Recruitment of Eph receptors into signaling clusters does not require ephrin contact

Eph receptors and their cell membrane-bound ephrin ligands regulate cell positioning and thereby establish or stabilize patterns of cellular organization. Although it is recognized that ephrin clustering is essential for Eph function, mechanisms that relay information of ephrin density into cell biological responses are poorly understood. We demonstrate by confocal time-lapse and fluorescence resonance energy transfer microscopy that within minutes of binding ephrin-A5-coated beads, EphA3 receptors assemble into large clusters. While remaining positioned around the site of ephrin contact, Eph clusters exceed the size of the interacting ephrin surface severalfold. EphA3 mutants with compromised ephrin-binding capacity, which alone are incapable of cluster formation or phosphorylation, are recruited effectively and become phosphorylated when coexpressed with a functional receptor. Our findings reveal consecutive initiation of ephrin-facilitated Eph clustering and cluster propagation, the latter of which is independent of ephrin contacts and cytosolic Eph signaling functions but involves direct Eph-Eph interactions.     

Toward the semisynthesis of multidomain transmembrane receptors: modification of Eph tyrosine kinases

Expressed protein ligation (EPL) is a protein engineering approach that allows the modification or assembly of a target protein from multiple recombinant and synthetic polypeptides. EPL has been previously used to modify intracellular proteins and small integral membrane proteins for structural and functional studies. Here we describe the semisynthetic site-specific modification of the complete, multidomain extracellular regions of both A and B classes of Eph receptor tyrosine kinases. We show that the ectodomains of these receptors can be ligated to different peptides under carefully established experimental conditions, while their biological activity is retained. This work extends the boundaries of the EPL technique for semisynthesis of multidomain, extracellular, disulfide-bonded, and glycosylated proteins and highlights its potential application for reconstituting entire single-pass transmembrane proteins.     

Oligodendrocytes in neurodegenerative diseases

Oligodendrocyte is a highly specialized glial cell type in the vertebrate central nervous system, which guarantees the long-distance transmission of action potential by producing myelin sheath wrapping adjacent axons. Disrupted myelin and oligodendrocytes are hallmarks of some devastating neurological diseases, such as multiple sclerosis, although their contribution to neurodegeneration in a given disease is still controversial. However, accumulating evidence from clinical studies and genetic animal models implicates oligodendrocyte dysfunction as one of major events in the processes of initiation and progression of neurodegeneration. In this article, we will review recent progress in understanding non-traditional function of oligodendrocytes in neuronal support and protection independent of myelin sheath and its possible contribution to neurodegeneration. Oligodendrocytes play a pivotal role in neurodegenerative diseases among which special emphasis is given to multiple system atrophy and Alzheimer’s disease in this review.     

Promiscuous and specific recognition among ephrins and Eph receptors

Eph–ephrin interactions control the signal transduction between cells and play an important role in carcinogenesis and other diseases. The interactions between Eph receptors and ephrins of the same subclass are promiscuous; there are cross-interactions between some subclasses, but not all. To understand how Eph–ephrin interactions can be both promiscuous and specific, we investigated sixteen energy landscapes of four Eph receptors (A2, A4, B2, and B4) interacting with four ephrin ligands (A1, A2, A5, and B2). We generated conformational ensembles and recognition energy landscapes starting from separated Eph and ephrin molecules and proceeding up to the formation of Eph–ephrin complexes. Analysis of the Eph–ephrin recognition trajectories and the co-evolution entropy of 400 ligand binding domains of Eph receptor and 241 ephrin ligands identified conserved residues during the recognition process. Our study correctly predicted the promiscuity and specificity of the interactions and provided insights into their recognition. The dynamic conformational changes during Eph–ephrin recognition can be described by progressive conformational selection and population shift events, with two dynamic salt bridges between EphB4 and ephrin-B2 contributing to the specific recognition. EphA3 cancer-related mutations lowered the binding energies. The specificity is not only controlled by the final stage of the interaction across the protein–protein interface, but also has large contributions from binding kinetics with the help of dynamic intermediates along the pathway from the separated Eph and ephrin to the Eph–ephrin complex.     

Ligand recognition by A‐class Eph receptors: crystal structures of the EphA2 ligand‐binding domain and the EphA2/ephrin‐A1 complex

Ephrin (Eph) receptor tyrosine kinases fall into two subclasses (A and B) according to preferences for their ephrin ligands. All published structural studies of Eph receptor/ephrin complexes involve B‐class receptors. Here, we present the crystal structures of an A‐class complex between EphA2 and ephrin‐A1 and of unbound EphA2. Although these structures are similar overall to their B‐class counterparts, they reveal important differences that define subclass specificity. The structures suggest that the A‐class Eph receptor/ephrin interactions involve smaller rearrangements in the interacting partners, better described by a ‘lock‐and‐key’‐type binding mechanism, in contrast to the ‘induced fit’ mechanism defining the B‐class molecules. This model is supported by structure‐based mutagenesis and by differential requirements for ligand oligomerization by the two subclasses in cell‐based Eph receptor activation assays. Finally, the structure of the unligated receptor reveals a homodimer assembly that might represent EphA2‐specific homotypic cell adhesion interactions.     

Sizes of interface residues account for cross‐class binding affinity patterns in Eph receptor–ephrin families

Eph receptors comprise the largest known family of receptor tyrosine kinases in mammals. They bind members of a second family, the ephrins. As both Eph receptors and ephrins are membrane bound, interactions permit unusual bidirectional cell–cell signaling. Eph receptors and ephrins each form two classes, A and B, based on sequences, structures, and patterns of affinity: Class A Eph receptors bind class A ephrins, and class B Eph receptors bind class B ephrins. The only known exceptions are the receptor EphA4, which can bind ephrinB2 and ephrinB3 in addition to the ephrin‐As (Bowden et al., Structure 2009;17:1386–1397); and EphB2, which can bind ephrin‐A5 in addition to the ephrin‐Bs (Himanen et al., Nat Neurosci 2004;7:501–509). A crystal structure is available of the interacting domains of the EphA4‐ephrin B2 complex (wwPDB entry 2WO2) (Bowden et al., Structure 2009;17:1386–1397). In this complex, the ligand‐binding domain of EphA4 adopts an EphB‐like conformation. To understand why other cross‐class EphA receptor–ephrinB complexes do not form, we modeled hypothetical complexes between (1) EphA4–ephrinB1, (2) EphA4–ephrinB3, and (3) EphA2–ephrinB2. We identify particular residues in the interface region, the size variations of which cause steric clashes that prevent formation of the unobserved complexes. The sizes of the sidechains of residues at these positions correlate with the pattern of binding affinity. Proteins 2014; 82:349–353. © 2013 Wiley Periodicals, Inc.     

The role of mitochondria in inherited neurodegenerative diseases

In the past decade, the genetic causes underlying familial forms of many neurodegenerative disorders, such as Huntington's disease, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, Friedreich ataxia, hereditary spastic paraplegia, dominant optic atrophy, Charcot-Marie-Tooth type 2A, neuropathy ataxia and retinitis pigmentosa, and Leber's hereditary optic atrophy have been elucidated. However, the common pathogenic mechanisms of neuronal death are still largely unknown. Recently, mitochondrial dysfunction has emerged as a potential 'lowest common denominator' linking these disorders. In this review, we discuss the body of evidence supporting the role of mitochondria in the pathogenesis of hereditary neurodegenerative diseases. We summarize the principal features of genetic diseases caused by abnormalities of mitochondrial proteins encoded by the mitochondrial or the nuclear genomes. We then address genetic diseases where mutant proteins are localized in multiple cell compartments, including mitochondria and where mitochondrial defects are likely to be directly caused by the mutant proteins. Finally, we describe examples of neurodegenerative disorders where mitochondrial dysfunction may be 'secondary' and probably concomitant with degenerative events in other cell organelles, but may still play an important role in the neuronal decay. Understanding the contribution of mitochondrial dysfunction to neurodegeneration and its pathophysiological basis will significantly impact our ability to develop more effective therapies for neurodegenerative diseases.     

Abnormal tau-containing filaments in neurodegenerative diseases

It has been known for some time that the neurofibrillary pathology in Alzheimer's disease consists of so-called paired helical and straight filaments made up of the microtubule-associated protein tau. The degree of dementia observed in the disease correlates better with the extent of neurofibrillary pathology than with the Abeta amyloid deposits, the other characteristic defining pathological fibrous deposit in Alzheimer's disease. However, no familial cases of Alzheimer's disease have been genetically linked to the tau protein locus. Recently a group of frontotemporal dementias with parkinsonism linked to chromosome 17 has been shown to be caused by mutations in the tau gene. Some are missense mutations giving altered tau proteins, whereas others affect the splicing of the pre-mRNA and change the balance between different tau isoforms. Histologically these diseases are all characterised by various kinds of filamentous tau protein deposits, mostly in the complete absence of Abeta deposits. The abnormal tau filaments show different morphologies, depending on the nature of the tau mutation. These diseases show that tau mutations can be a prime cause of inherited dementing illness and may throw some light on the pathological process in the much larger number of sporadic cases of Alzheimer's disease.     

Mechanisms of amyloid beta protein-induced modification in ion transport systems: implications for neurodegenerative diseases

1. Alzheimer's disease (AD) is a neurodegenerative disorder that affects the cognitive function of the brain. Pathological changes in AD are characterized by the formation of amyloid plaques and neurofibrillary tangles as well as extensive neuronal loss. Abnormal proteolytic processing of amyloid precursor protein (APP) is the central step that leads to formation of amyloid plaque, neurofibrillary tangles, and neuronal loss.2. The plaques, which accumulate extracellularly in the brain, are composed of aggregates and cause direct neurotoxic effects and/or increase neuronal vulnerability to excitotoxic insults. The aggregates consist of soluble pathologic amyloid beta peptides AP[1–42] and AP[1–43] and soluble nonpathologic AP[1–40]. Both APP and AP interact with ion transport systems. AP induces a wide range of effects as the result of activating a cascade of mechanisms.3. The major mechanisms proposed for AP-induced cytotoxicity involve the loss of Ca²⁺ homeostasis and the generation of reactive oxygen species (ROS). The changes in Ca²⁺ homeostasis could be the result of (1) changes in endogenous ion transport systems, e.g. Ca²⁺ and K⁺ channels and Na⁺/K⁺-ATPase, and membrane receptor proteins, such as ligand-driven ion channels and G-protein-driven releases of second messengers, and (2) formation of heterogeneous ion channels.4. The consequences of changes in Ca²⁺-homeostasis-induced generation of ROS are (a) direct modification of intrinsic ion transport systems and their regulatory mechanisms, and (b) indirect effects on ion transport systems via peroxidation of phospholipids in the membrane, inhibition of phosphorylation, and reduction of ATP levels and cytoplasmic pH.5. We propose that in AD, AP with its different conformations alters cell regulation by modifying several ion transport systems and also by forming heterogeneous ion channels. The changes in membrane transport systems are proposed as early steps in impairing neuronal function preceding plaque formation. We conclude that these changes damage the membrane by compromising its integrity and increasing its ion permeability. This mechanism of membrane damage is not only central for AD but also may explain other malfunctioned protein-processing–related pathologies.     

Nefarious NTRK oncogenic fusions in pediatric sarcomas: Too many to Trk

Neurotrophic Tyrosine Receptor Kinase (NTRK) genes undergo chromosomal translocations to create novel open reading frames coding for oncogenic fusion proteins; the N-terminal portion, donated by various partner genes, becomes fused to the tyrosine kinase domain of either NTRK1, NTRK2, or NTRK3. NTRK fusion proteins have been identified as driver oncogenes in a wide variety of tumors over the past three decades, including Pediatric Gliomas, Papillary Thyroid Carcinoma, Spitzoid Neoplasms, Glioblastoma, and additional tumors. Importantly, NTRK fusions function as drivers of pediatric sarcomas, accounting for approximately 15% of childhood cancers including Infantile Fibrosarcoma (IFS), a subset of pediatric soft tissue sarcoma (STS). While tyrosine kinase inhibitors (TKIs), such as larotrectinib and entrectinib, have demonstrated profound results against NTRK fusion-positive cancers, acquired resistance to these TKIs has resulted in the formation of gatekeeper, solvent-front, and compound mutations. We present a comprehensive compilation of oncogenic fusions involving NTRKs focusing specifically on pediatric STS, examining their biological signaling pathways and mechanisms of activation. The importance of an obligatory dimerization or multimerization domain, invariably donated by the N-terminal fusion partner, is discussed using characteristic fusions that occur in pediatric sarcomas. In addition, examples are presented of oncogenic fusion proteins in which the N-terminal partners may contribute additional biological activities beyond an oligomerization domain. Lastly, therapeutic approaches to the treatment of pediatric sarcoma will be presented, using first generation and second-generation agents such as selitrectinib and repotrectinib.     

Platelet-derived growth factors and their receptors: Structural and functional perspectives

The four types of platelet-derived growth factors (PDGFs) and the two types of PDGF receptors (PDGFRs, which belong to class III receptor tyrosine kinases) have important functions in the development of connective tissue cells. Recent structural studies have revealed novel mechanisms of PDGFs in propeptide loading and receptor recognition/activation. The detailed structural understanding of PDGF–PDGFR signaling has provided a template that can aid therapeutic intervention to counteract the aberrant signaling of this normally silent pathway, especially in proliferative diseases such as cancer. This review summarizes the advances in the PDGF system with a focus on relating the structural and functional understandings, and discusses the basic aspects of PDGFs and PDGFRs, the mechanisms of activation, and the insights into the therapeutic antagonism of PDGFRs. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases.     

Importance of Shank3 protein in regulating metabotropic glutamate receptor 5 (mGluR5) expression and signaling at synapses

Shank3/PROSAP2 gene mutations are associated with cognitive impairment ranging from mental retardation to autism. Shank3 is a large scaffold postsynaptic density protein implicated in dendritic spines and synapse formation; however, its specific functions have not been clearly demonstrated. We have used RNAi to knockdown Shank3 expression in neuronal cultures and showed that this treatment specifically reduced the synaptic expression of the metabotropic glutamate receptor 5 (mGluR5), but did not affect the expression of other major synaptic proteins. The functional consequence of Shank3 RNAi knockdown was impaired signaling via mGluR5, as shown by reduction in ERK1/2 and CREB phosphorylation induced by stimulation with (S)-3,5-dihydroxyphenylglycine (DHPG) as the agonist of mGluR5 receptors, impaired mGluR5-dependent synaptic plasticity (DHPG-induced long-term depression), and impaired mGluR5-dependent modulation of neural network activity. We also found morphological abnormalities in the structure of synapses (spine number, width, and length) and impaired glutamatergic synaptic transmission, as shown by reduction in the frequency of miniature excitatory postsynaptic currents (mEPSC). Notably, pharmacological augmentation of mGluR5 activity using 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)-benzamide as the positive allosteric modulator of these receptors restored mGluR5-dependent signaling (DHPG-induced phosphorylation of ERK1/2) and normalized the frequency of mEPSCs in Shank3-knocked down neurons. These data demonstrate that a deficit in mGluR5-mediated intracellular signaling in Shank3 knockdown neurons can be compensated by 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)-benzamide; this raises the possibility that pharmacological augmentation of mGluR5 activity represents a possible new therapeutic approach for patients with Shank3 mutations.     

The role of proteases in regulating Eph/ephrin signaling

Proteases regulate a myriad of cell functions, both in normal and disease states. In addition to protein turnover, they regulate a range of signaling processes, including those mediated by Eph receptors and their ephrin ligands. A variety of proteases is reported to directly cleave Ephs and/or ephrins under different conditions, to promote receptor and/or ligand shedding, and regulate receptor/ligand internalisation and signaling. They also cleave other adhesion proteins in response to Eph-ephrin interactions, to indirectly facilitate Eph-mediated functions. Proteases thus contribute to Eph/ephrin mediated changes in cell-cell and cell-matrix interactions, in cell morphology and in cell migration and invasion, in a manner which appears to be tightly regulated by, and co-ordinated with, Eph signaling. This review summarizes the current literature describing the function and regulation of protease activities during Eph/ephrin-mediated cell signaling.     

Eph receptors and their ligands: Promising molecular biomarkers and therapeutic targets in prostate cancer

Although at present, there is a high incidence of prostate cancer, particularly in the Western world, mortality from this disease is declining and occurs primarily only from clinically significant late stage tumors with a poor prognosis. A major current focus of this field is the identification of new biomarkers which can detect earlier, and more effectively, clinically significant tumors from those deemed “low risk”, as well as predict the prognostic course of a particular cancer. This strategy can in turn offer novel avenues for targeted therapies. The large family of Receptor Tyrosine Kinases, the Ephs, and their binding partners, the ephrins, has been implicated in many cancers of epithelial origin through stimulation of oncogenic transformation, tumor angiogenesis, and promotion of increased cell survival, invasion and migration. They also show promise as both biomarkers of diagnostic and prognostic value and as targeted therapies in cancer. This review will briefly discuss the complex roles and biological mechanisms of action of these receptors and ligands and, with regard to prostate cancer, highlight their potential as biomarkers for both diagnosis and prognosis, their application as imaging agents, and current approaches to assessing them as therapeutic targets. This review demonstrates the need for future studies into those particular family members that will prove helpful in understanding the biology and potential as targets for treatment of prostate cancer.     

Changes in adult neurogenesis in neurodegenerative diseases: cause or consequence?

This review addresses the role of adult hippocampal neurogenesis and stem cells in some of the most common neurodegenerative disorders and their related animal models. We discuss recent literature in relation to Alzheimer's disease and dementia, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, alcoholism, ischemia, epilepsy and major depression.     

Eph receptor and ephrin function in breast,gut, and skin epithelia

Epithelial cells are tightly coupled together through specialized intercellular junctions, including adherens junctions, desmosomes, tight junctions, and gap junctions. A growing body of evidence suggests epithelial cells also directly exchange information at cell-cell contacts via the Eph family of receptor tyrosine kinases and their membrane-associated ephrin ligands. Ligand-dependent and -independent signaling via Eph receptors as well as reverse signaling through ephrins impact epithelial tissue homeostasis by organizing stem cell compartments and regulating cell proliferation, migration, adhesion, differentiation, and survival. This review focuses on breast, gut, and skin epithelia as representative examples for how Eph receptors and ephrins modulate diverse epithelial cell responses in a context-dependent manner. Abnormal Eph receptor and ephrin signaling is implicated in a variety of epithelial diseases raising the intriguing possibility that this cell-cell communication pathway can be therapeutically harnessed to normalize epithelial function in pathological settings like cancer or chronic inflammation.     

Clinical implications from proteomic studies in neurodegenerative diseases: lessons from mitochondrial proteins

Mitochondria play a key role in eukaryotic cells, being mediators of energy, biosynthetic and regulatory requirements of these cells. Emerging proteomics techniques have allowed scientists to obtain the differentially expressed proteome or the proteomic redox status in mitochondria. This has unmasked the diversity of proteins with respect to subcellular location, expression and interactions. Mitochondria have become a research ‘hot spot’ in subcellular proteomics, leading to identification of candidate clinical targets in neurodegenerative diseases in which mitochondria are known to play pathological roles. The extensive efforts to rapidly obtain differentially expressed proteomes and unravel the redox proteomic status in mitochondria have yielded clinical insights into the neuropathological mechanisms of disease, identification of disease early stage and evaluation of disease progression. Although current technical limitations hamper full exploitation of the mitochondrial proteome in neurosciences, future advances are predicted to provide identification of specific therapeutic targets for neurodegenerative disorders.     

Eph receptor and ephrin function in breast,gut, and skin epithelia

Epithelial cells are tightly coupled together through specialized intercellular junctions, including adherens junctions, desmosomes, tight junctions, and gap junctions. A growing body of evidence suggests epithelial cells also directly exchange information at cell-cell contacts via the Eph family of receptor tyrosine kinases and their membrane-associated ephrin ligands. Ligand-dependent and -independent signaling via Eph receptors as well as reverse signaling through ephrins impact epithelial tissue homeostasis by organizing stem cell compartments and regulating cell proliferation, migration, adhesion, differentiation, and survival. This review focuses on breast, gut, and skin epithelia as representative examples for how Eph receptors and ephrins modulate diverse epithelial cell responses in a context-dependent manner. Abnormal Eph receptor and ephrin signaling is implicated in a variety of epithelial diseases raising the intriguing possibility that this cell-cell communication pathway can be therapeutically harnessed to normalize epithelial function in pathological settings like cancer or chronic inflammation.     

Altered Protein Tyrosine Phosphorylation in Alzheimer''s Disease

The activity of protein tyrosine kinase was determined in extracts from Alzheimer's disease brains and age- and postmortem time-matched control brains at autopsy using the synthetic peptide substrate poly(Glu4Tyr1). The specific activity of protein tyrosine kinases in the particulate fraction decreased roughly twofold (p less than 0.02) in Alzheimer's disease frontal cortex relative to unaffected control cortex. Cytosolic protein tyrosine kinase activity in Alzheimer's disease tissue was not significantly different from that in control tissue. In contrast to reduced particulate protein tyrosine kinase activity, analysis of Western blots of cytosolic and particulate fractions revealed increases in cytosolic antiphosphotyrosine immunoreactive polypeptides with molecular masses of 55 and 60 kDa. Quantitative immunohistochemistry and morphometry of frontal cortex sections with the antiphosphotyrosine antibody indicated increased antiphosphotyrosine staining in the neurons, although the number of antiphosphotyrosine-positive neurons per square millimeter decreased. Also, increased antiphosphotyrosine staining was observed in the hippocampal neurons. These results suggest that altered protein tyrosine kinases and protein tyrosine phosphorylation are involved in the pathology of Alzheimer's disease.