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Background: The molecular heterogeneity of Tau is a critical contributor to Alzheimer's disease (AD) progression and pathology. Currently, very little is known about the prevalence or impact of the diverse collection of Tau proteoforms. Additionally, individual proteoforms may have different interactions or frequencies within the broader proteome. Here, we detected and analyzed Tau proteoforms in the context of the broader proteome in model systems using a novel single‐molecule proteomic analysis platform with future applicability in AD samples. Method: We used a single‐molecule proteomic analysis platform that leverages a set of target isoform specific and target PTM specific affinity reagents combined with novel instrumentation, single‐molecule biochemistry, and machine learning bioinformatics to enable deep proteoform and broad proteome analysis. In this study, we first evaluated the proteoform distribution of Tau using commercially available antibody reagents. Proteoform heterogeneity was compared (specifically splicing and phosphorylation variants) in both healthy and diseased samples. Then, total protein counts were assessed across the proteome using Protein Identification by Short‐epitope Mapping (PrISM), which leverages proprietary multi‐affinity probes designed to recognize short epitopes and a machine learning algorithm that decodes binding of hundreds of multi‐affinity probes into protein quantifications. Result: The approach was evaluated by first measuring defined mixtures of recombinant Tau proteins. Next, we examined Tau enriched from induced pluripotent stem cell (iPSC)‐derived neurons and tau‐expressing cell lines. The platform revealed the molecular heterogeneity of Tau proteoforms missed by bulk measurements and peptide‐centric proteomics approaches. The proteoform data was then supplemented with broader proteome analysis in order to identify proteome‐wide changes – differences in proteins and pathways due to or in combination with specific Tau proteoforms and disease. Conclusion: Understanding the biology of complex diseases like AD and other Tauopathies depends on understanding both the specific Tau proteoforms that exist at baseline and in disease as well as the pathways and processes impacted in the proteome. Single‐molecule tools that can analyze proteoforms in depth and the proteome broadly and link molecular signatures to disease states are essential. These tools will enable improved biomarkers, improved understanding of disease, and improved therapies for Alzheimer's disease in the future. [ABSTRACT FROM AUTHOR]

Bor1p is a secondary transporter in yeast that is responsible for boron transport. Bor1p belongs to the SLC4 family which controls bicarbonate exchange and pH regulation in animals as well as borate uptake in plants. The SLC4 family is more distantly related to members of the Amino acid-Polyamine-organoCation (APC) superfamily, which includes well studied transporters such as LeuT, Mhp1, AdiC, vSGLT, UraA, SLC26Dg. Their mechanism generally involves relative movements of two domains: a core domain that binds substrate and a gate domain that in many cases mediates dimerization. To shed light on conformational changes governing transport by the SLC4 family, we grew helical membrane crystals of Bor1p from Saccharomyces mikatae and determined a structure at ∼6 Å resolution using cryo-electron microscopy. To evaluate the conformation of Bor1p in these crystals, a homology model was built based on the related anion exchanger from red blood cells (AE1). This homology model was fitted to the cryo-EM density map using the Molecular Dynamics (MD) Flexible Fitting method and then relaxed by all-atom MD simulation in explicit solvent and membrane. Mapping of water accessibility indicates that the resulting structure represents an inward-facing conformation. Comparisons of the resulting Bor1p model with the X-ray structure of AE1 in an outward-facing conformation, together with MD simulations of inward-facing and outward-facing Bor1p models, suggest rigid body movements of the core domain relative to the gate domain. These movements are consistent with the rocking-bundle transport mechanism described for other members of the APC superfamily. [ABSTRACT FROM AUTHOR]