scholarly journals Degradation of gap junction connexins is regulated by the interaction with Cx43-interacting protein of 75 kDa (CIP75)

2015 ◽  
Vol 466 (3) ◽  
pp. 571-585 ◽  
Author(s):  
Jennifer L. Kopanic ◽  
Barbara Schlingmann ◽  
Michael Koval ◽  
Alan F. Lau ◽  
Paul L. Sorgen ◽  
...  
Keyword(s):  
Gap Junction ◽  
Direct Interaction ◽  
Proteasomal Degradation ◽  
Degradation Mechanisms ◽  
Lysosomal Degradation ◽  
Interacting Protein ◽  
Gap Junction Channels ◽  
Uba Domain ◽  
Carboxyl Terminal ◽  
Connexin Proteins

Connexins are a family of transmembrane proteins that form gap junction channels. These proteins undergo both proteasomal and lysosomal degradation, mechanisms that serve to regulate connexin levels. Our previous work described CIP75 [connexin43 (Cx43)-interacting protein of 75 kDa], a protein involved in proteasomal degradation, as a novel Cx43-interacting protein. We have discovered two additional connexins, connexin40 (Cx40) and connexin45 (Cx45), that interact with CIP75. Nuclear magnetic resonance (NMR) analyses identified the direct interaction of the CIP75 UBA domain with the carboxyl-terminal (CT) domains of Cx40 and Cx45. Reduction in CIP75 by shRNA in HeLa cells expressing Cx40 or Cx45 resulted in increased levels of the connexins. Furthermore, treatment with trafficking inhibitors confirmed that both connexins undergo endoplasmic reticulum-associated degradation (ERAD), and that CIP75 preferentially interacts with the connexin proteins bound for proteasomal degradation from the ER. In addition, we have also discovered that CIP75 interacts with ER-localized Cx32 in a process that is likely mediated by Cx32 ubiquitination. Thus, we have identified novel interacting connexin proteins of CIP75, indicating a role for CIP75 in regulating the levels of connexins in general, through proteasomal degradation.

scholarly journals The Ubiquitin-associated Domain of hPLIC-2 Interacts with the Proteasome

2003 ◽  
Vol 14 (9) ◽  
pp. 3868-3875 ◽  
Author(s):  
Maurits F. Kleijnen ◽  
Rodolfo M. Alarcón ◽  
Peter M. Howley
Keyword(s):  
Proteasomal Degradation ◽  
Binding Domain ◽  
Full Length ◽  
Degradation Pathways ◽  
Wild Type ◽  
Amino Terminal ◽  
Uba Domain ◽  
Carboxyl Terminal ◽  
Protein Ligases

The ubiquitin-like hPLIC proteins can associate with proteasomes, and hPLIC overexpression can specifically interfere with ubiquitin-mediated proteolysis ( Kleijnen et al., 2000 ). Because the hPLIC proteins can also interact with certain E3 ubiquitin protein ligases, they may provide a link between the ubiquitination and proteasomal degradation machineries. The amino-terminal ubiquitin-like (ubl) domain is a proteasome-binding domain. Herein, we report that there is a second proteasome-binding domain in hPLIC-2: the carboxyl-terminal ubiquitin-associated (uba) domain. Coimmunoprecipitation experiments of wild-type and mutant hPLIC proteins revealed that the ubl and uba domains each contribute independently to hPLIC-2–proteasome binding. There is specificity for the interaction of the hPLIC-2 uba domain with proteasomes, because uba domains from several other proteins failed to bind proteasomes. Furthermore, the binding of uba domains to polyubiquitinated proteins does not seem to be sufficient for the proteasome binding. Finally, the uba domain is necessary for the ability of full-length hPLIC-2 to interfere with the ubiquitin-mediated proteolysis of p53. The PLIC uba domain has been reported to bind and affect the functions of proteins such as GABAAreceptor and presenilins. It is possible that the function of these proteins may be regulated or mediated through proteasomal degradation pathways.

CIP75 (connexin43-interacting protein of 75 kDa) mediates the endoplasmic reticulum dislocation of connexin43

2014 ◽  
Vol 458 (1) ◽  
pp. 57-67 ◽  
Author(s):  
Vivian Su ◽  
Christina Hoang ◽  
Dirk Geerts ◽  
Alan F. Lau
Keyword(s):  
Endoplasmic Reticulum ◽  
Gap Junction ◽  
Proteasomal Degradation ◽  
Endoplasmic Reticulum Membrane ◽  
Interacting Protein ◽  
Junction Protein ◽  
Gap Junction Protein

The proteasomal degradation of the gap junction protein connexin43 is facilitated by its interaction with the connexin43-interacting protein of 75 kDa at the endoplasmic reticulum membrane.

scholarly journals The Carboxyl Terminal Domain Regulates the Unitary Conductance and Voltage Dependence of Connexin40 Gap Junction Channels

2001 ◽  
Vol 88 (7) ◽  
pp. 666-673 ◽  
Author(s):  
Justus M. B. Anumonwo ◽  
Steven M. Taffet ◽  
Hong Gu ◽  
Marc Chanson ◽  
Alonso P. Moreno ◽  
...  
Keyword(s):  
Gap Junction ◽  
Voltage Dependence ◽  
Gap Junction Channels ◽  
Terminal Domain ◽  
Unitary Conductance ◽  
Carboxyl Terminal Domain ◽  
Carboxyl Terminal

scholarly journals Transfected connexin45 alters gap junction permeability in cells expressing endogenous connexin43.

1995 ◽  
Vol 130 (4) ◽  
pp. 987-995 ◽  
Author(s):  
M Koval ◽  
S T Geist ◽  
E M Westphale ◽  
A E Kemendy ◽  
R Civitelli ◽  
...  
Keyword(s):  
Gap Junction ◽  
Lucifer Yellow ◽  
Direct Interaction ◽  
Full Length ◽  
Osteosarcoma Cell ◽  
Dye Transfer ◽  
Fluorescent Reporter ◽  
Intercellular Transfer ◽  
Carboxyl Terminal ◽  
Gap Junctional

Many cells express multiple connexins, the gap junction proteins that interconnect the cytosol of adjacent cells. Connexin43 (Cx43) channels allow intercellular transfer of Lucifer Yellow (LY, MW = 443 D), while connexin45 (Cx45) channels do not. We transfected full-length or truncated chicken Cx45 into a rat osteosarcoma cell line ROS-17/2.8, which expresses endogenous Cx43. Both forms of Cx45 were expressed at high levels and colocalized with Cx43 at plasma membrane junctions. Cells transfected with full-length Cx45 (ROS/Cx45) and cells transfected with Cx45 missing the 37 carboxyl-terminal amino acids (ROS/Cx45tr) showed 30-60% of the gap junctional conductance exhibited by ROS cells. Intercellular transfer of three negatively charged fluorescent reporter molecules was examined. In ROS cells, microinjected LY was transferred to an average of 11.2 cells/injected cell, while dye transfer between ROS/Cx45 cells was reduced to 3.9 transfer between ROS/Cx45 cells was reduced to 3.9 cells. In contrast, ROS/Cx45tr cells transferred LY to > 20 cells. Transfer of calcein (MW = 623 D) was also reduced by approximately 50% in ROS/Cx45 cells, but passage of hydroxycoumarin carboxylic acid (HCCA; MW = 206 D) was only reduced by 35% as compared to ROS cells. Thus, introduction of Cx45 altered intercellular coupling between cells expressing Cx43, most likely the result of direct interaction between Cx43 and Cx45. Transfection of Cx45tr and Cx45 had different effects in ROS cells, consistent with a role of the carboxyl-terminal domain of Cx45 in determining gap junction permeability or interactions between connexins. These data suggest that coexpression of multiple connexins may enable cells to achieve forms of intercellular communication that cannot be attained by expression of a single connexin.

The origin of annular junctions: a mechanism of gap junction internalization

2001 ◽  
Vol 114 (4) ◽  
pp. 763-773 ◽  
Author(s):  
K. Jordan ◽  
R. Chodock ◽  
A.R. Hand ◽  
D.W. Laird
Keyword(s):  
Cell Surface ◽  
Gap Junction ◽  
Fluorescent Protein ◽  
Rat Kidney ◽  
Time Lapse ◽  
Gap Junctional Intercellular Communication ◽  
Gap Junction Channels ◽  
Normal Rat ◽  
Connexin Proteins ◽  
Green Fluorescent

Gap junctional intercellular communication is established when connexin proteins oligomerize into connexon hemichannels, which then pair at the cell surface with connexons from neighboring cells to form functional gap junction channels. Gap junction channels routinely cluster into gap junction plaques, which can exhibit dynamic characteristics while under the frequent processes of formation and removal from the cell surface. We have three lines of evidence to suggest that one mechanism of gap junction removal occurs when one of two contacting cells internalizes the gap junction contribution from both cells. First, in coculture experiments, green fluorescent protein-tagged connexin43 (Cx43-GFP) expressed in normal rat kidney (NRK) cells can be internalized into contacting cells that do not express Cx43-GFP, and the incidences of identifying these internalized structures increase in the presence of lysosomal inhibitors. Secondly, time-lapse imaging of live NRK cells revealed that large areas of gap junction plaques containing Cx43-GFP were internalized as vesicular-like structures into one of two adjacent cells. Finally, when live NRK cells that express endogenous Cx43 were microinjected with anti-Cx43 antibodies, antibody-tagged gap junctions were visualized in cells that contacted the microinjected cell within 3–6.5 hours. Together our results strongly suggest that one mechanism of gap junction removal from the cell surface involves a unique process in which the entire gap junction or a fragment of it is internalized into one of the two contacting cells as an annular junction.

scholarly journals Tick Importin-α Is Implicated in the Interactome and Regulome of the Cofactor Subolesin

Pathogens ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 457
Author(s):  
Sara Artigas-Jerónimo ◽  
Margarita Villar ◽  
Alejandro Cabezas-Cruz ◽  
Grégory Caignard ◽  
Damien Vitour ◽  
...  
Keyword(s):  
Rational Design ◽  
Fat Body ◽  
Animal Health ◽  
Direct Interaction ◽  
Vaccine Antigen ◽  
Tick Feeding ◽  
Interacting Protein ◽  
Expression Levels ◽  
Importin Α

Ticks and tick-borne diseases (TBDs) represent a burden for human and animal health worldwide. Currently, vaccines constitute the safest and most effective approach to control ticks and TBDs. Subolesin (SUB) has been identified as a vaccine antigen for the control of tick infestations and pathogen infection and transmission. The characterization of the molecular function of SUB and the identification of tick proteins interacting with SUB may provide the basis for the discovery of novel antigens and for the rational design of novel anti-tick vaccines. In the present study, we used the yeast two-hybrid system (Y2H) as an unbiased approach to identify tick SUB-interacting proteins in an Ixodes ricinus cDNA library, and studied the possible role of SUB as a chromatin remodeler through direct interaction with histones. The Y2H screening identified Importin-α as a potential SUB-interacting protein, which was confirmed in vitro in a protein pull-down assay. The sub gene expression levels in tick midgut and fat body were significantly higher in unfed than fed female ticks, however, the importin-α expression levels did not vary between unfed and fed ticks but tended to be higher in the ovary when compared to those in other organs. The effect of importin-α RNAi was characterized in I. ricinus under artificial feeding conditions. Both sub and importin-α gene knockdown was observed in all tick tissues and, while tick weight was significantly lower in sub RNAi-treated ticks than in controls, importin-α RNAi did not affect tick feeding or oviposition, suggesting that SUB is able to exert its function in the absence of Importin-α. Furthermore, SUB was shown to physically interact with histone 4, which was corroborated by protein pull-down and western blot analysis. These results confirm that by interacting with numerous tick proteins, SUB is a key cofactor of the tick interactome and regulome. Further studies are needed to elucidate the nature of the SUB-Importin-α interaction and the biological processes and functional implications that this interaction may have.

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