Characterization of Patterned Microbial Growth Dynamics in Aqueous Two-Phase Polymer Scaffolds.

Microbial growth confinement using liquid scaffolds based on an aqueous two-phase system (ATPS) is a promising technique to overcome the challenges in microbial-mammalian co-culture in vitro. To better understand the potential use of the ATPS in studying these complex interactions, the goal of this research was to characterize the effects of bacteria loading and biofilm maturation on the stability of a polyethylene glycol (PEG) and dextran (DEX) ATPS. Two ATPS formulations, consisting of 5% PEG/5% DEX and 10% PEG/10% DEX (w/v), were prepared. To test the containment limits of each ATPS formulation, Escherichia coli MG1655 overnight cultures were resuspended in DEX at optical densities (ODs) of 1, 0.3, 0.1, 0.03, and 0.01. Established E. coli colonies initially seeded at lower densities were contained within the DEX phase to a greater extent than E. coli colonies initially seeded at higher densities. Furthermore, the 10% PEG/10% DEX formulation demonstrated longer containment time of E. coli compared to the 5% PEG/5% DEX formulation. E. coli growth dynamics within the ATPS were found to be affected by the initial bacterial density, where colonies of lower initial seeding densities demonstrate more dynamic growth trends compared to colonies of higher initial seeding densities. However, the addition of DEX to the existing ATPS during the growth phase of the bacterial colony does not appear to disrupt the growth inertia of E. coli. We also observed that microbial growth can disrupt ATPS stability below the physical carrying capacity of the DEX droplets. In both E. coli and Streptococcus mutans UA159 colonies, the ATPS interfacial tensions are reduced, as suggested by the loss of fluorescein isothiocyanate (FITC)-DEX confinement and contact angel measurements, while the microbial colony remained well defined. In general, we observed that the stability of the ATPS microbial colony is proportional to polymer concentrations and inversely proportional to seeding density and culture time. These parameters can be combined as part of a toolset to control microbial growth in a heterotypic co-culture platform and should be considered in future work involving mammalian-microbial cell interactions.

Backbone resonance assignment of the cAMP-binding domains of the protein kinase A regulatory subunit Iα.

Protein kinase A (PKA) is the main receptor for the universal cAMP second messenger. PKA is a tetramer with two catalytic (C) and two regulatory (R) subunits, each including two tandem cAMP-binding domains, i.e. CBD-A and -B. Activation of the complex occurs with cAMP binding first to CBD-B, followed by a second molecule of cAMP binding to CBD-A, which causes the release of the active C-subunit. Unlike previous constructs for eukaryotic cAMP-binding domains (CBDs), the 29.5 kDa construct analyzed here [i.e. RIα (119-379)] spans the CBDs in full and provides insight into inter-domain communication. In this note we report the 1H, 13C, and 15 N backbone assignments of cAMP-bound RIα (119-379) CBDs (BMRB No. 50920).

Noncanonical protein kinase A activation by oligomerization of regulatory subunits as revealed by inherited Carney complex mutations.

Familial mutations of the protein kinase A (PKA) R1α regulatory subunit lead to a generalized predisposition for a wide range of tumors, from pituitary adenomas to pancreatic and liver cancers, commonly referred to as Carney complex (CNC). CNC mutations are known to cause overactivation of PKA, but the molecular mechanisms underlying such kinase overactivity are not fully understood in the context of the canonical cAMP-dependent activation of PKA. Here, we show that oligomerization-induced sequestration of R1α from the catalytic subunit of PKA (C) is a viable mechanism of PKA activation that can explain the CNC phenotype. Our investigations focus on comparative analyses at the level of structure, unfolding, aggregation, and kinase inhibition profiles of wild-type (wt) PKA R1α, the A211D and G287W CNC mutants, as well as the cognate acrodysostosis type 1 (ACRDYS1) mutations A211T and G287E. The latter exhibit a phenotype opposite to CNC with suboptimal PKA activation compared with wt. Overall, our results show that CNC mutations not only perturb the classical cAMP-dependent allosteric activation pathway of PKA, but also amplify significantly more than the cognate ACRDYS1 mutations nonclassical and previously unappreciated activation pathways, such as oligomerization-induced losses of the PKA R1α inhibitory function.

Molecular Mechanism for the (-)-Epigallocatechin Gallate-Induced Toxic to Nontoxic Remodeling of Aß Oligomers.

(-)-Epigallocatechin gallate (EGCG) effectively reduces the cytotoxicity of the Alzheimer's disease ß-amyloid peptide (Aß) by remodeling seeding-competent Aß oligomers into off-pathway seeding-incompetent Aß assemblies. However, the mechanism of EGCG-induced remodeling is not fully understood. Here we combine 15N and 1H dark-state exchange saturation transfer (DEST), relaxation, and chemical shift projection NMR analyses with fluorescence, dynamic light scattering, and electron microscopy to elucidate how EGCG remodels Aß oligomers. We show that the remodeling adheres to a Hill-Scatchard model whereby the Aß(1-40) self-association occurs cooperatively and generates Aß(1-40) oligomers with multiple independent binding sites for EGCG with a Kd ∼10-fold lower than that for the Aß(1-40) monomers. Upon binding to EGCG, the Aß(1-40) oligomers become less solvent exposed, and the ß-regions, which are involved in direct monomer-protofibril contacts in the absence of EGCG, undergo a direct-to-tethered contact shift. This switch toward less engaged monomer-protofibril contacts explains the seeding incompetency observed upon EGCG remodeling and suggests that EGCG interferes with secondary nucleation events known to generate toxic Aß assemblies. Unexpectedly, the N-terminal residues experience an opposite EGCG-induced shift from tethered to direct contacts, explaining why EGCG remodeling occurs without release of Aß(1-40) monomers. We also show that upon binding Aß(1-40) oligomers the relative positions of the EGCG B and D rings change with respect to that of ring A. These distinct structural changes occurring in both Aß(1-40) oligomers and EGCG during remodeling offer a foundation for understanding the molecular mechanism of EGCG as a neurotoxicity inhibitor. Furthermore, the results reported here illustrate the effectiveness of DEST-based NMR approaches in investigating the mechanism of low-molecular-weight amyloid inhibitors.

Atomic-resolution map of the interactions between an amyloid inhibitor protein and amyloid ß (Aß) peptides in the monomer and protofibril states.

Self-association of amyloid ß (Aß) peptides is a hallmark of Alzheimer's disease and serves as a general prototype for amyloid formation. A key endogenous inhibitor of Aß self-association is human serum albumin (HSA), which binds ∼90% of plasma Aß. However, the exact molecular mechanism by which HSA binds Aß monomers and protofibrils is not fully understood. Here, using dark-state exchange saturation transfer NMR and relaxation experiments complemented by morphological characterization, we mapped the HSA-Aß interactions at atomic resolution by examining the effects of HSA on Aß monomers and soluble high-molecular weight oligomeric protofibrils. We found that HSA binds both monomeric and protofibrillar Aß, but the affinity of HSA for Aß monomers is lower than for Aß protofibrils (Kd values are submillimolar rather than micromolar) yet physiologically relevant because of the ∼0.6-0.7 mm plasma HSA concentration. In both Aß protofibrils and monomers, HSA targets key Aß self-recognition sites spanning the ß strands found in cross-ß protofibril structures, leading to a net switch from direct to tethered contacts between the monomeric Aß and the protofibril surface. These HSA-Aß interactions are isoform-specific, because the HSA affinity of Aß monomers is lower for Aß(1-42) than for Aß(1-40). In addition, the HSA-induced perturbations of the monomer/protofibrils pseudo-equilibrium extend to the C-terminal residues in the Aß(1-42) isoform but not in Aß(1-40). These results provide an unprecedented view of how albumin interacts with Aß and illustrate the potential of dark-state exchange saturation transfer NMR in mapping the interactions between amyloid-inhibitory proteins and amyloidogenic peptides.

Allosteric Sensing of Fatty Acid Binding by NMR: Application to Human Serum Albumin.

Human serum albumin (HSA) serves not only as a physiological oncotic pressure regulator and a ligand carrier but also as a biomarker for pathologies ranging from ischemia to diabetes. Moreover, HSA is a biopharmaceutical with a growing repertoire of putative clinical applications from hypovolemia to Alzheimer's disease. A key determinant of the physiological, diagnostic, and therapeutic functions of HSA is the amount of long chain fatty acids (LCFAs) bound to HSA. Here, we propose to utilize (13)C-oleic acid for the NMR-based assessment of albumin-bound LCFA concentration (CONFA). (13)C-Oleic acid primes HSA for a LCFA-dependent allosteric transition that modulates the frequency separation between the two main (13)C NMR peaks of HSA-bound oleic acid (ΔνAB). On the basis of ΔνAB, the overall [(12)C-LCFA]Tot/[HSA]Tot ratio is reproducibly estimated in a manner that is only minimally sensitive to glycation, albumin concentration, or redox potential, unlike other methods to quantify HSA-bound LCFAs such as the albumin-cobalt binding assay.

Mapping the interactions between the Alzheimer's Aß-peptide and human serum albumin beyond domain resolution.

Human serum albumin (HSA) is a potent inhibitor of Aß self-association and this novel, to our knowledge, function of HSA is of potential therapeutic interest for the treatment of Alzheimer's disease. It is known that HSA interacts with Aß oligomers through binding sites evenly partitioned across the three albumin domains and with comparable affinities. However, as of this writing, no information is available on the HSA-Aß interactions beyond domain resolution. Here, we map the HSA-Aß interactions at subdomain and peptide resolution. We show that each separate subdomain of HSA domain 3 inhibits Aß self-association. We also show that fatty acids (FAs) compete with Aß oligomers for binding to domain 3, but the determinant of the HSA/Aß oligomer interactions are markedly distinct from those of FAs. Although salt bridges with the FA carboxylate determine the FA binding affinities, hydrophobic contacts are pivotal for Aß oligomer recognition. Specifically, we identified a site of Aß oligomer recognition that spans the HSA (494-515) region and aligns with the central hydrophobic core of Aß. The HSA (495-515) segment includes residues affected by FA binding and this segment is prone to self-associate into ß-amyloids, suggesting that sites involved in fibrilization may provide a lead to develop inhibitors of Aß self-association.