The membrane curvature-inducing REEP1-4 proteins generate an ER-derived vesicular compartment.

The endoplasmic reticulum (ER) is shaped by abundant membrane curvature-generating proteins that include the REEP family member REEP5. The REEP1 subfamily, consisting of four proteins in mammals (REEP1-4), is less abundant and lack a N-terminal region. Mutations in REEP1 and REEP2 cause Hereditary Spastic Paraplegia, but the function of these four REEP proteins remains enigmatic.

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.

Synthesis of Peptides and Proteins with Site-Specific Glutamate Arginylation.

Solid-phase peptide synthesis and protein semi-synthesis are powerful methods for site-specific modification of peptides and proteins. We describe protocols using these techniques for the syntheses of peptides and proteins bearing glutamate arginylation (E) at specific sites. These methods overcome challenges posed by enzymatic arginylation methods and allow for a comprehensive study of the effects of E on protein folding and interactions.

Molecular role of NAA38 in thermostability and catalytic activity of the human NatC N-terminal acetyltransferase.

N-terminal acetylation occurs on over 80% of human proteins and is catalyzed by a family of N-terminal acetyltransferases (NATs). All NATs contain a small catalytic subunit, while some also contain a large auxiliary subunit that facilitates catalysis and ribosome targeting for co-translational acetylation. NatC is one of the major NATs containing an NAA30 catalytic subunit, but uniquely contains two auxiliary subunits, large NAA35 and small NAA38.

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.

Molecular mechanism of N-terminal acetylation by the ternary NatC complex.

Protein N-terminal acetylation is predominantly a ribosome-associated modification, with NatA-E serving as the major enzymes. NatC is the most unusual of these enzymes, containing one Naa30 catalytic subunit and two auxiliary subunits, Naa35 and Naa38; and substrate selectivity profile that overlaps with NatE. Here, we report the cryoelectron microscopy structure of S.

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.

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.

Molecular basis for N-terminal alpha-synuclein acetylation by human NatB.

NatB is one of three major N-terminal acetyltransferase (NAT) complexes (NatA-NatC), which co-translationally acetylate the N-termini of eukaryotic proteins. Its substrates account for about 21% of the human proteome, including well known proteins such as actin, tropomyosin, CDK2, and α-synuclein (αSyn). Human NatB (hNatB) mediated N-terminal acetylation of αSyn has been demonstrated to play key roles in the pathogenesis of Parkinson's disease and as a potential therapeutic target for hepatocellular carcinoma.

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.

Structure and Mechanism of Acetylation by the N-Terminal Dual Enzyme NatA/Naa50 Complex.

NatA co-translationally acetylates the N termini of over 40% of eukaryotic proteins and can associate with another catalytic subunit, Naa50, to form a ternary NatA/Naa50 dual enzyme complex (also called NatE). The molecular basis of association between Naa50 and NatA and the mechanism for how their association affects their catalytic activities in yeast and human are poorly understood. Here, we determined the X-ray crystal structure of yeast NatA/Naa50 as a scaffold to understand coregulation of NatA/Naa50 activity in both yeast and human.

Cyclized NDGA modifies dynamic α-synuclein monomers preventing aggregation and toxicity.

Growing evidence implicates α-synuclein aggregation as a key driver of neurodegeneration in Parkinson's disease (PD) and other neurodegenerative disorders. Herein, the molecular and structural mechanisms of inhibiting α-synuclein aggregation by novel analogs of nordihydroguaiaretic acid (NDGA), a phenolic dibenzenediol lignan, were explored using an array of biochemical and biophysical methodologies. NDGA analogs induced modest, progressive compaction of monomeric α-synuclein, preventing aggregation into amyloid-like fibrils.

Using a FRET Library with Multiple Probe Pairs To Drive Monte Carlo Simulations of α-Synuclein.

We describe a strategy for experimentally-constraining computational simulations of intrinsically disordered proteins (IDPs), using α-synuclein, an IDP with a central role in Parkinson's disease pathology, as an example. Previously, data from single-molecule Förster Resonance Energy Transfer (FRET) experiments have been effectively utilized to generate experimentally constrained computational models of IDPs. However, the fluorophores required for single-molecule FRET experiments are not amenable to the study of short-range (<30 Å) interactions.

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo.

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting.