New Partners Identified by Mass Spectrometry Assay Reveal Functions of NCAM2 in Neural Cytoskeleton Organization

Neuronal cell adhesion molecule 2 (NCAM2) is a membrane protein with an important role in the morphological development of neurons. In the cortex and the hippocampus, NCAM2 is essential for proper neuronal differentiation, dendritic and axonal outgrowth and synapse formation. However, little is known about NCAM2 functional mechanisms and its interactive partners during brain development. Here we used mass spectrometry to study the molecular interactome of NCAM2 in the second postnatal week of the mouse cerebral cortex. We found that NCAM2 interacts with >100 proteins involved in numerous processes, including neuronal morphogenesis and synaptogenesis. We validated the most relevant interactors, including Neurofilaments (NEFs), Microtubule-associated protein 2 (MAP2), Calcium/calmodulin kinase II alpha (CaMKIIα), Actin and Nogo. An in silico analysis of the cytosolic tail of the NCAM2.1 isoform revealed specific phosphorylation site motifs with a putative affinity for some of these interactors. Our results expand the knowledge of NCAM2 interactome and confirm the key role of NCAM2 in cytoskeleton organization, neuronal morphogenesis and synaptogenesis. These findings are of interest in explaining the phenotypes observed in different pathologies with alterations in the NCAM2 gene.

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Ezrin interacts with the tumor suppressor CHL1 and promotes neuronal differentiation of human neuroblastoma

PLoS ONE ◽

10.1371/journal.pone.0244069 ◽

2020 ◽

Vol 15 (12) ◽

pp. e0244069

Author(s):

Marzia Ognibene ◽

Annalisa Pezzolo

Keyword(s):

Tumor Suppressor ◽

Cell Line ◽

Neuronal Differentiation ◽

Neuronal Cell ◽

Human Neuroblastoma ◽

Cytoskeleton Organization ◽

Neurite Retraction ◽

Neuronal Cell Adhesion Molecule ◽

Differentiation And Proliferation ◽

Low Expression

In a previous study, we demonstrated that CHL1, the neuronal cell adhesion molecule close homolog of L1, acts as a tumor suppressor in human neuroblastoma (NB), a still highly lethal childhood malignancy, influencing its differentiation and proliferation degree. Here we found that ezrin, one of the ERM (ezrin, radixin, moesin) proteins involved in cytoskeleton organization, strongly interacts with CHL1. The low expression of EZRIN, as well as the low expression of CHL1 and of the neuronal differentiation marker MAP2, correlates with poor outcome in NB patients. Knock-down of ezrin in HTLA-230 cell line induces neurite retraction, enhances cell proliferation and migration, and triggers anchorage-independent growth, with effects very similar to those already obtained by CHL1 silencing. Furthermore, lack of ezrin inhibits the expression of MAP2 and of the oncosuppressor molecule p53, whereas it enhances MAPK activation, all typical features of tumor aggressiveness. As already described, CHL1 overexpression in IMR-32 cell line provokes an opposite trend, but the co-silencing of ezrin reduces these effects, confirming the hypothesis that CHL1 acts in close connection with ezrin. Overall, our data show that ezrin reinforces the differentiating and oncosuppressive functions of CHL1, identifying this ERM protein as a new targetable molecule for NB therapy.

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The Actin-Binding Protein UNC-115/abLIM Controls Formation of Lamellipodia and Filopodia and Neuronal Morphogenesis in Caenorhabditis elegans

Molecular and Cellular Biology ◽

10.1128/mcb.25.12.5158-5170.2005 ◽

2005 ◽

Vol 25 (12) ◽

pp. 5158-5170 ◽

Cited By ~ 23

Author(s):

Yieyie Yang ◽

Erik A. Lundquist

Keyword(s):

Caenorhabditis Elegans ◽

Molecular Mechanisms ◽

Binding Protein ◽

Phosphorylation Site ◽

Serine Phosphorylation ◽

Actin Binding ◽

Axon Pathfinding ◽

Neuronal Morphogenesis ◽

Actin Binding Protein ◽

C Elegans

ABSTRACT The roles of actin-binding proteins in development and morphogenesis are not well understood. The actin-binding protein UNC-115 has been implicated in cytoskeletal signaling downstream of Rac in Caenorhabditis elegans axon pathfinding, but the cellular role of UNC-115 in this process remains undefined. Here we report that UNC-115 overactivity in C. elegans neurons promotes the formation of neurites and lamellipodial and filopodial extensions similar to those induced by activated Rac and normally found in C. elegans growth cones. We show that UNC-115 activity in neuronal morphogenesis is enhanced by two molecular mechanisms: when ectopically driven to the plasma membrane by the myristoylation sequence of c-Src, and by mutation of a putative serine phosphorylation site in the actin-binding domain of UNC-115. In support of the hypothesis that UNC-115 modulates actin cytoskeletal organization, we show that UNC-115 activity in serum-starved NIH 3T3 fibroblasts results in the formation of lamellipodia and filopodia. We conclude that UNC-115 is a novel regulator of the formation of lamellipodia and filopodia in neurons, possibly in the growth cone during axon pathfinding.

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Molecular Mechanism of Inhibition of Polysialyltransferase Domain (PSTD) by Heparin

Current Topics in Medicinal Chemistry ◽

10.2174/1568026621666210713165251 ◽

2021 ◽

Vol 21 ◽

Author(s):

Si-Min Liao ◽

Xue-Hui Liu ◽

Li-Xing Peng ◽

Bo Lu ◽

Ri-Bo Huang ◽

...

Keyword(s):

Polysialic Acid ◽

Neuronal Cell ◽

Selective Inhibition ◽

Migration And Invasion ◽

Heparin Binding ◽

Nmr Studies ◽

Clinical Prognosis ◽

Neuronal Cell Adhesion Molecule ◽

Mechanism Of Inhibition ◽

Therapeutic Opportunity

: Polysialic acid (polySia) is a unique carbohydrate polymer produced on the neuronal cell adhesion molecule (NCAM) in many cancer cells. It strongly correlates with the migration and invasion of tumor cells and aggressive, metastatic disease, and poor clinical prognosis in the clinic. Its synthesis is catalyzed by two polysialyltransferases (polySTs), ST8SiaIV (PST) and ST8SiaII (STX). Therefore, selective inhibition of polySTs presents a therapeutic opportunity to inhibit tumor invasion and metastasis due to NCAM polysialylation. It has been proposed that NCAM polysialylation could be inhibited by two types of heparin inhibitors, low molecular heparin (LMWH) and heparin tetrasaccharide (DP4). This review summarizes the interactions between Polysialyltransferase Domain (PSTD) in ST8SiaIV and CMP-Sia, and between the PSTD and polySia; and how LMWH and DP4 inhibit these interactions. Our NMR studies indicate that LMWH is a more effective inhibitor than DP4 for inhibition of NCAM polysialylation. The NMR identification of heparin-binding sites in the PSTD may provide insight into the design of specific inhibitors of polysialylation.

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Skimmer CID‒ion trap mass spectrometry of phosphotyrosine‒containing peptides

Spectroscopy An International Journal ◽

10.1155/2004/401436 ◽

2004 ◽

Vol 18 (3) ◽

pp. 441-451

Author(s):

Melissa D. Zolodz ◽

Karl V. Wood

Keyword(s):

Mass Spectrometry ◽

Ion Trap ◽

Phosphorylation Site ◽

Ion Trap Mass Spectrometry ◽

Cellular Functions ◽

Factors Affecting ◽

Parent Protein ◽

Phosphorylated Peptides ◽

Phosphorylated Peptide ◽

Analytical Tools

Proteomic analysis is becoming a popular field in science. Analysis of protein modifications is useful in deciphering cellular functions and errors in pathways that can result in disease. There has been increased interest in the phosphotyrosine proteome. Due to the difficulty in finding the location of the tyrosine phosphorylation site in the tyrosine phosphorylated peptide or even to verify that the parent protein is a phosphotyrosyl‒protein, new analytical tools are being developed. The phosphotyrosine immonium ion can be produced via skimmer CID for detection via ion trap mass spectrometry and is a useful marker for the indication of the presence of a phosphotyrosine residue. Skimmer CID analysis can also be used to differentiate phosphotyrosine‒containing peptides from other phosphorylated peptides. In this study, phosphotyrosine‒containing peptides were analyzed by skimmer CID in an ion trap mass spectrometer. The factors affecting the signal abundance of the phosphotyrosine immonium ion were investigated.

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Emergence of Non-Canonical Parvalbumin-Containing Interneurons in Hippocampus of a Murine Model of Type I Lissencephaly

10.1101/2020.08.21.262014 ◽

2020 ◽

Author(s):

Tyler G. Ekins ◽

Vivek Mahadevan ◽

Yajun Zhang ◽

James A. D’Amour ◽

Timothy Petros ◽

...

Keyword(s):

Synapse Formation ◽

Brain Structures ◽

Cellular Migration ◽

Morphological Development ◽

Type I ◽

Hippocampal Ca1 ◽

Migration Disorder ◽

Fast Spiking ◽

And Control ◽

The Impact

ABSTRACTType I lissencephaly is a neuronal migration disorder caused by haploinsuffiency of the LIS1 gene and is characterized in humans by agyria, mislamination of brain structures, developmental delays, and epilepsy. Here, we investigate the impact of LIS1 mutation on the cellular migration, morphophysiology, microcircuitry and genomics of mouse hippocampal CA1 parvalbumin-containing inhibitory interneurons (PV+INTs). We find that WT PV+INTs consist of two physiological subtypes (80% fast-spiking (FS), 20% non-fast-spiking (NFS)) and four morphological subtypes (basket, axo-axonic, bistratified, radiatum-targeting). We also discover that cell-autonomous mutations within interneurons disrupts morphological development of PV+INTs and results in the emergence of a non-canonical “intermediate spiking (IS)” subset of PV+INTs. In the GlobalLis mutant, IS/NFS cells become the dominant PV+INT subtypes (56%) and the percentage of FS cells shrinks to 44%. We also find that IS/NFS cells are prone to entering depolarizing block, causing them to temporarily lose the ability to initiate action potentials and control network excitation, potentially promoting seizures. Finally, single-cell nuclear RNAsequencing of PV+INTs revealed several misregulated genes related to morphogenesis, cellular excitability, and synapse formation.

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New insights into the phosphorylation of the threonine motif of the β1 integrin cytoplasmic domain

Life Science Alliance ◽

10.26508/lsa.202101301 ◽

2022 ◽

Vol 5 (4) ◽

pp. e202101301

Author(s):

Ralph T Böttcher ◽

Nico Strohmeyer ◽

Jonas Aretz ◽

Reinhard Fässler

Keyword(s):

Mass Spectrometry ◽

Ligand Binding ◽

Phosphorylation Site ◽

Cell Spreading ◽

Mouse Cell ◽

Β1 Integrin ◽

Β1 Integrins ◽

Integrin Ligand ◽

Human And Mouse ◽

Major Phosphorylation Site

Integrins require an activation step before ligand binding and signaling that is mediated by talin and kindlin binding to the β integrin cytosolic domain (β-tail). Conflicting reports exist about the contribution of phosphorylation of a conserved threonine motif in the β1-tail (β1-pT788/pT789) to integrin activation. We show that widely used and commercially available antibodies against β1-pT788/pT789 integrin do not detect specific β1-pT788/pT789 integrin signals in immunoblots of several human and mouse cell lysates but bind bi-phosphorylated threonine residues in numerous proteins, which were identified by mass spectrometry experiments. Furthermore, we found that fibroblasts and epithelial cells expressing the phospho-mimicking β1-TT788/789DD integrin failed to activate β1 integrins and displayed reduced integrin ligand binding, adhesion initiation and cell spreading. These cellular defects are specifically caused by the inability of kindlin to bind β1-tail polypeptides carrying a phosphorylated threonine motif or phospho-mimicking TT788/789DD substitutions. Our findings indicate that the double-threonine motif in β1-class integrins is not a major phosphorylation site but if phosphorylated would curb integrin function.

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