Specific Combinations of Physiological Tau Phosphorylation Regulate Tau-Microtubule Interactions in Developing Neurons.

Tau phosphorylation is a defining feature of Alzheimer's disease, yet it also plays an essential physiological role in stabilizing microtubules (MTs) during normal neuronal development. While individual phosphorylation sites have been well-studied in pathology, it remains largely unknown how combinatorial phosphorylation is regulated under physiological conditions. Here, we uncover distinct, site-specific phosphorylation patterns on tau in developing human neurons.

Genetic Code Expansion and Enzymatic Modifications as Accessible Methods for Studying Site-Specific Post-Translational Modifications of Alpha-Synuclein and Tau.

Alpha-synuclein (αS) and tau play important roles in the pathology of Parkinson's Disease and Alzheimer's Disease, respectively, as well as numerous other neurodegenerative diseases. Both proteins are classified as intrinsically disordered proteins (IDPs), as they have no stable structure that underlies their function in healthy tissue, and both proteins are prone to aggregation in disease states. There is substantial interest in understanding the roles that post-translational modifications (PTMs) play in regulating the structural dynamics and function of αS and tau monomers, as well as their propensity to aggregate.

Investigation of All Disease-Relevant Lysine Acetylation Sites in α-Synuclein Enabled by Non-canonical Amino Acid Mutagenesis.

Aggregates of α-synuclein (αS) are hallmarks of synucleinopathies, including Parkinson's Disease (PD) and Multiple System Atrophy (MSA). We have recently shown that αS lysine acetylation in the soluble monomer pool varies between healthy controls, PD, and MSA patients. To study the effects of lysine acetylation at all disease-relevant sites of αS, we first compared production of acetylated αS through either native chemical ligation or non-canonical amino acid (ncAA) mutagenesis.

Structural insights into the role of the proline rich region in tau function.

Tau plays an important role in modulating axonal microtubules in neurons, while intracellular tau aggregates are found in many neurodegenerative disorders. Tubulin binding sites are found in tau's proline-rich region (PRR), microtubule binding repeats (MTBRs), and pseudo-repeat (R'). Tau phosphorylation sites, which cluster with high frequency within the PRR, regulate tubulin interactions and correlates with disease.

Structural Insights into the Role of the Proline Rich Region in Tau Function.

Tau is a microtubule-associated protein that plays an important role in modulating axonal microtubules in neurons. Intracellular tau aggregates are found in a broad class of disorders, including Alzheimer's disease, termed tauopathies. Tau is an intrinsically disordered protein, and its structural disorder appears to be critical to its microtubule-related functions.

Multivalency drives interactions of alpha-synuclein fibrils with tau.

Age-related neurodegenerative disorders like Alzheimer's disease (AD) and Parkinson's disease (PD) are characterized by deposits of protein aggregates, or amyloid, in various regions of the brain. Historically, aggregation of a single protein was observed to be correlated with these different pathologies: tau in AD and α-synuclein (αS) in PD. However, there is increasing evidence that the pathologies of these two diseases overlap, and the individual proteins may even promote each other's aggregation.

Sequential CRISPR screening reveals partial NatB inhibition as a strategy to mitigate alpha-synuclein levels in human neurons.

Alpha-synuclein (αSyn) protein levels correlate with the risk and severity of Parkinson's disease and related neurodegenerative diseases. Lowering αSyn is being actively investigated as a therapeutic modality. Here, we systematically map the regulatory network that controls endogenous αSyn using sequential CRISPR-knockout and -interference screens in an αSyn gene ()-tagged cell line and induced pluripotent stem cell-derived neurons (iNeurons).

The Effects of Lipids on α-Synuclein Aggregation In Vitro.

The small neuronal protein α-synuclein (αS) is found in pre-synaptic terminals and plays a role in vesicle recycling and neurotransmission. Fibrillar aggregates of αS are the hallmark of Parkinson's disease and related neurodegenerative disorders. In both health and disease, interactions with lipids influence αS's structure and function, prompting much study of the effects of lipids on αS aggregation.

The C-terminus of α-Synuclein Regulates its Dynamic Cellular Internalization by Neurexin 1β.

The aggregation of the disordered neuronal protein, α-Synuclein (αS), is the primary pathological feature of Parkinson's disease. Current hypotheses favor cell-to-cell spread of αS species as underlying disease progression, driving interest in identifying the molecular species and cellular processes involved in cellular internalization of αS. Prior work from our lab identified the chemically specific interaction between αS and the presynaptic adhesion protein neurexin-1β (N1β) to be capable of driving cellular internalization of both monomer and aggregated forms of αS.

Combining non-canonical amino acid mutagenesis and native chemical ligation for multiply modifying proteins: A case study of α-synuclein post-translational modifications.

The Parkinson's disease associated protein α-synuclein (αS) has been found to contain numerous post-translational modifications (PTMs), in both physiological and pathological states. One PTM site of particular interest is serine 87, which is subject to both O-linked β-N-acetylglucosamine (gS) modification and phosphorylation (pS), with αS-pS enriched in Parkinson's disease. An often-overlooked aspect of these PTMs is their effect on the membrane-binding properties of αS, which are important to its role in regulating neurotransmitter release.

Semi-Synthetic CoA-α-Synuclein Constructs Trap N-Terminal Acetyltransferase NatB for Binding Mechanism Studies.

N-terminal acetylation is a chemical modification carried out by N-terminal acetyltransferases. A major member of this enzyme family, NatB, acts on much of the human proteome, including α-synuclein (αS), a synaptic protein that mediates vesicle trafficking. NatB acetylation of αS modulates its lipid vesicle binding properties and amyloid fibril formation, which underlies its role in the pathogenesis of Parkinson's disease.

Semi-synthetic CoA-α-Synuclein Constructs Trap N-terminal Acetyltransferase NatB for Binding Mechanism Studies.

N-terminal acetylation is a chemical modification carried out by N-terminal acetyltransferases (NATs). A major member of this enzyme family, NatB, acts on much of the human proteome, including α-synuclein (αS), a synaptic protein that mediates vesicle trafficking. NatB acetylation of αS modulates its lipid vesicle binding properties and amyloid fibril formation, which underlies its role in the pathogenesis of Parkinson's disease.

The N-terminal disease-associated R5L Tau mutation increases microtubule shrinkage rate due to disruption of microtubule-bound Tau patches.

Regulation of the neuronal microtubule cytoskeleton is achieved through the coordination of microtubule-associated proteins (MAPs). MAP-Tau, the most abundant MAP in the axon, functions to modulate motor motility, participate in signaling cascades, as well as directly mediate microtubule dynamics. Tau misregulation is associated with a class of neurodegenerative diseases, known as tauopathies, including progressive supranuclear palsy, Pick's disease, and Alzheimer's disease.

Cysteine-Based Mimic of Arginylation Reproduces Neuroprotective Effects of the Authentic Post-Translational Modification on α-Synuclein.

Arginylation is an understudied post-translational modification (PTM) involving the transfer of arginine to aspartate or glutamate sidechains in a protein. Among the targets of this PTM is α-synuclein (αS), a neuronal protein involved in regulating synaptic vesicles. The aggregation of αS is implicated in neurodegenerative diseases, particularly in Parkinson's disease, and arginylation has been found to protect against this pathological process.

α-Synuclein arginylation in the human brain.

Background: Alpha-synuclein (α-syn) exhibits pathological misfolding in many human neurodegenerative disorders. We previously showed that α-syn is arginylated in the mouse brain and that lack of arginylation leads to neurodegeneration in mice.

Methods: Here, we tested α-syn arginylation in human brain pathology using newly derived antibodies in combination with Western blotting, biochemical assays, and experiments in live neurons.

Potent inhibitors of toxic alpha-synuclein identified via cellular time-resolved FRET biosensors.

We have developed a high-throughput drug discovery platform, measuring fluorescence resonance energy transfer (FRET) with fluorescent alpha-synuclein (αSN) biosensors, to detect spontaneous pre-fibrillar oligomers in living cells. Our two αSN FRET biosensors provide complementary insight into αSN oligomerization and conformation in order to improve the success of drug discovery campaigns for the treatment of Parkinson's disease. We measure FRET by fluorescence lifetime, rather than traditional fluorescence intensity, providing a structural readout with greater resolution and precision.

Effects of Glutamate Arginylation on α-Synuclein: Studying an Unusual Post-Translational Modification through Semisynthesis.

A variety of post-translational modifications (PTMs) are believed to regulate the behavior and function of α-synuclein (αS), an intrinsically disordered protein that mediates synaptic vesicle trafficking. Fibrils of αS are implicated in neurodegenerative disorders such as Parkinson's disease. In this study, we used chemical synthesis and biophysical techniques to characterize the neuroprotective effects of glutamate arginylation, a hitherto little characterized PTM in αS.

Tau Avoids the GTP Cap at Growing Microtubule Plus-Ends.

Plus-end tracking proteins (+TIPs) associate with the growing end of microtubules and mediate important cellular functions. The majority of +TIPs are directed to the plus-end through a family of end-binding proteins (EBs), which preferentially bind the stabilizing cap of GTP-tubulin present during microtubule growth. One outstanding question is whether there may exist other microtubule-associated proteins (MAPs) that preferentially bind specific nucleotide states of tubulin.

Chemoenzymatic Semi-synthesis Enables Efficient Production of Isotopically Labeled α-Synuclein with Site-Specific Tyrosine Phosphorylation.

Post-translational modifications (PTMs) can affect the normal function and pathology of α-synuclein (αS), an amyloid-fibril-forming protein linked to Parkinson's disease. Phosphorylation of αS Tyr39 has recently been found to display a dose-dependent effect on fibril formation kinetics and to alter the morphology of the fibrils. Existing methods to access site-specifically phosphorylated αS for biochemical studies include total or semi-synthesis by native chemical ligation (NCL) as well as chemoenzymatic methods to phosphorylate peptides, followed by NCL.

Quantification of protein delivery in live cells using fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is a quantitative single-molecule method that measures the concentration and rate of diffusion of fluorophore-tagged molecules, both large and small, in vitro and within live cells, and even within discrete cellular compartments. FCS is exceptionally well-suited to directly quantify the efficiency of intracellular protein delivery-specifically, how well different "cell-penetrating" proteins and peptides guide proteinaceous materials into the cytosol and nuclei of live mammalian cells. This article provides an overview of the procedures necessary to execute robust FCS experiments and evaluate endosomal escape efficiencies: preparation of fluorophore-tagged proteins, incubation with mammalian cells and preparation of FCS samples, setup and execution of an FCS experiment, and a detailed discussion of and custom MATLAB® script for analyzing the resulting autocorrelation curves in the context of appropriate diffusion models.

Measuring Interactions Between Tau and Aggregation Inducers with Single-Molecule Förster Resonance Energy Transfer.

Tau is an intrinsically disordered protein implicated in the pathogenesis of Alzheimer's disease and other neurodegenerative disorders. Here we describe the application of single-molecule Förster resonance energy transfer (smFRET) for the characterization of the interactions between tau and polyphosphate, an intracellular polymer that accelerates tau aggregation. We describe the design of tau constructs, purification and fluorescent labeling of tau, and details of acquisition and analysis of smFRET data.

Single-Molecule FRET of Intrinsically Disordered Proteins.

Intrinsically disordered proteins (IDPs) are now widely recognized as playing critical roles in a broad range of cellular functions as well as being implicated in diverse diseases. Their lack of stable secondary structure and tertiary interactions, coupled with their sensitivity to measurement conditions, stymies many traditional structural biology approaches. Single-molecule Förster resonance energy transfer (smFRET) is now widely used to characterize the physicochemical properties of these proteins in isolation and is being increasingly applied to more complex assemblies and experimental environments.

Chemoenzymatic Semisynthesis of Phosphorylated α-Synuclein Enables Identification of a Bidirectional Effect on Fibril Formation.

Post-translational modifications (PTMs) impact the pathological aggregation of α-synuclein (αS), a hallmark of Parkinson's disease (PD). Here, we synthesize αS phosphorylated at tyrosine 39 (pY) through a novel route using enzymatic phosphorylation of a fragment followed by ligation to form the full-length protein. We can execute this synthesis in combination with unnatural amino acid mutagenesis to include two fluorescent labels for Förster resonance energy transfer (FRET) studies.