Dynamics of the peripheral membrane protein P2 from human myelin measured by neutron scattering - a comparison between wild-type protein and a hinge mutant

Myelin protein P2 is a fatty acid-binding structural component of the myelin sheath in the peripheral nervous system, and its function is related to its membrane binding capacity. Here, the link between P2 protein dynamics and structure and function was studied using elastic incoherent neutron scattering (EINS). The P38G mutation, at the hinge between the β barrel and the α-helical lid, increased the lipid stacking capacity of human P2 in vitro, and the mutated protein was also functional in cultured cells. The P38G mutation did not change the overall structure of the protein. For a deeper insight into P2 structure-function relationships, information on protein dynamics in the 10 ps to 1 ns time scale was obtained using EINS. Values of mean square displacements mainly from protein H atoms were extracted for wild-type P2 and the P38G mutant and compared. Our results show that at physiological temperatures, the P38G mutant is more dynamic than the wild-type P2 protein, especially on a slow 1-ns time scale. Molecular dynamics simulations confirmed the enhanced dynamics of the mutant variant, especially within the portal region in the presence of bound fatty acid. The increased softness of the hinge mutant of human myelin P2 protein is likely related to an enhanced flexibility of the portal region of this fatty acid-binding protein, as well as to its interactions with the lipid bilayer surface requiring conformational adaptations

Molecular mechanisms of CharcotMarie-Tooth neuropathy linked to mutations in human myelin protein P2

This article has an erratum: Doi 10.1038/s41598-017-18751-7Charcot-Marie-Tooth (CMT) disease is one of the most common inherited neuropathies. Recently, three CMT1-associated point mutations (I43N, T51P, and I52T) were discovered in the abundant peripheral myelin protein P2. These mutations trigger abnormal myelin structure, leading to reduced nerve conduction velocity, muscle weakness, and distal limb atrophy. P2 is a myelin-specific protein expressed by Schwann cells that binds to fatty acids and membranes, contributing to peripheral myelin lipid homeostasis. We studied the molecular basis of the P2 patient mutations. None of the CMT1-associated mutations alter the overall folding of P2 in the crystal state. P2 disease variants show increased aggregation tendency and remarkably reduced stability, T51P being most severe. In addition, P2 disease mutations affect protein dynamics. Both fatty acid binding by P2 and the kinetics of its membrane interactions are affected by the mutations. Experiments and simulations suggest opening of the beta barrel in T51P, possibly representing a general mechanism in fatty acid-binding proteins. Our findings demonstrate that altered biophysical properties and functional dynamics of P2 may cause myelin defects in CMT1 patients. At the molecular level, a few malformed hydrogen bonds lead to structural instability and misregulation of conformational changes related to ligand exchange and membrane binding.Peer reviewe

A multistage antimalarial targets the plasmepsins IX and X essential for invasion and egress.

: Regulated exocytosis by secretory organelles is important for malaria parasite invasion and egress. Many parasite effector proteins, including perforins, adhesins, and proteases, are extensively proteolytically processed both pre- and postexocytosis. Here we report the multistage antiplasmodial activity of the aspartic protease inhibitor hydroxyl-ethyl-amine-based scaffold compound 49c. This scaffold inhibits the preexocytosis processing of several secreted rhoptry and microneme proteins by targeting the corresponding maturases plasmepsins IX (PMIX) and X (PMX), respectively. Conditional excision of PMIX revealed its crucial role in invasion, and recombinantly active PMIX and PMX cleave egress and invasion factors in a 49c-sensitive manner.<br/

Structure and dynamics of a human myelin protein P2 portal region mutant indicate opening of the β barrel in fatty acid binding proteins

Abstract Background Myelin is a multilayered proteolipid sheath wrapped around selected axons in the nervous system. Its constituent proteins play major roles in forming of the highly regular membrane structure. P2 is a myelin-specific protein of the fatty acid binding protein (FABP) superfamily, which is able to stack lipid bilayers together, and it is a target for mutations in the human inherited neuropathy Charcot-Marie-Tooth disease. A conserved residue that has been proposed to participate in membrane and fatty acid binding and conformational changes in FABPs is Phe57. This residue is thought to be a gatekeeper for the opening of the portal region upon ligand entry and egress. Results We performed a structural characterization of the F57A mutant of human P2. The mutant protein was crystallized in three crystal forms, all of which showed changes in the portal region and helix α2. In addition, the behaviour of the mutant protein upon lipid bilayer binding suggested more unfolding than previously observed for wild-type P2. On the other hand, membrane binding rendered F57A heat-stable, similarly to wild-type P2. Atomistic molecular dynamics simulations showed opening of the side of the discontinuous β barrel, giving important indications on the mechanism of portal region opening and ligand entry into FABPs. The results suggest a central role for Phe57 in regulating the opening of the portal region in human P2 and other FABPs, and the F57A mutation disturbs dynamic cross-correlation networks in the portal region of P2. Conclusions Overall, the F57A variant presents similar properties to the P2 patient mutations recently linked to Charcot-Marie-Tooth disease. Our results identify Phe57 as a residue regulating conformational changes that may accompany membrane surface binding and ligand exchange in P2 and other FABPs

Raptor-Mediated Proteasomal Degradation of Deamidated 4E-BP2 Regulates Postnatal Neuronal Translation and NF-κB Activity

The translation initiation repressor 4E-BP2 is deamidated in the brain on asparagines N99/N102 during early postnatal brain development. This post-translational modification enhances 4E-BP2 association with Raptor, a central component of mTORC1 and alters the kinetics of excitatory synaptic transmission. We show that 4E-BP2 deamidation is neuron specific, occurs in the human brain, and changes 4E-BP2 subcellular localization, but not its disordered structure state. We demonstrate that deamidated 4E-BP2 is ubiquitinated more and degrades faster than the unmodified protein. We find that enhanced deamidated 4E-BP2 degradation is dependent on Raptor binding, concomitant with increased association with a Raptor-CUL4B E3 ubiquitin ligase complex. Deamidated 4E-BP2 stability is promoted by inhibiting mTORC1 or glutamate receptors. We further demonstrate that deamidated 4E-BP2 regulates the translation of a distinct pool of mRNAs linked to cerebral development, mitochondria, and NF-κB activity, and thus may be crucial for postnatal brain development in neurodevelopmental disorders, such as ASD

Critical assessment of the elemental composition of Corning archeological reference glasses by LA-ICP-MS

Corning archeological reference glasses A, B, C, and D have been made to simulate different historic technologies of glass production and are used as standards in historic glass investigations. In this work, nanoseconds (193, 266 nm) and femtosecond (800 nm) laser ablation were used to study the elemental composition of Corning glasses using laser ablation inductively coupled plasma mass spectrometry. The determined concentrations of 26 oxides (Li2O, B2O3, Na2O, MgO, Al2O3, SiO2, P2O5, K2O, CaO, TiO2, V2O5, Cr2O3, MnO, Fe2O3, CoO, NiO, CuO, ZnO, Rb2O, SrO, ZrO2, SnO2, Sb2O5, BaO, PbO, Bi2O3) are compared with values reported in the literature. Results show variable discrepancies between the data, with the largest differences found for Cr2O3 in Corning A; Li2O, B2O3, and Cr2O3 in Corning B; and MnO, Sb2O5, Cr2O3, and Bi2O3 in Corning C. The best agreement between the measured and literature values was found for Corning D. However, even for this reference, glass re-evaluation of the data was necessary and new values for PbO, BaO, and Bi2O3 are proposed

Enzymatic Characterization of a Human Acyltransferase Activity

Non-histone protein acylation is increasingly recognized as an important posttranslational modification, but little is known as to the biochemical properties of protein serine acylating enzymes.We here report that we have identified a metal-stimulated serine octanoyltransferase activity in microsomes from human erythroleukemic (HEL) cells. The HEL acylating enzyme was linear with respect to time and protein, exhibited a neutral pH optimum (stimulated by cobalt and zinc), and inhibited by chelating reagents. Hydroxylamine treatment removed most, but not all, of the attached radioactivity. A salt extract of microsomal membranes contained the major portion of enzyme activity, indicating that this acyltransferase is not an integral membrane protein. Sucrose density fractionation showed that the acyltransferase activity is concentrated in the endoplasmic reticulum. In competition experiments, the acyltransferase was well inhibited by activated forms of fatty acids containing at least eight to fourteen carbons, but not by acetyl CoA. The zinc-stimulated HEL acyltransferase did not octanoylate proenkephalin, proopiomelanocortin, His-tagged proghrelin, or proghrelin lacking the amino-terminal His-tag stub of Gly-Ala-Met. The peptides des-acyl ghrelin and ACTH were also not acylated; however, des-acyl ghrelin containing the N-terminal tripeptide Gly-Ala-Met was acylated. Mutagenesis studies indicated a requirement for serine five residues from the amino terminus, reminiscent of myristoyl transferase, but not of ghrelin acylation. However, recombinant myristoyl transferase could not recapitulate the hydroxylamine sensitivity, zinc-stimulation, nor EDTA inhibition obtained with HEL acyltransferase, properties preserved in the HEL cell enzyme purified through four sequential chromatographic steps.In conclusion, our data demonstrate the presence of a zinc-stimulated acyltransferase activity concentrated in the endoplasmic reticulum in HEL cells which is likely to contribute to medium-chain protein lipidation

Neuropathy-related mutations alter the membrane binding properties of the human myelin protein P0 cytoplasmic tail

Schwann cells myelinate selected axons in the peripheral nervous system (PNS) and contribute to fast saltatory conduction via the formation of compact myelin, in which water is excluded from between tightly adhered lipid bilayers. Peripheral neuropathies, such as Charcot-Marie-Tooth disease (CMT) and Dejerine-4 Sottas syndrome (DSS), are incurable demyelinating conditions that result in pain, decrease in muscle mass, and functional impairment. Many Schwann cell proteins, which are directly involved in the stability of compact myelin or its development, are subject to mutations linked to these neuropathies. The most abundant PNS myelin protein is protein zero (P0); point mutations in this transmembrane protein cause CMT subtype 8 1B and DSS. P0 tethers apposing lipid bilayers together through its extracellular immunoglobulin-like domain. Additionally, P0 contains a cytoplasmic tail (P0ct), which is membrane-associated and contributes to the physical properties of the lipid membrane. Six CMT- and DSS-associated missense mutations have been reported in P0ct. We generated recombinant disease mutant variants of P0ct and characterized them using biophysical methods. Compared to wild-type P0ct, some mutants have negligible differences in function and folding, while others highlight functionally important amino acids within P0ct. For example, the D224Y variant of P0ct induced tight membrane multilayer stacking. Our results show a putative molecular basis for the hypermyelinating phenotype observed in patients with this particular mutation and provide overall information on the effects of disease-linked mutations in a flexible, membrane-binding protein segment. Using neutron reflectometry, we additionally show that P0ct embeds deep into a lipid bilayer, explaining the observed effects of P0ct on the physical properties of the membrane

Differential Scanning Fluorometry Signatures as Indicators of Enzyme Inhibitor Mode of Action: Case Study of Glutathione S-Transferase

Differential scanning fluorometry (DSF), also referred to as fluorescence thermal shift, is emerging as a convenient method to evaluate the stabilizing effect of small molecules on proteins of interest. However, its use in the mechanism of action studies has received far less attention. Herein, the ability of DSF to report on inhibitor mode of action was evaluated using glutathione S-transferase (GST) as a model enzyme that utilizes two distinct substrates and is known to be subject to a range of inhibition modes. Detailed investigation of the propensity of small molecule inhibitors to protect GST from thermal denaturation revealed that compounds with different inhibition modes displayed distinct thermal shift signatures when tested in the presence or absence of the enzyme's native co-substrate glutathione (GSH). Glutathione-competitive inhibitors produced dose-dependent thermal shift trendlines that converged at high compound concentrations. Inhibitors acting via the formation of glutathione conjugates induced a very pronounced stabilizing effect toward the protein only when GSH was present. Lastly, compounds known to act as noncompetitive inhibitors exhibited parallel concentration-dependent trends. Similar effects were observed with human GST isozymes A1-1 and M1-1. The results illustrate the potential of DSF as a tool to differentiate diverse classes of inhibitors based on simple analysis of co-substrate dependency of protein stabilization

Concerted Action of Two Formins in Gliding Motility and Host Cell Invasion by Toxoplasma gondii

The invasive forms of apicomplexan parasites share a conserved form of gliding motility that powers parasite migration across biological barriers, host cell invasion and egress from infected cells. Previous studies have established that the duration and direction of gliding motility are determined by actin polymerization; however, regulators of actin dynamics in apicomplexans remain poorly characterized. In the absence of a complete ARP2/3 complex, the formin homology 2 domain containing proteins and the accessory protein profilin are presumed to orchestrate actin polymerization during host cell invasion. Here, we have undertaken the biochemical and functional characterization of two Toxoplasma gondii formins and established that they act in concert as actin nucleators during invasion. The importance of TgFRM1 for parasite motility has been assessed by conditional gene disruption. The contribution of each formin individually and jointly was revealed by an approach based upon the expression of dominant mutants with modified FH2 domains impaired in actin binding but still able to dimerize with their respective endogenous formin. These mutated FH2 domains were fused to the ligand-controlled destabilization domain (DD-FKBP) to achieve conditional expression. This strategy proved unique in identifying the non-redundant and critical roles of both formins in invasion. These findings provide new insights into how controlled actin polymerization drives the directional movement required for productive penetration of parasites into host cells