scholarly journals Phosphoserine acidic cluster motifs in the cytoplasmic domains of transmembrane proteins bind distinct basic regions on the μ subunits of clathrin adaptor protein complexes

2018 ◽  
Author(s):  
Rajendra Singh ◽  
Charlotte Stoneham ◽  
Christopher Lim ◽  
Xiaofei Jia ◽  
Javier Guenaga ◽  
...  
Keyword(s):  
Protein Trafficking ◽  
Transmembrane Proteins ◽  
Adaptor Protein ◽  
Cellular Protein ◽  
Membrane Lipid ◽  
Dependent Manner ◽  
Cytoplasmic Domains ◽  
Clathrin Adaptor ◽  
Hiv 1

AbstractProtein trafficking in the endosomal system involves the recognition of specific signals within the cytoplasmic domains (CDs) of transmembrane proteins by clathrin adaptors. One such signal is the phosphoserine acidic cluster (PSAC), the prototype of which is in the endoprotease Furin. How PSACs are recognized by clathrin adaptors has been controversial. We reported previously that HIV-1 Vpu, which modulates cellular immunoreceptors, contains a PSAC that binds to the µ subunits of clathrin adaptor protein (AP) complexes. Here, we show that the CD of Furin binds the µ subunits of AP-1 and AP-2 in a phosphorylation-dependent manner. Moreover, we identify a PSAC in a cytoplasmic loop of the cellular transmembrane Serinc3, an inhibitor of the infectivity of retroviruses. The two serines within the PSAC of Serinc3 are phosphorylated by casein kinase II and mediate interaction with the µ subunits in vitro. The sites of these serines vary among mammals in a manner consistent with host-pathogen conflict, yet the Serinc3-PSAC seems dispensible for anti-HIV activity and for counteraction by HIV-1 Nef. The CDs of Vpu, Furin, and the PSAC-containing loop of Serinc3 each bind the μ subunit of AP-2 (µ2) with similar affinities, but they appear to utilize different basic regions on µ2. The Serinc3 loop requires a region previously reported to bind the acidic plasma membrane lipid phosphatidylinositol-4,5-bisphosphate. These data suggest that the PSACs within different proteins recognize different basic regions on the µ surface, providing the potential to inhibit the activity of viral proteins without necessarily affecting cellular protein trafficking.

scholarly journals Recognition of dileucine-based sorting signals from HIV-1 Nef and LIMP-II by the AP-1 γ–σ1 and AP-3 δ–σ3 hemicomplexes

2003 ◽  
Vol 163 (6) ◽  
pp. 1281-1290 ◽  
Author(s):  
Katy Janvier ◽  
Yukio Kato ◽  
Markus Boehm ◽  
Jeremy R. Rose ◽  
José A. Martina ◽  
...  
Keyword(s):  
Transmembrane Proteins ◽  
Adaptor Protein ◽  
Integral Membrane Protein ◽  
Negative Factor ◽  
Sorting Signals ◽  
Immunodeficiency Virus ◽  
Sequence Requirements ◽  
Hiv 1

The sorting of transmembrane proteins to endosomes and lysosomes is mediated by signals present in the cytosolic tails of the proteins. A subset of these signals conform to the [DE]XXXL[LI] consensus motif and mediate sorting via interactions with heterotetrameric adaptor protein (AP) complexes. However, the identity of the AP subunits that recognize these signals remains controversial. We have used a yeast three-hybrid assay to demonstrate that [DE]XXXL[LI]-type signals from the human immunodeficiency virus negative factor protein and the lysosomal integral membrane protein II interact with combinations of the γ and σ1 subunits of AP-1 and the δ and σ3 subunits of AP-3, but not the analogous combinations of AP-2 and AP-4 subunits. The sequence requirements for these interactions are similar to those for binding to the whole AP complexes in vitro and for function of the signals in vivo. These observations reveal a novel mode of recognition of sorting signals involving the γ/δ and σ subunits of AP-1 and AP-3.

scholarly journals In VitroExposure to PC-1005 and Cervicovaginal Lavage Fluid from Women Vaginally Administered PC-1005 Inhibits HIV-1 and HSV-2 Infection in Human Cervical Mucosa

2016 ◽  
Vol 60 (9) ◽  
pp. 5459-5466 ◽  
Author(s):  
Guillermo Villegas ◽  
Giulia Calenda ◽  
Shimin Zhang ◽  
Olga Mizenina ◽  
Kyle Kleinbeck ◽  
...  
Keyword(s):  
Phase 1 ◽  
Lavage Fluid ◽  
Dependent Manner ◽  
Phase 1 Trial ◽  
Cervical Mucosa ◽  
Cervicovaginal Lavage ◽  
Dose Dependent ◽  
Anti Hiv ◽  
Hiv 1

ABSTRACTOur recent phase 1 trial demonstrated that PC-1005 gel containing 50 μM MIV-150, 14 mM zinc acetate dihydrate, and carrageenan (CG) applied daily vaginally for 14 days is safe and well tolerated. Importantly, cervicovaginal lavage fluid samples (CVLs) collected 4 or 24 h after the last gel application inhibited HIV-1 and human papillomavirus (HPV) in cell-based assays in a dose-dependent manner (MIV-150 for HIV-1 and CG for HPV). Herein we aimed to determine the anti-HIV and anti-herpes simplex virus 2 (anti-HSV-2) activity of PC-1005 in human cervical explants afterin vitroexposure to the gel and to CVLs from participants in the phase 1 trial. Single HIV-1BaLinfection and HIV-1BaL–HSV-2 coinfection explant models were utilized. Coinfection with HSV-2 enhanced tissue HIV-1BaLinfection.In vitroexposure to PC-1005 protected cervical mucosa against HIV-1BaL(up to a 1:300 dilution) in single-challenge and cochallenge models. CG gel (PC-525) provided some barrier effect against HIV-1BaLat the 1:100 dilution in a single-challenge model but not in the cochallenge model. Both PC-1005 and PC-525 at the 1:100 dilution inhibited HSV-2 infection, pointing to a CG-mediated protection. MIV-150 and CG in CVLs inhibited HIV (single-challenge or cochallenge models) and HSV-2 infections in explants in a dose-dependent manner (P< 0.05). Stronger inhibition of HIV-1 infection by CVLs collected 4 h after the last gel administration was observed compared to infection detected in the presence of baseline CVLs. The anti-HIV and anti-HSV-2 activity of PC-1005 gelin vitroand CVLs in human ectocervical explants supports the further development of PC-1005 gel as a broad-spectrum on-demand microbicide.

scholarly journals In Vitro Formation of Recycling Vesicles from Endosomes Requires Adaptor Protein-1/Clathrin and Is Regulated by Rab4 and the Connector Rabaptin-5

2004 ◽  
Vol 15 (11) ◽  
pp. 4990-5000 ◽  
Author(s):  
Adriana Pagano ◽  
Pascal Crottet ◽  
Cristina Prescianotto-Baschong ◽  
Martin Spiess
Keyword(s):  
Brefeldin A ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Vesicle Formation ◽  
Dependent Manner ◽  
Nucleotide Exchange ◽  
Vitro Assay ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

The involvement of clathrin and associated adaptor proteins in receptor recycling from endosomes back to the plasma membrane is controversial. We have used an in vitro assay to identify the molecular requirements for the formation of recycling vesicles. Cells expressing the asialoglycoprotein receptor H1, a typical recycling receptor, were surface biotinylated and then allowed to endocytose for 10 min. After stripping away surface-biotin, the cells were permeabilized and the cytosol washed away. In a temperature-, cytosol-, and nucleotide-dependent manner, the formation of sealed vesicles containing biotinylated H1 could be reconstituted. Vesicle formation was strongly inhibited upon immunodepletion of adaptor protein (AP)-1, but not of AP-2 or AP-3, from the cytosol, and was restored by readdition of purified AP-1. Vesicle formation was stimulated by supplemented clathrin, but inhibited by brefeldin A, consistent with the involvement of ARF1 and a brefeldin-sensitive guanine nucleotide exchange factor. The GTPase rab4, but not rab5, was required to generate endosome-derived vesicles. Depletion of rabaptin-5/rabex-5, a known interactor of both rab4 and γ-adaptin, stimulated and addition of the purified protein strongly inhibited vesicle production. The results indicate that recycling is mediated by AP-1/clathrin-coated vesicles and regulated by rab4 and rabaptin-5/rabex-5.

Adaptor Protein Lnk Inhibits C-Fms Mediated Macrophage Function

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3555-3555
Author(s):  
Saskia Gueller ◽  
Helen S. Goodridge ◽  
Hongtao Xing ◽  
Sigal Gery ◽  
Hubert Serve ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Adaptor Protein ◽  
Inhibitory Effect ◽  
Sh2 Domain ◽  
Boyden Chamber ◽  
Tyrosine Kinase Domain ◽  
Pleckstrin Homology Domain ◽  
Dependent Manner ◽  
Knock Out

Abstract The macrophage colony-stimulating factor receptor (c-Fms) plays an important role in proliferation, differentiation and survival of macrophages and is involved in the regulation of distinct macrophage functions. Interaction with the ligand M-CSF results in activation of the intracellular tyrosine kinase domain and phosphorylation of tyrosine residues, thereby creating binding sites for several molecules containing Src homology 2 (SH2) domains. One such protein is the adaptor Lnk that negatively regulates several hematopoietic cytokine receptors including MPL, EpoR and c-Kit. Lnk belongs to a family of proteins sharing several structural motifs including a SH2 domain and a pleckstrin homology domain. The SH2 domain is known to be essential for its inhibitory effect which can be abolished by the point mutation R392E. In this study, we investigated the ability of Lnk to interact and modulate the function of c-Fms. In order to determine if Lnk can bind to c-Fms, immunoprecipitation was performed with lysates from 293T cells co-transfected with the cDNAs for c-Fms and Lnk. Only after exposure to M-CSF, Lnk bound to c-Fms, and binding was dependent on an intact SH2 domain. To elucidate further if Lnk exhibits biological and functional effects on macrophages, we examined both in-vitro differentiated macrophages derived from the bone marrow and also macrophages harvested from peritoneum from Lnk deleted (KO) and wild type (WT) mice. These cells appeared to be at a similar stage of differentiation because expression levels of myeloid and macrophage surface markers such as F4/80, CD11b and CD11c were the same in both bone marrow-derived and peritoneum-derived macrophages from Lnk KO and WT mice. Clonogenic assays demonstrated that the number of M-CFUs in the bone marrow were elevated in Lnk KO as compared to WT mice. Furthermore, the M-CSF-induced phosphorylation of AKT in these Lnk KO macrophages was increased and prolonged compared to WT macrophages. This was associated with prominent up-regulation of c-Fms in macrophages from Lnk KO mice. We found that Lnk additionally had several functional effects on bone marrow-derived macrophages. Production of reactive oxygen species (ROS) was dramatically increased in a M-CSF-dependent manner in Lnk KO macrophages upon stimulation with zymosan. In addition, knock-out of Lnk led to altered cytokine production of macrophages: Stimulation with zymosan caused increased levels of TNFalpha and IL-6 in the KO cells, while bacterial lipoproteins (Pam3CSK4) decreased levels of TNFalpha in KO compared to WT macrophages. Last, Lnk inhibited M-CSF-induced migration of macrophages in the Boyden chamber as Lnk KO macrophages showed a significantly higher migration capacity than WT macrophages. In summary, we show for the first time that Lnk can bind to c-Fms and can blunt the stimulation of M-CSF. Modulation of levels of Lnk in macrophages may provide a unique therapeutic approach to increase innate host defenses.

scholarly journals Testicular membrane lipid damage by complex mixture of leachate from municipal battery recycling site as indication of idiopathic male infertility in rat

2013 ◽  
Vol 6 (4) ◽  
pp. 192-197 ◽  
Author(s):  
Jacob K. Akintunde ◽  
Ganiyu Oboh ◽  
Akintunde A. Akindahunsi
Keyword(s):  
Male Infertility ◽  
Lipid Membrane ◽  
Complex Mixture ◽  
Membrane Lipid ◽  
Metal Exposure ◽  
Dependent Manner ◽  
Mixed Metal ◽  
Lipid Damage ◽  
Battery Recycling

ABSTRACT Leachate from a municipal battery recycling site is a potent source of mixed-metal released into the environment. The present study investigated the degree at which mixed-metal exposure to the municipal auto-battery leachate (MABL) and to the Elewi Odo municipal auto-battery recycling site leachate (EOMABRL) affected the lipid membrane of the testes in in vitro experiment. The results showed elevated level of mixed-metals over the permissible levels in drinking water, as recommended by regulatory authorities. In the leachate samples, the levels of malondialdehyde (MDA), a biomarker of lipid damage, was significantly (p<0.05) increased in rat testes in a dose-dependent manner. MDA induced by the municipal auto-battery leachate (MABL) was significantly (p<0.05) higher than the leachate from Elewi Odo municipal auto-battery recycling site (EOMABRL). The testicular lipid membrane capacity was compromised following treatment with leachate from the municipal battery recycling site, implicating mixed-metal exposure as the causative agent of testicular damage and male infertility.

scholarly journals The interaction of HIV-1 and HIV-2 with cellular protein trafficking pathways

Retrovirology ◽  
2013 ◽  
Vol 10 (Suppl 1) ◽  
pp. P2
Author(s):  
Justine Alford ◽  
Robert Spooner ◽  
Michela Marongiu ◽  
Emma Anderson
Keyword(s):  
Protein Trafficking ◽  
Cellular Protein ◽  
Hiv 1

scholarly journals Di-Leucine Signals Mediate Targeting of Tyrosinase and Synaptotagmin to Synaptic-like Microvesicles within PC12 Cells

1999 ◽  
Vol 10 (11) ◽  
pp. 3979-3990 ◽  
Author(s):  
Anastasiya D. Blagoveshchenskaya ◽  
Eric W. Hewitt ◽  
Daniel F. Cutler
Keyword(s):  
Plasma Membrane ◽  
Pc12 Cells ◽  
Brefeldin A ◽  
Adaptor Protein ◽  
Dependent Manner ◽  
Protein Traffic ◽  
C Terminus ◽  
Targeting Signal

One pathway in forming synaptic-like microvesicles (SLMV) involves direct budding from the plasma membrane, requires adaptor protein 2 (AP2) and is brefeldin A (BFA) resistant. A second route leads from the plasma membrane to an endosomal intermediate from which SLMV bud in a BFA-sensitive, AP3-dependent manner. Because AP3 has been shown to bind to a di-leucine targeting signal in vitro, we have investigated whether this major class of targeting signals is capable of directing protein traffic to SLMV in vivo. We have found that a di-leucine signal within the cytoplasmic tail of human tyrosinase is responsible for the majority of the targeting of HRP-tyrosinase chimeras to SLMV in PC12 cells. Furthermore, we have discovered that a Met-Leu di-hydrophobic motif within the extreme C terminus of synaptotagmin I supports 20% of the SLMV targeting of a CD4-synaptotagmin chimera. All of the traffic to the SLMV mediated by either di-Leu or Met-Leu is BFA sensitive, strongly suggesting a role for AP3 and possibly for an endosomal intermediate in this process. The differential reduction in SLMV targeting for HRP-tyrosinase and CD4-synaptotagmin chimeras by di-alanine substitutions or BFA treatment implies that different proteins use the two routes to the SLMV to differing extents.

Effects of membrane lipid and fluidity modifications on HIV-1 infectibility of primate lymphocytes in vitro

1990 ◽  
Vol 10 (3) ◽  
pp. 263-270 ◽  
Author(s):  
J. Pascal Zimmer ◽  
Hans A. Lehr ◽  
Christoph Hübner ◽  
Stephan G. Lindner ◽  
Ralf Ramsperger ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Fluidity ◽  
Lipid Composition ◽  
Membrane Lipid ◽  
Serum Factors ◽  
Membrane Lipid Composition ◽  
Plasma Membrane Lipid ◽  
Hiv 1

Although most non-human primates, except the chimpanzee and the gibbon in vivo are not infectible by HIV-1, lymphocytes of several of these species can be infected by HIV-1 in vitro.In order to investigate whether the in vitro infectibility of primate lymphocytes might be attributed to plasma membrane adaptation processes or to serum factors, we compared HIV-1 infectibility of cultivated peripheral blood lymphocytes of macaques and of baboons on day one and on day ten of cultivation. These data were correlated to plasma membrane lipid composition and membrane fluidity.We found a correlation between increased HIV-1 in vitro infectibility and changes in plasma membrane lipid composition resulting in decreased membrane fluidity of cultured primate lymphocytes.

scholarly journals A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Edward A Partlow ◽  
Richard W Baker ◽  
Gwendolyn M Beacham ◽  
Joshua S Chappie ◽  
Andres E Leschziner ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Proteins ◽  
Active Conformation ◽  
Genetic Screens ◽  
Structural Mechanism ◽  
C Elegans ◽  
Dependent Inactivation ◽  
Clathrin Adaptor

Endocytosis of transmembrane proteins is orchestrated by the AP2 clathrin adaptor complex. AP2 dwells in a closed, inactive state in the cytosol, but adopts an open, active conformation on the plasma membrane. Membrane-activated complexes are also phosphorylated, but the significance of this mark is debated. We recently proposed that NECAP negatively regulates AP2 by binding open and phosphorylated complexes (Beacham et al., 2018). Here, we report high-resolution cryo-EM structures of NECAP bound to phosphorylated AP2. The site of AP2 phosphorylation is directly coordinated by residues of the NECAP PHear domain that are predicted from genetic screens in C. elegans. Using membrane mimetics to generate conformationally open AP2, we find that a second domain of NECAP binds these complexes and cryo-EM reveals both domains of NECAP engaging closed, inactive AP2. Assays in vitro and in vivo confirm these domains cooperate to inactivate AP2. We propose that phosphorylation marks adaptors for inactivation.

scholarly journals A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex

2019 ◽  
Author(s):  
Edward A. Partlow ◽  
Richard W. Baker ◽  
Gwendolyn M. Beacham ◽  
Joshua S. Chappie ◽  
Andres E. Leschziner ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Proteins ◽  
Active Conformation ◽  
Genetic Screens ◽  
Structural Mechanism ◽  
C Elegans ◽  
Dependent Inactivation ◽  
Clathrin Adaptor

AbstractEndocytosis of transmembrane proteins is orchestrated by the AP2 clathrin adaptor complex. AP2 dwells in a closed, inactive state in the cytosol, but adopts an open, active conformation on the plasma membrane. Membrane-activated complexes are also phosphorylated, but the significance of this mark is debated. We recently proposed that NECAP negatively regulates AP2 by binding open and phosphorylated complexes (Beacham et al., 2018). Here, we report high-resolution cryo-EM structures of NECAP bound to phosphorylated AP2. The site of AP2 phosphorylation is directly coordinated by residues of the NECAP PHear domain that are predicted from genetic screens in C. elegans. Using membrane mimetics to generate conformationally open AP2, we find that a second domain of NECAP binds these complexes and cryo-EM reveals both domains of NECAP engaging closed, inactive AP2. Assays in vitro and in vivo confirm these domains cooperate to inactivate AP2. We propose that phosphorylation marks adaptors for inactivation.

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