.2 ± 0.8, respectively. Significantly poorer fits (RMSD > 0.008) resulted when n was set to equal 5 or 6.[12] The AU data collected for Acid-VY fit optimally to a tetramer where n = 3.94 with an RMSD value of 0.00861 (Figure 1C). The Acid-VY data was fit to a variety of other oligomeric assemblies, all of which afforded poorer fits to the AU experimental data and corresponding increased RMSD values (Figure S2).[12] We next used wavelength-dependent circular dichroism (CD) spectroscopy to characterize concentration and temperature-dependent changes in the secondary structures of Zwit-VY and Acid-VY (Figures 2 and S3). Zwit-VY underwent a concentration-dependent increase in 314-helical structure (as judged by the molar residue ellipticity at 209 nm, MRE209[13]) between 12 and 100 µM (Figure 2A), consistent with a concentration-dependent equilibrium between a partially structured monomer and a folded oligomer. This behavior mimics the behavior of previously characterized β-peptide bundles Zwit-1F, Zwit-1F*, and Acid-1Y.[5, 6] A plot of MRE209 as a function of Zwit-VY concentration fit well to a monomer-tetramer equilibrium with ln Ka = 38.3 ± 0.5 (Figure 2A), in agreement with the value calculated from the AU data (38.2 ± 0.8). Acid-VY also displayed a concentration-dependent increase in 314-helical structure (as judged by the MRE minimum at 209), fitting best to a monomer-tetramer equilibrium with a ln Ka of 39.3 ± 0.5 (Figure S3).[12] In addition, the temperature dependent CD spectra of both Zwit-VY and Acid-VY show concentration-dependent increases in Tm that imply self-association (Figures 2B and S4C).[14] The Tm (defined as the maximum in a plot of δMRE209•δT−1) of a 50 µM Zwit-VY solution (88% folded) is 85 °C, and for a 80 µM (88% folded) solution of Acid-VY is also 85 °C. Unexpectedly, these values are higher than the Tm values of 70 °C and 78 °C previously observed for Zwit-1F (100 µM, 65% folded) and Acid-1Y (100 µM, 92% folded), respectively.[5] In summary, analytical ultracentrifugation measurements, as well as wavelength and temperature-dependent CD experiments indicate that β-peptides Zwit-VY and Acid-VY assemble into a 314-helical tetramer. Figure 2 Zwit-VY self-association monitored by circular dichroism spectroscopy (CD). (A) Plot of MRE209 as a function of [Zwit-VY]monomer fit to a monomer-tetramer equilibrium. (B) Plot of δMRE209•δT−1 for the concentrations of ... We used 1-anilino-8-naphthalenesulfonate (ANS) to further probe features of the tetrameric Zwit-VY and Acid-VY hydrophobic cores. The fluorescence of ANS increases upon binding to hydrophobic surfaces.[15] A significant increase in ANS fluorescence (between 60 and 100 fold) upon addition of protein indicates a loosely folded, exposed hydrophobic core as shown for the α-lactalbumin molten globule.[16] Most well-folded or unfolded proteins do not provide favorable ANS binding sites and minimal fluorescence changes are observed in these cases.[16] The relative fluorescence of 10 µM ANS increased from a value of 1.1 at 12.5 µM Zwit-VY (79% tetramer) to a value of 1.6 at 350 µM Zwit-VY (98% tetramer, Figure S5). The increase in relative ANS fluorescence upon addition of Acid-VY ranged from 1.1 at 25 µM (91% tetramer) to 1.2 at 300 µM (98% tetramer). Taken with the CD data, the minimal concentration-dependent increase in ANS fluorescence upon addition of Zwit-VY and Acid-VY suggests that both tetramers possess minimally exposed hydrophobic cores. Finally, we used hydrogen/deuterium exchange NMR to characterize the kinetic stability of the Zwit-VY and Acid-VY tetramers.[17] The 1H NMR spectra of both samples show dispersion of the amide N-H resonances (0.8 ppm) that is higher than that observed for the β-peptide monomer Acid-1YL2A,L,11A (Figures 3 and S6)[5] although smaller than that observed for Zwit-1F (1.4 ppm) and Acid-1Y (1.5 ppm). As was observed for the isosteric octameric bundles Zwit-1F and Acid-1Y,[4, 6] the pattern of the amide N-H resonances of Zwit-VY and Acid-VY are nearly identical, suggesting that the quaternary structures of the two bundles are similar.[5, 6] Figure 3 Kinetic stability of the Zwit-VY tetramer as determined by hydrogen/deuterium exchange analysis. (A) 500 MHz 1H NMR spectra of 0.6 mM Zwit-VY acquired at the indicated times after a lyophilized Zwit-VY sample was reconstituted in phosphate-buffered D ... The amide (N-H) exchange rate constants (kex) derived from the deuterium exchange NMR experiment correlate with the availability of amide protons to exchange with bulk solvent. Slow exchange rate constants are found for amide protons that are protected from exchange due to participation in stabilizing hydrogen bond interactions. The rate of disappearance of peaks a – d in a 600 µM sample of Zwit-VY (Figure 3A) fit a first order kinetic model with rate constants (kex) between 1.6 × 10−3 and 5.5 × 10−4 s−1 (Figure 3B). Comparison of these values to the rate constant for exchange of an amide N-H in poly-β-homoglycine (krc) allows the calculation of a protection factor (P = krc/kex)[18] that facilitates comparisons between protein and β-peptide quaternary systems.[5, 18, 19] The protection factors calculated for a 600 µM (98% folded) sample of Zwit-VY fall between 4 × 103 and 1.8 × 104.[12] These values are comparable to the range calculated for a 1.5 mM (97% folded) solution of Zwit-1F of 3.4 × 103 and 2 × 104.[5, 6] A similar trend in protection factors was also observed for Acid-VY.[12] The minimal difference in protection factors of Zwit-VY and Zwit-1F provides additional evidence that the valine β-peptide derivatives assemble into a discrete tetrameric structure possessing kinetically stable hydrophobic cores.[5, 17, 20, 21] In summary, here we show that β-peptide bundle stoichiometry can be controlled by the identity of side chains buried at the subunit interface in a manner analogous to that observed in natural coiled coil proteins.[10] These results provide a second, critical step in the “bottom-up” assembly of β-peptide assemblies possessing defined sizes, reproducible structures, and sophisticated function.[22]

Enhancing β3‐Peptide Bundle Stability by Design

We reported recently that certain β3‐peptides self‐assemble in aqueous solution into discrete bundles of unique structure and defined stoichiometry. The first β‐peptide bundle reported was the octameric Zwit‐1F, whose fold is characterized by a well‐packed, leucine‐rich core and a salt‐bridge‐rich surface. Close inspection of the Zwit‐1F structure revealed four nonideal interhelical salt‐bridge interactions whose heavy atom–heavy atom distances were longer than found in natural proteins of known structure. Here we demonstrate that the thermodynamic stability of a β‐peptide bundle can be enhanced by optimizing the length of these four interhelical salt bridges. Combined with previous work on the role of internal packing residues, these results provide another critical step in the “bottom‐up” formation of β‐peptide assemblies with defined sizes, reproducible structures, and sophisticated function.

Remodeling a β-peptide bundle

Natural biopolymers fold with fidelity, burying diverse side chains into well-packed cores and protecting their backbones from solvent. Certain β-peptide oligomers assemble into bundles of defined octameric stoichiometry that resemble natural proteins in many respects. These β-peptide bundles are thermostable, fold cooperatively, exchange interior amide N–H protons slowly, exclude hydrophobic dyes, and can be characterized at high resolution using X-ray crystallography – just like many proteins found in nature. But unlike natural proteins, all octameric β-peptide bundles contain a sequence-uniform hydrophobic core composed of 32 leucine side chains. Here we apply rational design principles, including the Rosetta computational design methodology, to introduce sequence diversity into the bundle core while retaining the characteristic β-peptide bundle fold. Using circular dichroism spectroscopy and analytical ultracentrifugation, we confirmed the prediction that an octameric bundle still assembles upon a major remodelling of its core: the mutation of sixteen core β-homo-leucine side chains into sixteen β-homo-phenylalanine side chains. Nevertheless, the bundle containing a partially β-homo-phenylalanine core poorly protects interior amide protons from exchange, suggesting molten-globule-like properties. We further improve stability by the incorporation of eight β-homo-pentafluorophenyalanine side chains, giving an assembly with amide protection factors comparable to prior well-structured bundles. By demonstrating that their cores tolerate significant sequence variation, the β-peptide bundles reported here represent a starting point for the “bottom-up” construction of β-peptide assemblies possessing both structure and sophisticated function.