AAGAB is an assembly chaperone regulating AP1 and AP2 clathrin adaptors

2021 ◽  
Author(s):  
Chun Wan ◽  
Lauren Crisman ◽  
Bing Wang ◽  
Yuan Tian ◽  
Shifeng Wang ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Synergistic Effects ◽  
Binding Protein ◽  
Surface Protein ◽  
Protein Homeostasis ◽  
Loss Of Function ◽  
Intracellular Membrane ◽  
Proteomic Analyses ◽  
The Way

Multimeric cargo adaptors such as AP2 play central roles in intracellular membrane trafficking. We recently discovered that the assembly of AP2 adaptor, a key player in clathrin-mediated endocytosis, is a highly organized process controlled by alpha and gamma adaptin binding protein (AAGAB, also known as p34). In this work, we demonstrate that besides AP2, AAGAB also regulates the assembly of AP1, a cargo adaptor involved in clathrin-mediated transport between the trans-Golgi and the endosome. AAGAB, however, is not involved in the formation of other adaptor complexes including AP3. AAGAB promotes AP1 assembly by binding and stabilizing the γ and σ subunits of AP1, and its mutation abolishes AP1 assembly and disrupts AP1-mediated cargo trafficking. Comparative proteomic analyses indicate that AAGAB mutation massively alters surface protein homeostasis and its loss-of-function phenotypes reflect the synergistic effects of AP1 and AP2 deficiency. Together, these findings establish AAGAB as an assembly chaperone for both AP1 and AP2 adaptors and pave the way for understanding the pathogenesis of AAGAB-linked diseases.

scholarly journals Regulation of ClC-2 Chloride Channel Proteostasis by Molecular Chaperones: Correction of Leukodystrophy-Associated Defect

2021 ◽  
Vol 22 (11) ◽  
pp. 5859
Author(s):  
Ssu-Ju Fu ◽  
Meng-Chun Hu ◽  
Cheng-Tsung Hsiao ◽  
An-Ting Cheng ◽  
Tsung-Yu Chen ◽  
...  
Keyword(s):  
Heat Shock ◽  
Membrane Trafficking ◽  
Molecular Chaperones ◽  
Therapeutic Potential ◽  
Critical Role ◽  
Ion Homeostasis ◽  
Surface Protein ◽  
Loss Of Function ◽  
Er Quality Control ◽  
Fk506 Binding Protein

The ClC-2 channel plays a critical role in maintaining ion homeostasis in the brain and the testis. Loss-of-function mutations in the ClC-2-encoding human CLCN2 gene are linked to the white matter disease leukodystrophy. Clcn2-deficient mice display neuronal myelin vacuolation and testicular degeneration. Leukodystrophy-causing ClC-2 mutant channels are associated with anomalous proteostasis manifesting enhanced endoplasmic reticulum (ER)-associated degradation. The molecular nature of the ER quality control system for ClC-2 protein remains elusive. In mouse testicular tissues and Leydig cells, we demonstrated that endogenous ClC-2 co-existed in the same protein complex with the molecular chaperones heat shock protein 90β (Hsp90β) and heat shock cognate protein (Hsc70), as well as the associated co-chaperones Hsp70/Hsp90 organizing protein (HOP), activator of Hsp90 ATPase homolog 1 (Aha1), and FK506-binding protein 8 (FKBP8). Further biochemical analyses revealed that the Hsp90β-Hsc70 chaperone/co-chaperone system promoted mouse and human ClC-2 protein biogenesis. FKBP8 additionally facilitated membrane trafficking of ClC-2 channels. Interestingly, treatment with the Hsp90-targeting small molecule 17-allylamino-17-demethoxygeldanamycin (17-AAG) substantially boosted ClC-2 protein expression. Also, 17-AAG effectively increased both total and cell surface protein levels of leukodystrophy-causing loss-of-function ClC-2 mutant channels. Our findings highlight the therapeutic potential of 17-AAG in correcting anomalous ClC-2 proteostasis associated with leukodystrophy.

scholarly journals Active and Passive Intranasal Immunizations with Streptococcal Surface Protein C5a Peptidase Prevent Infection of Murine Nasal Mucosa-Associated Lymphoid Tissue, a Functional Homologue of Human Tonsils

2005 ◽  
Vol 73 (12) ◽  
pp. 7878-7886 ◽  
Author(s):  
Hae-Sun Park ◽  
P. Patrick Cleary
Keyword(s):  
Nasal Mucosa ◽  
Lymphoid Tissue ◽  
Streptococcal Infection ◽  
Surface Protein ◽  
Effective Vaccine ◽  
Infection Model ◽  
Loss Of Function ◽  
Intranasal Immunization ◽  
Endemic Disease ◽  
Group B

ABSTRACT C5a peptidase, also called SCPA (surface-bound C5a peptidase), is a surface-bound protein on group A streptococci (GAS), etiologic agents for a variety of human diseases including pharyngitis, impetigo, toxic shock, and necrotizing fasciitis, as well as the postinfection sequelae rheumatic fever and rheumatic heart disease. This protein is highly conserved among different serotypes and is also expressed in human isolates of group B, C, and G streptococci. Human tonsils are the primary reservoirs for GAS, maintaining endemic disease across the globe. We recently reported that GAS preferentially target nasal mucosa-associated lymphoid tissue (NALT) in mice, a tissue functionally analogous to human tonsils. Experiments using a C5a peptidase loss-of-function mutant and an intranasal infection model showed that this protease is required for efficient colonization of NALT. An effective vaccine should prevent infection of this secondary lymphoid tissue; therefore, the potential of anti-SCPA antibodies to protect against streptococcal infection of NALT was investigated. Experiments showed that GAS colonization of NALT was significantly reduced following intranasal immunization of mice with recombinant SCPA protein administered alone or with cholera toxin, whereas a high degree of GAS colonization of NALT was observed in control mice immunized with phosphate-buffered saline only. Moreover, administration of anti-SCPA serum by the intranasal route protected mice against streptococcal infection. These results suggest that intranasal immunization with SCPA would prevent colonization and infection of human tonsils, thereby eliminating potential reservoirs that maintain endemic disease.

scholarly journals The PKD-Dependent Biogenesis of TGN-to-Plasma Membrane Transport Carriers

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1618
Author(s):  
Yuichi Wakana ◽  
Felix Campelo
Keyword(s):  
Cell Surface ◽  
Membrane Trafficking ◽  
Surface Protein ◽  
Membrane Contact Sites ◽  
Tissue Organization ◽  
Constitutive Secretion ◽  
Membrane Contact ◽  
Golgi Network ◽  
Organelle Membrane ◽  
And Control

Membrane trafficking is essential for processing and transport of proteins and lipids and to establish cell compartmentation and tissue organization. Cells respond to their needs and control the quantity and quality of protein secretion accordingly. In this review, we focus on a particular membrane trafficking route from the trans-Golgi network (TGN) to the cell surface: protein kinase D (PKD)-dependent pathway for constitutive secretion mediated by carriers of the TGN to the cell surface (CARTS). Recent findings highlight the importance of lipid signaling by organelle membrane contact sites (MCSs) in this pathway. Finally, we discuss our current understanding of multiple signaling pathways for membrane trafficking regulation mediated by PKD, G protein-coupled receptors (GPCRs), growth factors, metabolites, and mechanosensors.

scholarly journals The EF-Hand Ca2+-binding Protein p22 Plays a Role in Microtubule and Endoplasmic Reticulum Organization and Dynamics with Distinct Ca2+-binding Requirements

2004 ◽  
Vol 15 (2) ◽  
pp. 481-496 ◽  
Author(s):  
Josefa Andrade ◽  
Hu Zhao ◽  
Brian Titus ◽  
Sandra Timm Pearce ◽  
Margarida Barroso
Keyword(s):  
Endoplasmic Reticulum ◽  
Conformational Changes ◽  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Network Formation ◽  
Binding Protein ◽  
Dependent Manner ◽  
Microtubule Depolymerization ◽  
Independent Manner ◽  
Ef Hand

We have reported that p22, an N-myristoylated EF-hand Ca2+-binding protein, associates with microtubules and plays a role in membrane trafficking. Here, we show that p22 also associates with membranes of the early secretory pathway membranes, in particular endoplasmic reticulum (ER). On binding of Ca2+, p22's ability to associate with membranes increases in an N-myristoylation-dependent manner, which is suggestive of a nonclassical Ca2+-myristoyl switch mechanism. To address the intracellular functions of p22, a digitonin-based “bulk microinjection” assay was developed to load cells with anti-p22, wild-type, or mutant p22 proteins. Antibodies against a p22 peptide induce microtubule depolymerization and ER fragmentation; this antibody-mediated effect is overcome by preincubation with the respective p22 peptide. In contrast, N-myristoylated p22 induces the formation of microtubule bundles, the accumulation of ER structures along the bundles as well as an increase in ER network formation. An N-myristoylated Ca2+-binding p22 mutant, which is unable to undergo Ca2+-mediated conformational changes, induces microtubule bundling and accumulation of ER structures along the bundles but does not increase ER network formation. Together, these data strongly suggest that p22 modulates the organization and dynamics of microtubule cytoskeleton in a Ca2+-independent manner and affects ER network assembly in a Ca2+-dependent manner.

scholarly journals Crosstalk of CREB and Fos/Jun on a single cis-element: transcriptional repression of the steroidogenic acute regulatory protein gene

2007 ◽  
Vol 39 (4) ◽  
pp. 261-277 ◽  
Author(s):  
Pulak R Manna ◽  
Douglas M Stocco
Keyword(s):  
Transcriptional Repression ◽  
Protein Gene ◽  
Binding Protein ◽  
Regulatory Protein ◽  
Ectopic Expression ◽  
Fine Tuning ◽  
Creb Binding Protein ◽  
Loss Of Function ◽  
Cis Element ◽  
Steroidogenic Acute Regulatory

AbstractTranscriptional regulation of the steroidogenic acute regulatory (StAR) protein gene by cAMP-dependent mechanisms occurs in the absence of a consensus cAMP-response element (CRE; TGACGTCA) and is mediated by several sequence-specific transcription factors. We previously identified three CRE-like sites (within the −151/−1 bp cAMP-responsive region of the mouse StAR gene), of which the CRE2 site overlaps with an activator protein-1 (AP-1) motif (TGACTGA, designated as CRE2/AP-1) that can bind both CRE and AP-1 DNA-binding proteins. The present studies were aimed at exploring the functional crosstalk between CREB (CRE-binding protein) and cFos/cJun (AP-1 family members) on the CRE2/AP-1 element and its role in regulating transcription of the StAR gene. Using MA-10 mouse Leydig tumor cells, we demonstrate that the CRE and AP-1 families of proteins interact with the CRE2/AP-1 sequence. CREB, cFos, and cJun proteins were found to bind to the CRE2/AP-1 motif but not the CRE1 and CRE3 sites. Treatment with the cAMP analog (Bu)2cAMP augmented phosphorylation of CREB (Ser133), cFos (Thr325), and cJun (ser73). Chromatin immunoprecipitation studies revealed that the induction of CREB, cFos, and cJun by (Bu)2cAMP was correlated with protein–DNA interactions and recruitment of the coactivator CREB-binding protein (CBP) to the StAR promoter. EMSA studies employing CREB and cFos/cJun proteins demonstrated competition between these factors for binding to the CRE2/AP-1 motif. Transfection of cells containing the −151/−1 StAR reporter with CREB and cFos/cJun resulted in trans-repression of the StAR gene, an event tightly associated with CBP, demonstrating that both CREB and Fos/Jun compete with each other for binding with limited amounts of intracellular CBP. Overexpression of adenovirus E1A, which binds and inactivates CBP, markedly suppressed StAR gene expression. Ectopic expression of CBP eliminated the repression of the StAR gene by E1A and potentiated the activity of CREB and cFos/cJun on StAR promoter responsiveness. These findings identify molecular events involved in crosstalk between CREB and cFos/cJun, which confer both gain and loss of function on a single cis-element in fine-tuning of the regulatory events involved in transcription of the StAR gene.

Identification and functional characterisation of a novel KCNJ2 mutation, Val302del, causing Andersen–Tawil syndrome

2015 ◽  
Vol 93 (7) ◽  
pp. 569-575 ◽  
Author(s):  
Balázs Ördög ◽  
Lidia Hategan ◽  
Mária Kovács ◽  
György Seprényi ◽  
Zsófia Kohajda ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Genetic Disorder ◽  
Clinical Manifestations ◽  
Clinical Importance ◽  
Inhibitory Effect ◽  
Periodic Paralysis ◽  
Inward Rectification ◽  
Loss Of Function ◽  
Gene Encoding ◽  
Functional Characterisation

Loss-of-function mutations of the KCNJ2 gene encoding for the inward rectifier potassium channel subunit Kir2.1 cause Andersen–Tawil Syndrome (ATS), a rare genetic disorder characterised by periodic paralysis, ventricular arrhythmias, and dysmorphic features. Clinical manifestations of the disease appear to vary greatly with the nature of mutation, therefore, functional characterisation of ATS-causing mutations is of clinical importance. In this study, we describe the identification and functional analysis of a novel KCNJ2 mutation, Val302del, identified in a patient with ATS. Heterologously expressed wild type (WT) and Val302del mutant alleles showed similar subcellular distribution of the Kir2.1 protein with high intensity labelling from the membrane region, demonstrating normal membrane trafficking of the Val302del Kir2.1 variant. Cells transfected with the WT allele displayed a robust current with strong inward rectification, while no current above background was detected in cells expressing the Val302del Kir2.1 subunit. Co-transfection of CHO cells with the WT and the Val302del Kir2.1 revealed a dose-dependent inhibitory effect of the Val302del Kir2.1 mutant subunit on WT Kir2.1 currents. These observations indicate that the WT and the Val302del mutant subunits co-assemble in the cell membrane and that the mutation affects potassium conductivity and (or) gating of the WT/Val302del heteromeric Kir2.1 channels.

The stem-loop binding protein CDL-1 is required for chromosome condensation, progression of cell death and morphogenesis inCaenorhabditis elegans

Development ◽  
2002 ◽  
Vol 129 (1) ◽  
pp. 187-196 ◽  
Author(s):  
Yuki Kodama ◽  
Joel H. Rothman ◽  
Asako Sugimoto ◽  
Masayuki Yamamoto
Keyword(s):  
Cell Death ◽  
Structural Changes ◽  
Binding Protein ◽  
Core Histone ◽  
Loss Of Function ◽  
Histone Proteins ◽  
Stem Loop ◽  
C Elegans ◽  
Morphological Defects ◽  
In The Wild

Histones play important roles not only in the structural changes of chromatin but also in regulating gene expression. Expression of histones is partly regulated post-transcriptionally by the stem-loop binding protein (SLBP)/hairpin binding protein (HBP). We report the developmental function of CDL-1, the C. elegans homologue of SLBP/HBP. In the C. elegans cdl-1 mutants, cell corpses resulting from programmed cell death appear later and persist much longer than those in the wild type. They also exhibit distinct morphological defects in body elongation and movement of the pharyngeal cells toward the buccal opening. The CDL-1 protein binds to the stem-loop structures in the 3′-UTR of C. elegans core histone mRNAs, and the mutant forms of this protein show reduced binding activities. A decrease in the amount of core histone proteins phenocopied the cdl-1 mutant embryos, suggesting that CDL-1 contributes to the proper expression of core histone proteins. We propose that loss-of-function of cdl-1 causes aberrant chromatin structure, which affects the cell cycle and cell death, as well as transcription of genes essential for morphogenesis.

Crystal structure of the GTP-binding protein-like domain of AGAP1

2021 ◽  
Vol 77 (4) ◽  
Author(s):  
Nuo Cheng ◽  
Hao Zhang ◽  
Shiyan Zhang ◽  
Xiaodan Ma ◽  
Guoyu Meng
Keyword(s):  
Membrane Trafficking ◽  
Protein Transport ◽  
Structural Information ◽  
Binding Protein ◽  
Lymphoblastic Leukemia ◽  
Diffusion Method ◽  
Gtp Binding Protein ◽  
Aberrant Expression ◽  
Cell Parameters ◽  
Gtp Binding

AGAP1 is often considered to regulate membrane trafficking, protein transport and actin cytoskeleton dynamics. Recent studies have shown that aberrant expression of AGAP1 is associated with many diseases, including neurodevelopmental disorders and acute lymphoblastic leukemia. It has been proposed that the GTP-binding protein-like domain (GLD) is involved in the binding of cofactors and thus regulates the catalytic activity of AGAP1. To obtain a better understanding of the pathogenic mechanism underpinning AGAP1-related diseases, it is essential to obtain structural information. Here, the GLD (residues 70–235) of AGAP1 was overexpressed in Escherichia coli BL21 (DE3) cells. Affinity and gel-filtration chromatography were used to obtain AGAP1GLD with high purity for crystallization. Using the hanging-drop vapor-diffusion method with the protein at a final concentration of 20 mg ml−1, AGAP1GLD protein crystals of suitable size were obtained. The crystals were found to diffract to 3.0 Å resolution and belonged to space group I4, with unit-cell parameters a = 100.39, b = 100.39, c = 48.08 Å. The structure of AGAP1GLD exhibits the highly conserved functional G1–G5 loops and is generally similar to other characterized ADP-ribosylation factor (Arf) GTPase-activating proteins (GAPs), implying an analogous function to Arf GAPs. Additionally, this study indicates that AGAP1 could be classified as a type of NTPase, the activity of which might be regulated by protein partners or by its other domains. Taken together, these results provide insight into the regulatory mechanisms of AGAP1 in cell signaling.

Lipoprotein lipase binding to adipocytes: evidence for the presence of a heparin-sensitive binding protein

1993 ◽  
Vol 265 (6) ◽  
pp. E880-E888 ◽  
Author(s):  
A. Sasaki ◽  
P. Sivaram ◽  
I. J. Goldberg
Keyword(s):  
Cell Surface ◽  
Lipoprotein Lipase ◽  
Binding Protein ◽  
Surface Protein ◽  
Surface Binding ◽  
Cell Membrane Proteins ◽  
Adipocyte Cell ◽  
Capillary Endothelial Cells ◽  
Fold Excess ◽  
Ligand Blot

Lipoprotein lipase (LPL) is synthesized by adipocytes, associated with the cell surface, and released from the cells when they are treated with heparin. Release of LPL from the adipocyte is required for LPL to migrate to its physiological site of action on the luminal surface of capillary endothelial cells. To better understand this process, we studied the interaction of LPL with adipocyte cell membrane proteins. With the use of a ligand blot method, LPL specifically bound to a heparin-releasable, 116-kDa protein on mouse-derived brown fat adipose cell (BFC-1 beta) and rat adipocyte membranes. A 116-kDa cell surface protein was metabolically labeled with [35S]methionine and bound to LPL-Sepharose. This suggested that the LPL-binding protein was synthesized by the cells. When BFC-1 beta were treated with heparin to eliminate heparin-sensitive cell surface binding sites, LPL binding to the cells decreased and release of newly synthesized LPL activity increased. 125I-labeled LPL binding to control cells was reduced (> 70%) by a 50-fold excess of unlabeled LPL. The residual LPL binding to heparin-treated cells was, however, not decreased by the addition of unlabeled LPL. These data imply that specific adipocyte surface LPL binding involves heparin-sensitive sites. We hypothesize that the heparin-releasable, 116-kDa LPL-binding protein mediates specific LPL binding to adipocytes and that LPL activity within adipose tissue is regulated, in part, by the interaction of LPL with this binding protein.

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