scholarly journals Direct binding of the flexible C-terminal segment of periaxin to β4 integrin suggests a molecular basis for CMT4F

2019 ◽  
Author(s):  
Arne Raasakka ◽  
Helen Linxweiler ◽  
Peter J Brophy ◽  
Diane L Sherman ◽  
Petri Kursula
Keyword(s):  
Schwann Cell ◽  
Basal Lamina ◽  
Protein Complexes ◽  
Terminal Segment ◽  
Terminal Region ◽  
Membrane Receptors ◽  
Charcot Marie Tooth Disease ◽  
Cell Cytoskeleton ◽  
C Terminus ◽  
Β4 Integrin

The process of myelination in the nervous system requires coordinated formation of both transient and stable supramolecular complexes. Myelin-specific proteins play key roles in these assemblies, which may link membranes to each other or connect the myelinating cell cytoskeleton to the extracellular matrix. The myelin protein periaxin is known to play an important role in linking the Schwann cell cytoskeleton to the basal lamina through membrane receptors, such as the dystroglycan complex. Mutations that truncate periaxin from the C terminus cause demyelinating peripheral neuropathy, Charcot-Marie-Tooth disease type 4F, indicating a function for the periaxin C-terminal region in myelination. We identified the cytoplasmic domain of β4 integrin as a specific high-affinity binding partner for periaxin. The C-terminal region of periaxin remains unfolded and flexible when bound to the third fibronectin type III domain of β4 integrin. Our data suggest that periaxin is able to link the Schwann cell cytoplasm to the basal lamina through a two-pronged interaction via different membrane protein complexes, which bind close to the N and C terminus of this elongated, flexible molecule.

scholarly journals A murine model of Charcot-Marie-Tooth disease 4F reveals a role for the C-terminus of periaxin in the formation and stabilization of Cajal bands

2018 ◽  
Vol 3 ◽  
pp. 20 ◽  
Author(s):  
Diane L. Sherman ◽  
Peter J. Brophy
Keyword(s):  
Plasma Membrane ◽  
Schwann Cell ◽  
Laminin Receptor ◽  
Cell Plasma Membrane ◽  
Charcot Marie Tooth ◽  
Charcot Marie Tooth Disease ◽  
C Terminus ◽  
Cell Plasma ◽  
Inherited Neuropathies ◽  
Tooth Disease

Charcot-Marie-Tooth (CMT) disease comprises up to 80 monogenic inherited neuropathies of the peripheral nervous system (PNS) that collectively result in demyelination and axon degeneration. The majority of CMT disease is primarily either dysmyelinating or demyelinating in which mutations affect the ability of Schwann cells to either assemble or stabilize peripheral nerve myelin. CMT4F is a recessive demyelinating form of the disease caused by mutations in the Periaxin (PRX) gene. Periaxin (Prx) interacts with Dystrophin Related Protein 2 (Drp2) in an adhesion complex with the laminin receptor Dystroglycan (Dag). In mice the Prx/Drp2/Dag complex assembles adhesive domains at the interface between the abaxonal surface of the myelin sheath and the cytoplasmic surface of the Schwann cell plasma membrane. Assembly of these appositions causes the formation of cytoplasmic channels called Cajal bands beneath the surface of the Schwann cell plasma membrane. Loss of either Periaxin or Drp2 disrupts the appositions and causes CMT in both mouse and man. In a mouse model of CMT4F, complete loss of Periaxin first prevents normal Schwann cell elongation resulting in abnormally short internodal distances which can reduce nerve conduction velocity, and subsequently precipitates demyelination. Distinct functional domains responsible for Periaxin homodimerization and interaction with Drp2 to form the Prx/Drp2/Dag complex have been identified at the N-terminus of Periaxin. However, CMT4F can also be caused by a mutation that results in the truncation of Periaxin at the extreme C-terminus with the loss of 391 amino acids. By modelling this in mice, we show that loss of the C-terminus of Periaxin results in a surprising reduction in Drp2. This would be predicted to cause the observed instability of both appositions and myelin, and contribute significantly to the clinical phenotype in CMT4F.

Abstract 52: The Role of Kindlin-2 in Integrin Activation Depends on a Short C-Terminal Region

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Jamila Hirbawi ◽  
Kamila Bledzka ◽  
Yan Qing Ma ◽  
Jun Qin ◽  
Edward F Plow
Keyword(s):  
Amino Acids ◽  
Point Mutation ◽  
Single Point ◽  
Terminal Region ◽  
Membrane Receptors ◽  
Single Point Mutation ◽  
Loss Of Function ◽  
Integrin Activation ◽  
C Terminus

Integrins are heterodimeric cell membrane receptors that regulate cell adhesion, migration, and survival. The kindlins are known to be key regulators of integrin activation, the transition from a low affinity, default state to a high affinity state for ligand. This function depends on their binding, together with talin, to the cytoplasmic tails (CT) of the β subunit of integrins. Kindlins are FERM domain containing proteins, and it is its F3 (PTB) subdomain of the FERM that is the primary binding site for integrin β CT. At its very C-terminus, beyond the F3, is a short extension of 21 amino acids, K2 660-680, and we have focused on the role of this region in the co-activator function of kindlin-2 (K2). For this analysis, we performed PAC-1 (antibody to detect activated αIIbβ3 integrin) binding assays in CHO cells stably expressing integrin α IIb β 3 that were transiently transfected with talin head domain and K2 mutants. Expression levels of all proteins were verified to be similar by western blotting and FACS. Truncation of K2 at residue 660 essentially eliminated the co-activator function of K2. Deletion of smaller segments also reduced co-activator activity by 50% to 100%. Deletion of just the last two amino acids in the sequence, W 679 V 680 , resulted in a 50% reduction in co-activator activity and a single point mutation of Y 673 A also led to a 50% loss of function. A combination mutant consisting of the W 679 V 680 deletion and the Y 673 point mutation resulted in 100% loss of kindlin-2 co-activator activity. Pull-down experiments performed using GST tagged β 3 CT and CHO lysates transfected with GFP-kindlin-2 forms suggested that the C-terminal deletion did not disrupt binding to β 3 CT. This observation was corroborated by surface plasmon resonance studies in which the binding of full-length K2 and K2Δ666C (Δ666) was compared, and their K D values for immobilized β3 CT were found to be essentially the same. Overall, these data establish an important and unanticipated role of the carboxy-terminal region of kindlin-2 in its integrin co-activator function that is not dependent of its binding to integrin.

scholarly journals Adenovirus Type 5 E1A and E6 Proteins of Low-Risk Cutaneous Beta-Human Papillomaviruses Suppress Cell Transformation through Interaction with FOXK1/K2 Transcription Factors

2010 ◽  
Vol 84 (6) ◽  
pp. 2719-2731 ◽  
Author(s):  
Jessica Komorek ◽  
Mohan Kuppuswamy ◽  
T. Subramanian ◽  
S. Vijayalingam ◽  
Elena Lomonosova ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Transcription Factors ◽  
Cell Transformation ◽  
Protein Complexes ◽  
Cellular Protein ◽  
Human Papillomaviruses ◽  
Terminal Region ◽  
Oncogenic Transformation ◽  
Exon 2 ◽  
C Terminus

ABSTRACT The adenovirus (Adv) oncoprotein E1A stimulates cell proliferation and inhibits differentiation. These activities are primarily linked to the N-terminal region (exon 1) of E1A, which interacts with multiple cellular protein complexes. The C terminus (exon 2) of E1A antagonizes these processes, mediated in part through interaction with C-terminal binding proteins 1 and 2 (CtBP1/2). To identify additional cellular E1A targets that are involved in the modulation of E1A C-terminus-mediated activities, we undertook tandem affinity purification of E1A-associated proteins. Through mass spectrometric analysis, we identified several known E1A-interacting proteins as well as novel E1A targets, such as the forkhead transcription factors, FOXK1/K2. We identified a Ser/Thr-containing sequence motif in E1A that mediated interaction with FOXK1/K2. We demonstrated that the E6 proteins of two beta-human papillomaviruses (HPV14 and HPV21) associated with epidermodysplasia verruciformis also interacted with FOXK1/K2 through a motif similar to that of E1A. The E1A mutants deficient in interaction with FOXK1/K2 induced enhanced cell proliferation and oncogenic transformation. The hypertransforming activity of the mutant E1A was suppressed by HPV21 E6. An E1A-E6 chimeric protein containing the Ser/Thr domain of the E6 protein in E1A interacted efficiently with FOXK1/K2 and inhibited cell transformation. Our results suggest that targeting FOXK1/K2 may be a common mechanism for certain beta-HPVs and Adv5. E1A exon 2 mutants deficient in interaction with the dual-specificity kinases DYRK1A/1B and their cofactor HAN11 also induced increased cell proliferation and transformation. Our results suggest that the E1A C-terminal region may suppress cell proliferation and oncogenic transformation through interaction with three different cellular protein complexes: FOXK1/K2, DYRK(1A/1B)/HAN11, and CtBP1/2.

scholarly journals Distinct roles for the Charcot-Marie-Tooth disease-causing endosomal regulators Mtmr5 and Mtmr13 in axon radial sorting and Schwann cell myelination

2019 ◽  
Author(s):  
Anna E. Mammel ◽  
Katherine C. Delgado ◽  
Andrea L. Chin ◽  
Alec F. Condon ◽  
Jo Q. Hill ◽  
...  
Keyword(s):  
Schwann Cell ◽  
Protein Complexes ◽  
Endosomal Trafficking ◽  
Rab Gtpases ◽  
Charcot Marie Tooth ◽  
Charcot Marie Tooth Disease ◽  
Tooth Type ◽  
Perinatal Lethality ◽  
Radial Sorting ◽  
Redundant Functions

ABSTRACTThe form of Charcot-Marie-Tooth type 4B (CMT4B) disease caused by mutations in myotubularin-related 5 (MTMR5; also called SET Binding Factor 1; SBF1) shows a spectrum of axonal and demyelinating nerve phenotypes. This contrasts with the CMT4B subtypes caused by MTMR2 or MTMR13 (SBF2) mutations, which are characterized by myelin outfoldings and classic demyelination. Thus, it is unclear whether MTMR5 plays an analogous or distinct role from that of its homolog, MTMR13, in the peripheral nervous system (PNS). MTMR5 and MTMR13 are pseudophosphatases predicted to regulate endosomal trafficking by activating Rab GTPases and binding to the phosphoinositide 3-phosphatase MTMR2. In the mouse PNS, Mtmr2 was required to maintain wild type levels of Mtmr5 and Mtmr13, suggesting that these factors function in discrete protein complexes. Genetic elimination of both Mtmr5 and Mtmr13 in mice led to perinatal lethality, indicating that the two proteins have partially redundant functions during embryogenesis. Loss of Mtmr5 in mice did not cause CMT4B-like myelin outfoldings. However, adult Mtmr5-/- mouse nerves contained fewer myelinated axons than control nerves, likely as a result of axon radial sorting defects. Mtmr5 levels were highest during axon radial sorting, whereas Mtmr13 levels rose as myelin formed, and remained high through adulthood. Our findings suggest that Mtmr5 and Mtmr13 ensure proper axon radial sorting and Schwann cell myelination, respectively, perhaps through their direct interactions with Mtmr2. This study enhances our understanding of the non-redundant roles of the endosomal regulators MTMR5 and MTMR13 during normal peripheral nerve development and disease.

E3 ubiquitin ligases

2005 ◽  
Vol 41 ◽  
pp. 15-30 ◽  
Author(s):  
Helen C. Ardley ◽  
Philip A. Robinson
Keyword(s):  
Protein Complexes ◽  
Loss Of Function ◽  
Direct Role ◽  
Cellular Processes ◽  
Substrate Protein ◽  
Protein Ubiquitination ◽  
C Terminus ◽  
Full Complement ◽  
Spatio Temporal ◽  
Eukaryotic Organisms

The selectivity of the ubiquitin–26 S proteasome system (UPS) for a particular substrate protein relies on the interaction between a ubiquitin-conjugating enzyme (E2, of which a cell contains relatively few) and a ubiquitin–protein ligase (E3, of which there are possibly hundreds). Post-translational modifications of the protein substrate, such as phosphorylation or hydroxylation, are often required prior to its selection. In this way, the precise spatio-temporal targeting and degradation of a given substrate can be achieved. The E3s are a large, diverse group of proteins, characterized by one of several defining motifs. These include a HECT (homologous to E6-associated protein C-terminus), RING (really interesting new gene) or U-box (a modified RING motif without the full complement of Zn2+-binding ligands) domain. Whereas HECT E3s have a direct role in catalysis during ubiquitination, RING and U-box E3s facilitate protein ubiquitination. These latter two E3 types act as adaptor-like molecules. They bring an E2 and a substrate into sufficiently close proximity to promote the substrate's ubiquitination. Although many RING-type E3s, such as MDM2 (murine double minute clone 2 oncoprotein) and c-Cbl, can apparently act alone, others are found as components of much larger multi-protein complexes, such as the anaphase-promoting complex. Taken together, these multifaceted properties and interactions enable E3s to provide a powerful, and specific, mechanism for protein clearance within all cells of eukaryotic organisms. The importance of E3s is highlighted by the number of normal cellular processes they regulate, and the number of diseases associated with their loss of function or inappropriate targeting.

Affinity Tags for Protein Purification

2020 ◽  
Vol 21 (8) ◽  
pp. 821-830
Author(s):  
Vibhor Mishra
Keyword(s):  
Protein Purification ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Protein Domain ◽  
Affinity Tag ◽  
Small Peptides ◽  
Affinity Tags ◽  
C Terminus ◽  
Solubility Enhancers ◽  
As Solubility

The affinity tags are unique proteins/peptides that are attached at the N- or C-terminus of the recombinant proteins. These tags help in protein purification. Additionally, some affinity tags also serve a dual purpose as solubility enhancers for challenging protein targets. By applying a combinatorial approach, carefully chosen affinity tags designed in tandem have proven to be very successful in the purification of single proteins or multi-protein complexes. In this mini-review, the key features of the most commonly used affinity tags are discussed. The affinity tags have been classified into two significant categories, epitope tags, and protein/domain tags. The epitope tags are generally small peptides with high affinity towards a chromatography resin. The protein/domain tags often perform double duty as solubility enhancers as well as aid in affinity purification. Finally, protease-based affinity tag removal strategies after purification are discussed.

scholarly journals Mechanical force effect on the two-state equilibrium of the hyaluronan-binding domain of CD44 in cell rolling

2015 ◽  
Vol 112 (22) ◽  
pp. 6991-6996 ◽  
Author(s):  
Takashi Suzuki ◽  
Miho Suzuki ◽  
Shinji Ogino ◽  
Ryo Umemoto ◽  
Noritaka Nishida ◽  
...  
Keyword(s):  
Shear Stress ◽  
Conformational Change ◽  
Mechanical Force ◽  
Binding Domain ◽  
Terminal Region ◽  
Hematopoietic Progenitor Cell ◽  
High Shear ◽  
Cell Rolling ◽  
High Affinity ◽  
C Terminus

CD44 is the receptor for hyaluronan (HA) and mediates cell rolling under fluid shear stress. The HA-binding domain (HABD) of CD44 interconverts between a low-affinity, ordered (O) state and a high-affinity, partially disordered (PD) state, by the conformational change of the C-terminal region, which is connected to the plasma membrane. To examine the role of tensile force on CD44-mediated rolling, we used a cell-free rolling system, in which recombinant HABDs were attached to beads through a C-terminal or N-terminal tag. We found that the rolling behavior was stabilized only at high shear stress, when the HABD was attached through the C-terminal tag. In contrast, no difference was observed for the beads coated with HABD mutants that constitutively adopt either the O state or the PD state. Steered molecular dynamics simulations suggested that the force from the C terminus disrupts the interaction between the C-terminal region and the core of the domain, thus providing structural insights into how the mechanical force triggers the allosteric O-to-PD transition. Based on these results, we propose that the force applied from the C terminus enhances the HABD–HA interactions by inducing the conformational change to the high-affinity PD transition more rapidly, thereby enabling CD44 to mediate lymphocyte trafficking and hematopoietic progenitor cell homing under high-shear conditions.

scholarly journals Interaction of the Aspergillus nidulans Microtubule-Organizing Center (MTOC) Component ApsB with Gamma-Tubulin and Evidence for a Role of a Subclass of Peroxisomes in the Formation of Septal MTOCs

2010 ◽  
Vol 9 (5) ◽  
pp. 795-805 ◽  
Author(s):  
Nadine Zekert ◽  
Daniel Veith ◽  
Reinhard Fischer
Keyword(s):  
Aspergillus Nidulans ◽  
Protein Complexes ◽  
Spindle Pole ◽  
Eukaryotic Cells ◽  
Microtubule Organizing Center ◽  
Body Protein ◽  
Content Type ◽  
C Terminus ◽  
Peroxisomal Targeting ◽  
Gamma Tubulin

ABSTRACT Peroxisomes are a diverse class of organelles involved in different physiological processes in eukaryotic cells. Although proteins imported into peroxisomes carry a peroxisomal targeting sequence at the C terminus (PTS1) or an alternative one close to the N terminus (PTS2), the protein content of peroxisomes varies drastically. Here we suggest a new class of peroxisomes involved in microtubule (MT) formation. Eukaryotic cells assemble MTs from distinct points in the cell. In the fungus Aspergillus nidulans, septum-associated microtubule-organizing centers (sMTOCs) are very active in addition to the spindle pole bodies (SPBs). Previously, we identified a novel MTOC-associated protein, ApsB (Schizosaccharomyces pombe mto1), whose absence affected MT formation from sMTOCs more than from SPBs, suggesting that the two protein complexes are organized differently. We show here that sMTOCs share at least two further components, gamma-tubulin and GcpC (S. pombe Alp6) with SPBs and found that ApsB interacts with gamma-tubulin. In addition, we discovered that ApsB interacts with the Woronin body protein HexA and is targeted to a subclass of peroxisomes via a PTS2 peroxisomal targeting sequence. The PTS2 motif was necessary for function but could be replaced with a PTS1 motif at the C terminus of ApsB. These results suggest a novel function for a subclass of peroxisomes in cytoskeletal organization.

Limitations of Allotopic Expression of Mitochondrial Genes in Mammalian Cells

Genetics ◽  
2003 ◽  
Vol 165 (2) ◽  
pp. 707-720
Author(s):  
Jose Oca-Cossio ◽  
Lesley Kenyon ◽  
Huiling Hao ◽  
Carlos T Moraes
Keyword(s):  
Mitochondrial Dna ◽  
Mammalian Cells ◽  
Mitochondrial Genes ◽  
Terminal Segment ◽  
Universal Genetic Code ◽  
Apocytochrome B ◽  
Dna Mutations ◽  
C Terminus ◽  
Cytoplasmic Compartment ◽  
Allotopic Expression

Abstract The possibility of expressing mitochondrial DNA-coded genes in the nuclear-cytoplasmic compartment provides an attractive system for genetic treatment of mitochondrial disorders associated with mitochondrial DNA mutations. In theory, by recoding mitochondrial genes to adapt them to the universal genetic code and by adding a DNA sequence coding for a mitochondrial-targeting sequence, one could achieve correct localization of the gene product. Such transfer has occurred in nature, and certain species of algae and plants express a number of polypeptides that are commonly coded by mtDNA in the nuclear-cytoplasmic compartment. In the present study, allotopic expression of three different mtDNA-coded polypeptides (ATPase8, apocytochrome b, and ND4) into COS-7 and HeLa cells was analyzed. Among these, only ATPase8 was correctly expressed and localized to mitochondria. The full-length, as well as truncated forms, of apocytochrome b and ND4 decorated the periphery of mitochondria, but also aggregated in fiber-like structures containing tubulin and in some cases also vimentin. The addition of a hydrophilic tail (EGFP) to the C terminus of these polypeptides did not change their localization. Overexpression of molecular chaperones also did not have a significant effect in preventing aggregations. Allotopic expression of apocytochrome b and ND4 induced a loss of mitochondrial membrane potential in transfected cells, which can lead to cell death. Our observations suggest that only a subset of mitochondrial genes can be replaced allotopically. Analyses of the hydrophobic patterns of different polypeptides suggest that hydrophobicity of the N-terminal segment is the main determinant for the importability of peptides into mammalian mitochondria.

scholarly journals The C Terminus of the Ribosomal-Associated Protein LrtA is an Intrinsically Disordered Oligomer

2018 ◽  
Vol 19 (12) ◽  
pp. 3902 ◽  
Author(s):  
José L. Neira ◽  
A. Marcela Giudici ◽  
Felipe Hornos ◽  
Arantxa Arbe ◽  
Bruno Rizzuti
Keyword(s):  
Computational Modelling ◽  
Terminal Region ◽  
Physiological Conditions ◽  
Intrinsically Disordered ◽  
Disordered Protein ◽  
C Terminus ◽  
Conformational Preferences ◽  
Self Association ◽  
Association Equilibrium ◽  
Post Stress

The 191-residue-long LrtA protein of Synechocystis sp. PCC 6803 is involved in post-stress survival and in stabilizing 70S ribosomal particles. It belongs to the hibernating promoting factor (HPF) family, intervening in protein synthesis. The protein consists of two domains: The N-terminal region (N-LrtA, residues 1101), which is common to all the members of the HPF, and seems to be well-folded; and the C-terminal region (C-LrtA, residues 102-191), which is hypothesized to be disordered. In this work, we studied the conformational preferences of isolated C-LrtA in solution. The protein was disordered, as shown by computational modelling, 1D-1H NMR, steady-state far- UV circular dichroism (CD) and chemical and thermal denaturations followed by fluorescence and far-UV CD. Moreover, at physiological conditions, as indicated by several biochemical and hydrodynamic techniques, isolated C-LrtA intervened in a self-association equilibrium, involving several oligomerization reactions. Thus, C-LrtA was an oligomeric disordered protein.

Sign in / Sign up

Export Citation Format

Share Document