scholarly journals The carboxy terminal coiled-coil modulates Orai1 internalization during meiosis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Rawad Hodeify ◽  
Maya Dib ◽  
Ethel Alcantara-Adap ◽  
Raphael Courjaret ◽  
Nancy Nader ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Coiled Coil ◽  
Structural Motif ◽  
Deletion Mutants ◽  
Binding Motif ◽  
Terminal Sequence ◽  
Molecular Determinant ◽  
C Terminus ◽  
The Stability ◽  
Carboxy Terminal

AbstractRegulation of Ca2+ signaling is critical for the progression of cell division, especially during meiosis to prepare the egg for fertilization. The primary Ca2+ influx pathway in oocytes is Store-Operated Ca2+ Entry (SOCE). SOCE is tightly regulated during meiosis, including internalization of the SOCE channel, Orai1. Orai1 is a four-pass membrane protein with cytosolic N- and C-termini. Orai1 internalization requires a caveolin binding motif (CBM) in the N-terminus as well as the C-terminal cytosolic domain. However, the molecular determinant for Orai1 endocytosis in the C-terminus are not known. Here we show that the Orai1 C-terminus modulates Orai1 endocytosis during meiosis through a structural motif that is based on the strength of the C-terminal intersubunit coiled coil (CC) domains. Deletion mutants show that a minimal C-terminal sequence after transmembrane domain 4 (residues 260–275) supports Orai1 internalization. We refer to this region as the C-terminus Internalization Handle (CIH). Access to CIH however is dependent on the strength of the intersubunit CC. Mutants that increase the stability of the coiled coil prevent internalization independent of specific mutation. We further used human and Xenopus Orai isoforms with different propensity to form C-terminal CC and show a strong correlation between the strength of the CC and Orai internalization. Furthermore, Orai1 internalization does not depend on clathrin, flotillin or PIP2. Collectively these results argue that Orai1 internalization requires both the N-terminal CBM and C-terminal CIH where access to CIH is controlled by the strength of intersubunit C-terminal CC.

scholarly journals Engineering of Adenovirus Vectors Containing Heterologous Peptide Sequences in the C Terminus of Capsid Protein IX

2002 ◽  
Vol 76 (14) ◽  
pp. 6893-6899 ◽  
Author(s):  
Igor P. Dmitriev ◽  
Elena A. Kashentseva ◽  
David T. Curiel
Keyword(s):  
Heparan Sulfate ◽  
Capsid Protein ◽  
Minor Component ◽  
Cell Types ◽  
Binding Motif ◽  
Flag Epitope ◽  
C Terminus ◽  
Protein Ix ◽  
A Minor ◽  
Carboxy Terminal

ABSTRACT The utility of the present generation of adenovirus (Ad) vectors for gene therapy applications could be improved by restricting native viral tropism to selected cell types. In order to achieve modification of Ad tropism, we proposed to exploit a minor component of viral capsid, protein IX (pIX), for genetic incorporation of targeting ligands. Based on the proposed structure of pIX, we hypothesized that its C terminus could be used as a site for incorporation of heterologous peptide sequences. We engineered recombinant Ad vectors containing modified pIX carrying a carboxy-terminal Flag epitope along with a heparan sulfate binding motif consisting of either eight consecutive lysines or a polylysine sequence. Using an anti-Flag antibody, we have shown that modified pIXs are incorporated into virions and display Flag-containing C-terminal sequences on the capsid surface. In addition, both lysine octapeptide and polylysine ligands were accessible for binding to heparin-coated beads. In contrast to virus bearing lysine octapeptide, Ad vector displaying a polylysine was capable of recognizing cellular heparan sulfate receptors. We have demonstrated that incorporation of a polylysine motif into the pIX ectodomain results in a significant augmentation of Ad fiber knob-independent infection of CAR-deficient cell types. Our data suggest that the pIX ectodomain can serve as an alternative to the fiber knob, penton base, and hexon proteins for incorporation of targeting ligands for the purpose of Ad tropism modification.

‘Shed’ furin: mapping of the cleavage determinants and identification of its C-terminus

2001 ◽  
Vol 354 (3) ◽  
pp. 689-695 ◽  
Author(s):  
Barbara PLAIMAUER ◽  
Gabriele MOHR ◽  
Waltraud WERNHART ◽  
Michèle HIMMELSPACH ◽  
Friedrich DORNER ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Deletion Mutants ◽  
Amino Acid Substitutions ◽  
Individual Amino Acid ◽  
Internal Deletion ◽  
Bioactive Proteins ◽  
C Terminus ◽  
Golgi System ◽  
Prime Determinant ◽  
Precursor Molecules

The human endoprotease furin is involved in the proteolytic maturation of the precursor molecules of a wide variety of bioactive proteins. Despite its localization in the membranes of the trans-Golgi system by means of a transmembrane domain, it has repeatedly been reported to form a C-terminally truncated, naturally secreted form referred to as ‘shed’ furin. In order to identify the cleavage site, internal deletion mutants of increasing size, N-terminal to Leu708, and subsequently individual amino acid substitutions were introduced, and Arg683 was identified as the prime determinant for shedding. MS analysis determined Ser682 as the C-terminus of shed furin, suggesting that monobasic cleavage may occur N-terminal to Arg683. Alteration of Arg683 directs the shedding mechanism to alternative cleaving sites previously unused.

scholarly journals The Dense-Core Vesicle Maturation Protein CCCP-1 Binds RAB-2 and Membranes through its C-terminal Domain

2017 ◽  
Author(s):  
Jérôme Cattin-Ortolá ◽  
Irini Topalidou ◽  
Annie Dosey ◽  
Alexey J. Merz ◽  
Michael Ailion
Keyword(s):  
Structure Function ◽  
Function Analysis ◽  
Coiled Coil ◽  
Lipid Binding ◽  
Small Gtpase ◽  
Dense Core ◽  
Binding Motif ◽  
Dense Core Vesicle ◽  
Terminal Domain ◽  
C Terminus

AbstractDense-core vesicles (DCVs) are secretory organelles that store and release modulatory neurotransmitters from neurons and endocrine cells. Recently, the conserved coiled-coil protein CCCP-1 was identified as a component of the DCV biogenesis pathway in the nematode C. elegans. CCCP-1 binds the small GTPase RAB-2 and colocalizes with it at the trans-Golgi. Here we report a structure-function analysis of CCCP-1 to identify domains of the protein important for its localization, binding to RAB-2, and function in DCV biogenesis. We find that the CCCP-1 C-terminal domain (CC3) has multiple activities. CC3 is necessary and sufficient for CCCP-1 localization and for binding to RAB-2, and is required for the function of CCCP-1 in DCV biogenesis. Additionally, CCCP-1 binds membranes directly through its CC3 domain, indicating that CC3 may comprise a previously uncharacterized lipid-binding motif. We conclude that CCCP-1 is a coiled-coil protein that binds an activated Rab and localizes to the Golgi via its C-terminus, properties similar to members of the golgin family of proteins. CCCP-1 also shares biophysical features with golgins; it has an elongated shape and forms oligomers.Synopsis statementCCCP-1 is a coiled-coil protein important for dense-core vesicle (DCV) biogenesis. A structure-function analysis of CCCP-1 shows that its C-terminal domain is required for (1) localization to membrane compartments near the trans-Golgi, (2) binding to activated RAB-2, (3) function in DCV biogenesis, and (4) direct binding to membranes. CCCP-1 has an elongated shape and forms oligomers. These findings suggest that CCCP-1 resembles members of the golgin family of proteins that act as membrane tethers.

scholarly journals The Vaccinia Virus 14-Kilodalton (A27L) Fusion Protein Forms a Triple Coiled-Coil Structure and Interacts with the 21-Kilodalton (A17L) Virus Membrane Protein through a C-Terminal α-Helix

1998 ◽  
Vol 72 (12) ◽  
pp. 10126-10137 ◽  
Author(s):  
María-Isabel Vázquez ◽  
German Rivas ◽  
David Cregut ◽  
Luis Serrano ◽  
Mariano Esteban
Keyword(s):  
Amino Acids ◽  
Vaccinia Virus ◽  
Structural Model ◽  
Analytical Ultracentrifugation ◽  
Transmembrane Domain ◽  
Coiled Coil ◽  
C Terminus ◽  
Α Helix ◽  
Mutant Forms ◽  
Helical Region

ABSTRACT The vaccinia virus 14-kDa protein (encoded by the A27L gene) plays an important role in the biology of the virus, acting in virus-to-cell and cell-to-cell fusions. The protein is located on the surface of the intracellular mature virus form and is essential for both the release of extracellular enveloped virus from the cells and virus spread. Sequence analysis predicts the existence of four regions in this protein: a structureless region from amino acids 1 to 28, a helical region from residues 29 to 37, a triple coiled-coil helical region from residues 44 to 72, and a Leu zipper motif at the C terminus. Circular dichroism spectroscopy, analytical ultracentrifugation, and chemical cross-linking studies of the purified wild-type protein and several mutant forms, lacking one or more of the above regions or with point mutations, support the above-described structural division of the 14-kDa protein. The two contiguous cysteine residues at positions 71 and 72 are not responsible for the formation of 14-kDa protein trimers. The location of hydrophobic residues at the a and d positions on a helical wheel and of charged amino acids in adjacent positions, e and g, suggests that the hydrophobic and ionic interactions in the triple coiled-coil helical region are involved in oligomer formation. This conjecture was supported by the construction of a three-helix bundle model and molecular dynamics. Binding assays with purified proteins expressed in Escherichia coli and cytoplasmic extracts from cells infected with a virus that does not produce the 14-kDa protein during infection (VVindA27L) show that the 21-kDa protein (encoded by the A17L gene) is the specific viral binding partner and identify the putative Leu zipper, the predicted third α-helix on the C terminus of the 14-kDa protein, as the region involved in protein binding. These findings were confirmed in vivo, following transfection of animal cells with plasmid vectors expressing mutant forms of the 14-kDa protein and infected with VVindA27L. We find the structural organization of 14kDa to be similar to that of other fusion proteins, such as hemagglutinin of influenza virus and gp41 of human immunodeficiency virus, except for the presence of a protein-anchoring domain instead of a transmembrane domain. Based on our observations, we have established a structural model of the 14-kDa protein.

The Stability of Wild-type and Deletion Mutants of Human C-terminus Hsp70-interacting Protein (CHIP)

2013 ◽  
Vol 20 (5) ◽  
pp. 524-529 ◽  
Author(s):  
Isabel C.R. Millan ◽  
Ana L.A. Squillace ◽  
Lisandra M. Gava ◽  
Carlos H.I. Ramos
Keyword(s):  
Protein Chip ◽  
Deletion Mutants ◽  
Wild Type ◽  
Interacting Protein ◽  
C Terminus ◽  
The Stability

scholarly journals The proximal C-terminus of α1C subunits is necessary for junctional membrane targeting of cardiac L-type calcium channels

2012 ◽  
Vol 448 (2) ◽  
pp. 221-231 ◽  
Author(s):  
Tsutomu Nakada ◽  
Bernhard E. Flucher ◽  
Toshihide Kashihara ◽  
Xiaona Sheng ◽  
Toshihide Shibazaki ◽  
...  
Keyword(s):  
Calcium Channels ◽  
Action Potential ◽  
Fluorescent Protein ◽  
Ryanodine Receptors ◽  
Structural Motif ◽  
Deletion Mutants ◽  
C Terminus ◽  
Specific Localization ◽  
Α Helix ◽  
Green Fluorescent

In cardiac myocytes, LTCCs (L-type calcium channels) form a functional signalling complex with ryanodine receptors at the JM (junctional membrane). Although the specific localization of LTCCs to the JM is critical for excitation–contraction coupling, their targeting mechanism is unclear. Transient transfection of GFP (green fluorescent protein)–α1S or GFP–α1C, but not P/Q-type calcium channel α1A, in dysgenic (α1S-null) GLT myotubes results in correct targeting of these LTCCs to the JMs and restoration of action-potential-induced Ca2+ transients. To identify the sequences of α1C responsible for JM targeting, we generated a range of α1C–α1A chimaeras, deletion mutants and alanine substitution mutants and studied their targeting properties in GLT myotubes. The results revealed that amino acids L1681QAGLRTL1688 and P1693EIRRAIS1700, predicted to form two adjacent α-helices in the proximal C-terminus, are necessary for the JM targeting of α1C. The efficiency of restoration of action-potential-induced Ca2+ transients in GLT myotubes was significantly decreased by mutations in the targeting motif. JM targeting was not disrupted by the distal C-terminus of α1C which binds to the second α-helix. Therefore we have identified a new structural motif in the C-terminus of α1C that mediates the targeting of cardiac LTCCs to JMs independently of the interaction between proximal and distal C-termini of α1C.

scholarly journals Structure of a truncated form of leucine zipper II of JIP3 reveals an unexpected antiparallel coiled-coil arrangement

2016 ◽  
Vol 72 (3) ◽  
pp. 198-206 ◽  
Author(s):  
Paola Llinas ◽  
Mélanie Chenon ◽  
T. Quyen Nguyen ◽  
Catia Moreira ◽  
Annélie de Régibus ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Molecular Motors ◽  
Map Kinases ◽  
Leucine Zipper ◽  
Coiled Coil ◽  
Structural Rearrangement ◽  
Structural Motif ◽  
Scaffolding Proteins ◽  
Truncated Form ◽  
The Stability

JIP3 and JIP4, two highly related scaffolding proteins for MAP kinases, are binding partners for two molecular motors as well as for the small G protein ARF6. The leucine zipper II (LZII) region of JIP3/4 is the binding site for these three partners. Previously, the crystal structure of ARF6 bound to JIP4 revealed LZII in a parallel coiled-coil arrangement. Here, the crystal structure of an N-terminally truncated form of LZII of JIP3 alone shows an unexpected antiparallel arrangement. Using molecular dynamics and modelling, the stability of this antiparallel LZII arrangement, as well as its specificity for ARF6, were investigated. This study highlights that N-terminal truncation of LZII can change its coiled-coil orientation without affecting its overall stability. Further, a conserved buried asparagine residue was pinpointed as a possible structural determinant for this dramatic structural rearrangement. Thus, LZII of JIP3/4 is a versatile structural motif, modifications of which can impact partner recognition and thus biological function.

scholarly journals TRPM Channels in Human Diseases

Cells ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 2604
Author(s):  
Ivanka Jimenez ◽  
Yolanda Prado ◽  
Felipe Marchant ◽  
Carolina Otero ◽  
Felipe Eltit ◽  
...  
Keyword(s):  
Transient Receptor Potential ◽  
Transmembrane Domain ◽  
Current Knowledge ◽  
Coiled Coil ◽  
Receptor Potential ◽  
Point Of View ◽  
Human Diseases ◽  
Diagnostic Strategies ◽  
Transient Receptor Potential Melastatin ◽  
C Terminus

The transient receptor potential melastatin (TRPM) subfamily belongs to the TRP cation channels family. Since the first cloning of TRPM1 in 1989, tremendous progress has been made in identifying novel members of the TRPM subfamily and their functions. The TRPM subfamily is composed of eight members consisting of four six-transmembrane domain subunits, resulting in homomeric or heteromeric channels. From a structural point of view, based on the homology sequence of the coiled-coil in the C-terminus, the eight TRPM members are clustered into four groups: TRPM1/M3, M2/M8, M4/M5 and M6/M7. TRPM subfamily members have been involved in several physiological functions. However, they are also linked to diverse pathophysiological human processes. Alterations in the expression and function of TRPM subfamily ion channels might generate several human diseases including cardiovascular and neurodegenerative alterations, organ dysfunction, cancer and many other channelopathies. These effects position them as remarkable putative targets for novel diagnostic strategies, drug design and therapeutic approaches. Here, we review the current knowledge about the main characteristics of all members of the TRPM family, focusing on their actions in human diseases.

scholarly journals Functional Analysis of Carboxy-Terminal Deletion Mutants of c-Myb

1999 ◽  
Vol 73 (7) ◽  
pp. 5875-5886 ◽  
Author(s):  
Duen-Mei Wang ◽  
John W. Dubendorff ◽  
Colleen H. Woo ◽  
Joseph S. Lipsick
Keyword(s):  
Reporter Gene ◽  
Transcriptional Activation ◽  
Terminal Deletion ◽  
Deletion Mutants ◽  
Transactivation Domain ◽  
Proliferating Cells ◽  
Native Form ◽  
C Terminus ◽  
Differentiation And Proliferation ◽  
Carboxy Terminal

ABSTRACT The c-myb gene is implicated in the differentiation and proliferation of hematopoietic cells. Truncations of the N and/or C terminus of c-Myb, found in v-Myb, can potentiate its transforming ability. Two negative regulatory subregions, located in the C terminus, were mapped previously by using GAL4–c-Myb fusion proteins in transient transfection assays for the transcriptional activation of a GAL4-responsive reporter gene. To dissect the C terminus of c-Myb in terms of its involvement in transcriptional activation and oncogenic transformation, a series of C-terminal deletion mutants of c-Myb were analyzed. In addition, linker insertion mutants within the transactivation domain and/or heptad leucine repeat of c-Myb were examined along with those deletion mutants. In this study, we demonstrated that the removal of both of the two previously mapped negative regulatory subregions from the native form of c-Myb not only supertransactivates a Myb-responsive reporter gene but also potentiates its transforming ability in culture. However, in contrast to previous results, cells transformed by all of the mutants analyzed here except v-Myb itself exhibited the same phenotype as those transformed by c-Myb. The proliferating cells were bipotenial and differentiated into both the granulocytic and monocytic lineages. This result implies that the C terminus of c-Myb alone has no effect on the lineage determination. Finally, the transactivation activities of these mutants correlated with their transforming activities when a mim-1reporter gene was used but not when a model promoter containing five tandem Myb-binding sites was used. In particular, a very weakly transforming mutant with a linker insertion in the heptad leucine repeat superactivated the model promoter but not the mim-1reporter gene.

scholarly journals Properties of the desmin tail domain: studies using synthetic peptides and antipeptide antibodies.

1990 ◽  
Vol 111 (5) ◽  
pp. 2063-2075 ◽  
Author(s):  
L Birkenberger ◽  
W Ip
Keyword(s):  
Polyclonal Antibodies ◽  
Coiled Coil ◽  
Ultrastructural Level ◽  
Structural Motif ◽  
Label Free ◽  
Tail Domain ◽  
Amino Terminal ◽  
Filament Structure ◽  
Carboxy Terminal ◽  
High Degree

Intermediate filament (IF) proteins have a common structural motif consisting of an alpha-helical rod domain flanked by non-alpha-helical amino-terminal head and carboxy-terminal tail domains. Coiled-coil interaction between neighboring rod domains is though to generate the backbone of the 10-nm filament. There must also be other interactions between subunits to bring them into alignment and to effect elongation of the filament, but these are poorly understood. To examine the involvement of the tail domain in filament structure and stabilization, we have studied the interaction between a synthetic peptide corresponding to residues 442-450 of avian desmin, and authentic desmin protein. The potential importance of this region lies in its hydrophilic nature and its high degree of homology among the Type III IF proteins and cytokeratins 8 and 18. The peptide, D442-450, binds to a 27-residue region between lys-436 and leu-463, the carboxy terminus. The presence of the peptide during assembly causes the filaments to appear much more loosely packed than normal desmin IF. We have also generated polyclonal antibodies against this peptide and attempted to localize this portion of the tailpiece along desmin IFs by immunological procedures. By immunoblotting, we found that anti-D442-450 antibodies recognize desmin and only those proteolytic fragments that contain the tailpiece. In contrast, the antibodies do not label any structure in adult gizzard smooth muscle and skeletal muscle myofibrils in immunofluorescence experiments during which conventional antidesmin antibodies do. At the ultrastructural level, anti-D442-450 antibodies label free desmin tetramers but not desmin IFs. These results show that, as part of an assembled IF, the epitope of anti-D442-450 is inaccessible to the antibodies, and suggest that either the tailpiece of an IF protein may not be entirely peripheral to the filament backbone, or the interaction between end domains during assembly masks this particular region of the IF molecule.

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