Cryo-EM, X-ray diffraction, and atomistic simulations reveal determinants for the formation of a supramolecular myelin-like proteolipid lattice.

Myelin protein P2 is a peripheral membrane protein of the fatty acid-binding protein family that functions in the formation and maintenance of the peripheral nerve myelin sheath. Several P2 gene mutations cause human Charcot-Marie-Tooth neuropathy, but the mature myelin sheath assembly mechanism is unclear. Here, cryo-EM of myelin-like proteolipid multilayers revealed an ordered three-dimensional (3D) lattice of P2 molecules between stacked lipid bilayers, visualizing supramolecular assembly at the myelin major dense line. The data disclosed that a single P2 layer is inserted between two bilayers in a tight intermembrane space of ∼3 nm, implying direct interactions between P2 and two membrane surfaces. X-ray diffraction from P2-stacked bicelle multilayers revealed lateral protein organization, and surface mutagenesis of P2 coupled with structure-function experiments revealed a role for both the portal region of P2 and its opposite face in membrane interactions. Atomistic molecular dynamics simulations of P2 on model membrane surfaces suggested that Arg-88 is critical for P2-membrane interactions, in addition to the helical lid domain. Negatively charged lipid headgroups stably anchored P2 on the myelin-like bilayer surface. Membrane binding may be accompanied by opening of the P2 ß-barrel structure and ligand exchange with the apposing bilayer. Our results provide an unprecedented view into an ordered, multilayered biomolecular membrane system induced by the presence of a peripheral membrane protein from human myelin. This is an important step toward deciphering the 3D assembly of a mature myelin sheath at the molecular level.

Trapped in the epineurium: early entry into the endoneurium is restricted to neuritogenic T cells in experimental autoimmune neuritis.

BACKGROUND: Autoimmune polyneuropathies are acquired inflammatory disorders of the peripheral nervous system (PNS) characterized by inflammation, demyelination, and axonal degeneration. Although the pathogenesis has not been fully elucidated, T cells recognizing self-antigens are believed to initiate inflammation in a subgroup of patients. However, the route and time of T cell entry into the PNS have not yet been described in detail. In this study, we analyzed both kinetics as well as localization of retrovirally transfected green fluorescent protein (GFP)-expressing neuritogenic T lymphocytes in experimental autoimmune neuritis (EAN). METHODS: T lymphocytes obtained from rats following EAN induction by immunization with peripheral nerve protein peptide P255-78 were retrovirally engineered to express GFP. Non-specific T cells were negatively selected by in vitro restimulation, whereas GFP-expressing neuritogenic T cells (reactive to P255-78) were adoptively transferred into healthy rats (AT-EAN). Antigen-specific T cell tracking and localization was performed by flow cytometry and immunohistochemistry during the course of disease. RESULTS: After induction of autoimmune neuritis, P2-reactive T cells were detectable in the liver, spleen, lymph nodes, lung, peripheral blood, and the sciatic nerves with distinct kinetics. A significant number of GFP+ T cells appeared early in the lung with a peak at day four. In the peripheral nerves within the first days, GFP-negative T cells rapidly accumulated and exceeded the number of GFP-expressing cells, but did not enter the endoneurium. Very early after adoptive transfer, T cells are found in proximity to peripheral nerves and in the epineurium. However, only GFP-expressing neuritogenic T cells are able to enter the endoneurium from day five after transfer. CONCLUSIONS: Our findings suggest that neuritogenic T cells invade the PNS early in the course of disease. However, neuritogenic T cells cross the blood-nerve barrier with a certain delay without preference to dorsal roots. Further understanding of the pathophysiological role of autoagressive T cells may help to improve therapeutic strategies in immune-mediated neuropathies.

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

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 ß 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.

A role of peripheral myelin protein 2 in lipid homeostasis of myelinating Schwann cells.

Peripheral myelin protein 2 (Pmp2, P2 or Fabp8), a member of the fatty acid binding protein family, was originally described together with myelin basic protein (Mbp or P1) and myelin protein zero (Mpz or P0) as one of the most abundant myelin proteins in the peripheral nervous system (PNS). Although Pmp2 is predominantly expressed in myelinated Schwann cells, its role in glia is currently unknown. To study its function in PNS biology, we have generated a complete Pmp2 knockout mouse (Pmp2(-/-) ). Comprehensive characterization of Pmp2(-/-) mice revealed a temporary reduction in their motor nerve conduction velocity (MNCV). While this change was not accompanied by any defects in general myelin structure, we detected transitory alterations in the myelin lipid profile of Pmp2(-/-) mice. It was previously proposed that Pmp2 and Mbp have comparable functions in the PNS suggesting that the presence of Mbp can partially mask the Pmp2(-/-) phenotype. Indeed, we found that Mbp lacking Shi(-/-) mice, similar to Pmp2(-/-) animals, have preserved myelin structure and reduced MNCV, but this phenotype was not aggravated in Pmp2(-/-) /Shi(-/-) mutants indicating that Pmp2 and Mbp do not substitute each other's functions in the PNS. These data, together with our observation that Pmp2 binds and transports fatty acids to membranes, uncover a role for Pmp2 in lipid homeostasis of myelinating Schwann cells.

Atomic resolution view into the structure-function relationships of the human myelin peripheral membrane protein P2.

P2 is a fatty acid-binding protein expressed in vertebrate peripheral nerve myelin, where it may function in bilayer stacking and lipid transport. P2 binds to phospholipid membranes through its positively charged surface and a hydrophobic tip, and accommodates fatty acids inside its barrel structure. The structure of human P2 refined at the ultrahigh resolution of 0.93 Šallows detailed structural analyses, including the full organization of an internal hydrogen-bonding network. The orientation of the bound fatty-acid carboxyl group is linked to the protonation states of two coordinating arginine residues. An anion-binding site in the portal region is suggested to be relevant for membrane interactions and conformational changes. When bound to membrane multilayers, P2 has a preferred orientation and is stabilized, and the repeat distance indicates a single layer of P2 between membranes. Simulations show the formation of a double bilayer in the presence of P2, and in cultured cells wild-type P2 induces membrane-domain formation. Here, the most accurate structural and functional view to date on P2, a major component of peripheral nerve myelin, is presented, showing how it can interact with two membranes simultaneously while going through conformational changes at its portal region enabling ligand transfer.

Effects of Lid Domain Structural Changes on the Interactions between Peripheral Myelin Protein 2 and a Lipid Bilayer.

Peripheral myelin protein 2 (P2) plays an important role in the stacking of the myelin membrane and lipid transport. Here we investigate the interactions between P2 and a model myelin membrane using molecular dynamics simulations, focusing on the effect of the L27D mutation and conformational changes in the α2-helix in the lid domain of P2. The L27D mutation weakens the binding of the lid domain of P2 on the membrane. The α2-helix is either folded or unfolded on the membrane. Compared with the α2-helix structure in water, the membrane stabilizes the structure of the α2-helix, whereas the unfolding of the α2-helix reduces the binding affinity of P2 on the membrane. These findings reveal the energetics of the mutant and the structural changes of P2 on the interactions between the protein and the lipid bilayer and help us to understand the microscopic mechanism of the formation of the myelin sheath structure and some neurological disorders.

B1-induced caspase-independent apoptosis in MCF-7 cells is mediated by down-regulation of Bcl-2 via p53 binding to P2 promoter TATA box.

The Bcl-2 family contains a panel of proteins which are conserved regulators of apoptosis in mammalian cells, like the anti-apoptotic protein Bcl-2. According to its significant role in altering susceptibility to apoptosis, the deciphering of the mechanism of Bcl-2 expression modulation may be crucial for identifying therapeutics strategies for cancer. Treatment with naphthalimide-based DNA intercalators, including M2-A and R16, generally leads to a decrease in Bcl-2 intracellular amounts. Whereas the interest for these chemotherapeutics is accompanied by advances in the fundamental understanding of their anticancer properties, the molecular mechanism underlying changes in Bcl-2 expression remains poorly understood. We report here that p53 contributes to Bcl-2 down-regulation induced by B1, a novel naphthalimide-based DNA intercalating agent. Indeed, the decrease in Bcl-2 protein levels observed during B1-induced apoptosis was correlated to the decrease in mRNA levels, as a result of the inhibition of Bcl-2 transcription and promoter activity. In this context, we evaluated p53 contribution in the Bcl-2 transcriptional down-regulation. We found a significant increase of p53 binding to P(2) promoter TATA box in MCF7 cells by chromatin immunoprecipitation. These data suggest that B1-induced caspase-independent apoptosis in MCF-7 cells is associated with the activation of p53 and the down-regulation of Bcl-2. Our study strengthens the links between p53 and Bcl-2 at a transcriptional level, upon naphthalimide-based DNA intercalator treatment.

PMP2/FABP8 induces PI(4,5)P2-dependent transbilayer reorganization of sphingomyelin in the plasma membrane.

Sphingomyelin (SM) is a mammalian lipid mainly distributed in the outer leaflet of the plasma membrane (PM). We show that peripheral myelin protein 2 (PMP2), a member of the fatty-acid-binding protein (FABP) family, can localize at the PM and controls the transbilayer distribution of SM. Genetic screening with genome-wide small hairpin RNA libraries identifies PMP2 as a protein involved in the transbilayer movement of SM. A biochemical assay demonstrates that PMP2 is a phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-binding protein. PMP2 induces the tubulation of model membranes in a PI(4,5)P2-dependent manner, accompanied by the modification of the transbilayer membrane distribution of lipids. In the PM of PMP2-overexpressing cells, inner-leaflet SM is increased whereas outer-leaflet SM is reduced. PMP2 is a causative protein of Charcot-Marie-Tooth disease (CMT). A mutation in PMP2 associated with CMT increases its affinity for PI(4,5)P2, inducing membrane tubulation and the subsequent transbilayer movement of lipids.

Human myelin protein P2: from crystallography to time-lapse membrane imaging and neuropathy-associated variants.

Peripheral myelin protein 2 (P2) is a fatty acid-binding protein expressed in vertebrate peripheral nervous system myelin, as well as in human astrocytes. Suggested functions of P2 include membrane stacking and lipid transport. Mutations in the PMP2 gene, encoding P2, are associated with Charcot-Marie-Tooth disease (CMT). Recent studies have revealed three novel PMP2 mutations in CMT patients. To shed light on the structure and function of these P2 variants, we used X-ray and neutron crystallography, small-angle X-ray scattering, circular dichroism spectroscopy, computer simulations and lipid binding assays. The crystal and solution structures of the I50del, M114T and V115A variants of P2 showed minor differences to the wild-type protein, whereas their thermal stability was reduced. Vesicle aggregation assays revealed no change in membrane stacking characteristics, while the variants showed altered fatty acid binding. Time-lapse imaging of lipid bilayers indicated formation of double-membrane structures induced by P2, which could be related to its function in stacking of two myelin membrane surfaces in vivo. In order to better understand the links between structure, dynamics and function, the crystal structure of perdeuterated P2 was refined from room temperature data using neutrons and X-rays, and the results were compared to simulations and cryocooled crystal structures. Our data indicate similar properties for all known human P2 CMT variants; while crystal structures are nearly identical, thermal stability and function of CMT variants are impaired. Our data provide new insights into the structure-function relationships and dynamics of P2 in health and disease.

Myelin basic protein and myelin protein 2 act synergistically to cause stacking of lipid bilayers.

Saltatory conduction of nerve impulses along axonal membranes depends on the presence of a multilayered membrane, myelin, that wraps around the axon. Myelin basic protein (MBP) and myelin protein 2 (P2) are intimately involved in the generation of the myelin sheath. They are also implicated in a number of neurological diseases, including autoimmune diseases of both the central and peripheral nervous systems. Here, we have used atomic force microsopy (AFM) to study the effects of MBP and P2 on lipid bilayers. MBP in association with a mica substrate appeared unstructured, and tended to coat the mica surface in the form of a monolayer. In contrast, P2 appeared as discrete particles, with molecular volumes consistent with the formation of both monomers and dimers. Either MBP or P2, at micromolar concentrations, caused stacking of brain lipid bilayers. This stacking effect was significantly potentiated when both proteins were added together. Bilayers composed of phosphatidylcholine (PC) and phosphatidylserine (PS) were stacked by MBP, provided that cholesterol was also present; in contrast, P2 did not stack PC/PS/cholesterol bilayers. Hence, the bilayer stacking effects of the two proteins have different lipid requirements.

Porcine P2 myelin protein primary structure and bound fatty acids determined by mass spectrometry.

Complementary collision-induced/electron capture dissociation Fourier-transform ion cyclotron resonance mass spectrometry was used to fully sequence the protein P2 myelin basic protein. It is an antigenic fatty-acid-binding protein that can induce experimental autoimmune neuritis: an animal model of Guillain-Barré syndrome, a disorder similar in etiology to multiple sclerosis. Neither the primary structure of the porcine variant, nor the fatty acids bound by the protein have been well established to date. A 1.8-A crystal structure shows but a bound ligand could not be unequivocally identified. A protocol for ligand extraction from protein crystals has been developed with subsequent gas chromatography MS analysis allowing determination that oleic, stearic, and palmitic fatty acids are associated with the protein. The results provide unique and general evidence of the utility of mass spectrometry for characterizing proteins from natural sources and generating biochemical information that may facilitate attempts to elucidate the causes for disorders such as demyelination.

Uncoupling of obesity from insulin resistance through a targeted mutation in aP2, the adipocyte fatty acid binding protein.

Fatty acid binding proteins (FABPs) are small cytoplasmic proteins that are expressed in a highly tissue-specific manner and bind to fatty acids such as oleic and retinoic acid. Mice with a null mutation in aP2, the gene encoding the adipocyte FABP, were developmentally and metabolically normal. The aP2-deficient mice developed dietary obesity but, unlike control mice, they did not develop insulin resistance or diabetes. Also unlike their obese wild-type counterparts, obese aP2-/- animals failed to express in adipose tissue tumor necrosis factor-alpha (TNF-alpha), a molecule implicated in obesity-related insulin resistance. These results indicate that aP2 is central to the pathway that links obesity to insulin resistance, possibly by linking fatty acid metabolism to expression of TNF-alpha.

A Quasielastic Neutron Scattering Investigation on the Molecular Self-Dynamics of Human Myelin Protein P2.

The human myelin protein P2 is a membrane binding protein believed to maintain correct lipid composition and organization in peripheral nerve myelin. Its function is related to its ability to stack membranes, and this function can be enhanced by the P38G mutation, whereby the overall protein structure does not change but the molecular dynamics increase. Mutations in P2 are linked to human peripheral neuropathy. Here, the dynamics of wild-type P2 and the P38G variant were studied using quasielastic neutron scattering on time scales from 10 ps to 1 ns at 300 K. The results suggest that the mutant protein dynamics are increased on both the fastest and the slowest measured time scales, by increasing the dynamics amplitude and/or the portion of atoms participating in the movement.

The myelin protein PMP2 is regulated by SOX10 and drives melanoma cell invasion.

The transcription factor sex determining region Y-box 10 (SOX10) plays a key role in the development of melanocytes and glial cells from neural crest precursors. SOX10 is involved in melanoma initiation, proliferation, invasion, and survival. However, specific mediators which impart its oncogenic properties remain widely unknown. To identify target genes of SOX10, we performed RNA sequencing after ectopic expression of SOX10 in human melanoma cells. Among nine differentially regulated genes, peripheral myelin protein 2 (PMP2) was consistently upregulated in several cell lines. Direct regulation of PMP2 by SOX10 was shown by chromatin immunoprecipitation, electrophoretic mobility shift, and luciferase reporter assays. Moreover, a coregulation of PMP2 by SOX10 and early growth response 2 in melanoma cells was found. Phenotypical investigation demonstrated that PMP2 expression can increase melanoma cell invasion. As PMP2 protein was detected only in a subset of melanoma cell lines, it might contribute to melanoma heterogeneity.

Changes in gene expression in small bowel neuroendocrine tumors associated with progression to metastases.

BACKGROUND: Small bowel neuroendocrine tumors (SBNETs) present frequently with metastases, yet little is known about the molecular basis of this progression. This study sought to identify the serial differential expression of genes between normal small bowel, primary small bowel neuroendocrine tumors, and liver metastases. METHODS: RNA isolated from matched normal small bowel tissue, primary small bowel neuroendocrine tumors, and liver metastases in 12 patients was analyzed with whole transcriptome expression microarrays and RNA-Seq. Changes in gene expression between primary small bowel neuroendocrine tumors and normal small bowels, and liver metastases versus primary small bowel neuroendocrine tumors were calculated. Common genes that were differentially expressed serially (increasing or decreasing from normal small bowel to primary small bowel neuroendocrine tumors to liver metastases) were identified, and 10 were validated using qPCR. RESULTS: Use of 2 transcriptome platforms allowed for a robust discrimination of genes important in small bowel neuroendocrine tumors progression. Serial differential expression was validated in 7/10 genes, all of which had been described previously in abdominal cancers, and with several interacting with members of the AKT, MYC, or MAPK3 pathways. Liver metastases had consistent underexpression of PMP22, while high expression of SERPINA10 and SYT13 was characteristic of both pSBTs and liver metastases. CONCLUSION: Identification of the serial differential expression of genes from normal tissues to primary tumors to metastases lends insight into important pathways for SBNETs progression. Differential expression of various genes, including PMP22, SYT13 and SERPINA10, are associated with the progression of SBNETs and warrant further investigation.

Molecular mechanisms of Charcot-Marie-Tooth neuropathy linked to mutations in human myelin protein P2.

Charcot-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 ß 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.

Loss of adipocyte-type fatty acid binding protein and other protein biomarkers is associated with progression of human bladder transitional cell carcinomas.

Multifocal recurrent papillary tumors provide a unique model system to study the molecular mechanisms underlying the steps involved in transitional cell carcinoma progression and offer a valuable source of material to search for biomarkers that may form the basis for diagnosis, prognosis, and treatment. We have examined the protein expression profiles of normal bladder urothelium and of 63 transitional cell carcinomas of various histopathological grades and T stages using high-resolution, two-dimensional gel electrophoresis, microsequencing, mass spectrometry, and a two-dimensional gel protein database approach for polypeptide identification (http://biobase.dk/cgi-bin/celis). In general, the results revealed a striking similarity between the overall qualitative expression patterns of papillary tumors of all grades, as well as of papillary and solid tumors of grade III. With few exceptions, tumors of grades I-III expressed, albeit at different levels, all of the keratins (7, 8, 13, 17, 18, 19, and 20) found in the normal urothelium. Grade IV tumors lacked or expressed reduced levels of keratin 13 but most resembled low-grade tumors. One invasive grade IV tumor, however, expressed a fibroblast-like protein phenotype. Four proteins that were expressed by normal urothelium and were lost at various stages of progression were identified as glutathione S-transferase mu, prostaglandin dehydrogenase (PGDH), a fatty acid binding protein with homology to the adipocyte isoform (A-FABP), and keratin 13. The percentage of tumors expressing A-FABP was very high in low-grade lesions but decreased drastically (P = 0.0006) in grade III and IV neoplasms. In addition, low-grade tumors contained more A-FABP than their high-grade counterparts. The stage of the disease was also statistically (P = 0.0269) related to the presence or absence of A-FABP in grade III tumors. Similar analysis of glutathione S-transferase mu and PGDH showed a statistically significant decrease of these proteins in high-grade (grades III and IV) tumors (P = 0.0026 and P = 0.0044, respectively). Only PGDH showed a suggestive correlation (P = 0.0775) with the stage of the disease in grade III tumors. Keratin 13 showed a drastic decrease in grade IV tumors. In addition to identifying biomarkers that may have prognostic value, our studies have suggested that A-FABP is an important component of the pathway(s) leading to bladder cancer development.

Expression of fatty acid binding protein in the liver during pregnancy and lactation in the rat.

Expression of the liver fatty acid binding protein (L-FABP) has been studied in the liver of pregnant and lactating rats. The L-FABP concentration found in the cytosol by immuno-enzymatic assay (ELISA) was consistently higher in the dams during the pregnancy and the lactation than in the age-matched virgin females. Paradoxically, a decrease in the L-FABP mRNA level occurred in the maternal liver during the last days of the gestation. This level remained low on days 7 and 14 of the lactation. Since the transcription rate of the L-FABP gene was unchanged in the maternal liver, these data suggest a post-transcriptional regulation of the L-FABP during pregnancy and lactation in the rat. The nutritional adaptations occurring during pregnancy and lactation are not involved in this regulation since a chronic maternal food-restriction failed to correct these modifications. The mechanism of this regulation is presently unknown, but possibilities include hormonally mediated effects.

Structural properties of the adipocyte lipid binding protein.

The adipocyte lipid binding protein, ALBP (also adipocyte fatty acid binding protein, A-FABP, 422 protein, aP2, and p15 protein), is one of the most studied of the intracellular lipid binding protein family. Here we sequentially compare the different sources of ALBP and describe the idea that one-third of the amino acid side chains near the N-terminal end appear to play a major role in conformational dynamics and in ligand transfer. Crystallographic data for mouse ALBP are summarized and the ligand binding cavity analyzed in terms of the overall surface and conformational dynamics. The region of the proposed ligand portal is described. Amino acid side chains critical to cavity formation and fatty acid interactions are analyzed by comparing known crystal structures containing a series of different hydrophobic ligands. Finally, we address ALBP ligand binding affinity and thermodynamic studies.