scholarly journals De novo macrocyclic peptides for inhibiting, stabilising and probing the function of the Retromer endosomal trafficking complex

2020 ◽  
Author(s):  
Kai-En Chen ◽  
Qian Guo ◽  
Yi Cui ◽  
Amy K. Kendall ◽  
Timothy A. Hill ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Cyclic Peptides ◽  
De Novo ◽  
Late Onset ◽  
Endosomal Trafficking ◽  
Endosomal Membrane ◽  
Cellular Localisation ◽  
Macrocyclic Peptides ◽  
In Cells

ABSTRACTThe Retromer complex (Vps35-Vps26-Vps29) is essential for endosomal membrane trafficking and signalling. Mutations in Retromer cause late-onset Parkinson’s disease, while viral and bacterial pathogens can hijack the complex during cellular infection. To modulate and probe its function we have created a novel series of macrocyclic peptides that bind Retromer with high affinity and specificity. Crystal structures show the majority of cyclic peptides bind to Vps29 via a Pro-Leu-containing sequence, structurally mimicking known interactors such as TBC1D5, and blocking their interaction with Retromer in vitro and in cells. By contrast, macrocyclic peptide RT-L4 binds Retromer at the Vps35-Vps26 interface and is a more effective molecular chaperone than reported small molecules, suggesting a new therapeutic avenue for targeting Retromer. Finally, tagged peptides can be used to probe the cellular localisation of Retromer and its functional interactions in cells, providing novel tools for studying Retromer function.

scholarly journals Connecdenn 3/DENND1C binds actin linking Rab35 activation to the actin cytoskeleton

2012 ◽  
Vol 23 (1) ◽  
pp. 163-175 ◽  
Author(s):  
Andrea L. Marat ◽  
Maria S. Ioannou ◽  
Peter S. McPherson
Keyword(s):  
Actin Cytoskeleton ◽  
Membrane Trafficking ◽  
Small Gtpase ◽  
Actin Binding ◽  
Binding Motif ◽  
Endosomal Trafficking ◽  
Nucleotide Exchange ◽  
Endosomal Membrane ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors

The small GTPase Rab35 regulates endosomal membrane trafficking but also recruits effectors that modulate actin assembly and organization. Differentially expressed in normal and neoplastic cells (DENN)–domain proteins are a newly identified class of Rab guanine-nucleotide exchange factors (GEFs) that are grouped into eight families, each activating a common Rab. The members of one family, connecdenn 1–3/DENND1A–C, are all GEFs for Rab35. Why Rab35 requires multiple GEFs is unknown. We demonstrate that connecdenn 3 uses a unique C-terminal motif, a feature not found in connecdenn 1 or 2, to directly bind actin. This interaction couples Rab35 activation to the actin cytoskeleton, resulting in dramatic changes in cell shape, notably the formation of protrusive membrane extensions. These alterations are specific to Rab35 activated by connecdenn 3 and require both the actin-binding motif and N-terminal DENN domain, which harbors the GEF activity. It was previously demonstrated that activated Rab35 recruits the actin-bundling protein fascin to actin, but the relevant GEF for this activity was unknown. We demonstrate that connecdenn 3 and Rab35 colocalize with fascin and actin filaments, suggesting that connecdenn 3 is the relevant GEF. Thus, whereas connecdenn 1 and 2 activate Rab35 for endosomal trafficking, connecdenn 3 uniquely activates Rab35 for its role in actin regulation.

99 EXTRACELLULAR VESICLES OF BOVINE OVIDUCTAL FLUID MODIFY THE GENE EXPRESSION ON BOVINE IN VITRO-DERIVED EMBRYOS

2016 ◽  
Vol 28 (2) ◽  
pp. 179 ◽  
Author(s):  
R. Lopera-Vasquez ◽  
M. Hamdi ◽  
V. Maillo ◽  
C. Nunez ◽  
M. Yanez-Mo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
Extracellular Vesicles ◽  
Membrane Trafficking ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Expression Patterns ◽  
Water Channel ◽  
Developmental Competence

Extracellular vesicles (EVs) act as intercellular communicators through their protein, lipid, and mRNA content. The interaction of EVs from oviducal environment and the first stages of embryo development is currently an enigma. The aim of the present study was to evaluate the developmental competence and the expression profile of bovine blastocysts cultured with previously purified EVs recovered from ampullary and isthmic oviducal fluid (OF) under different centrifugal forces. OF-EVs recovered from oviducts of slaughtered heifers in early luteal phase were quantified with a nanoparticle tracking analysis system, and their integrity and size were assessed by electron microscopy. In vitro-produced zygotes were cultured in SOF+3 mg mL–1 BSA (C–), C– with 3 × 105 OF-EVs/mL from the ampulla (A) and isthmus (I) isolated at 1 × 103 (A10k and I10k, respectively) and 1 × 105 (A100k and I100k, respectively) × g. A control culture group of SOF+5% FCS (C+) was included. Blastocyst development was recorded on Day 7, 8, and 9 (D0: day of fertilization). Blastocysts on Days 7/8 cultured in C–, C+, I10k, and I100k were used to measure the relative mRNA expression of genes related with membrane trafficking (AQP3, AQP11, and ATP1A1), metabolism (LDLR and LDHA), and epigenetics (DNMT3A, IGF2R, GRB10, and SNRPN) by RT-qPCR. One-way ANOVA was used for statistical analysis. The size of ampullary and isthmic OF-EVs was similar with a mean of 220 nm. The concentration of I10k was significantly lower compared with A100k (3.6 × 108 v. 10.5 × 108 EVs/mL, respectively; P < 0.05); however, no differences were found in the rest of the groups with a mean concentration of 7.6 × 108 EVs/mL. EVs and C– groups showed a delayed embryo development at Day 7 compared with C+ (range: 12.0–13.8 v. 20.6%, respectively, P < 0.05); however, it was compensated at Days 8 and 9 (Day 9 range: 28.5–30.8%). The water channel related protein AQP3, associated with blastocoel formation, water, and cryoprotectant movement during cryopreservation, was up-regulated in I10k and I100k blastocysts compared with C+. The lipid receptor LDLR, proposed as a regulator of lipid uptake in blastocysts, was significantly down-regulated in C+ compared with the other groups, a possible consequence of a higher concentration of lipids in the C+ group. The de novo DNA methyltransferase DNMT3A and the imprinting gene SNRPN were down-regulated in the C+ compared with I100k, suggesting alterations in imprinting. In conclusion, bovine isthmic OF-EVs supplementation in in vitro embryo culture has a positive effect on gene expression patterns of developmental related genes compared with serum supplementation, suggesting an association between the oviducal environment and the developing embryo. Funded by the Spanish Ministry of Science and Innovation (AGL2012–37510 and AGL2012–39652-C02–01).

scholarly journals Cyclic peptides with a distinct arginine-fork motif recognize the HIV trans-activation response RNA in vitro and in cells

2021 ◽  
pp. 101390
Author(s):  
Sai Shashank Chavali ◽  
Sachitanand M. Mali ◽  
Rachel Bonn ◽  
Abhijith Saseendran ◽  
Ryan P. Bennett ◽  
...  
Keyword(s):  
Cyclic Peptides ◽  
Activation Response ◽  
In Cells

scholarly journals Computational Approaches to Investigate and Design Lipid-binding Domains for Membrane Biosensing

2021 ◽  
Vol 75 (12) ◽  
pp. 1031-1036
Author(s):  
Sriraksha Srinivasan ◽  
Stefano Vanni
Keyword(s):  
Membrane Trafficking ◽  
De Novo ◽  
Md Simulations ◽  
Lipid Binding ◽  
Broad Perspective ◽  
Computational Approaches ◽  
Binding Domains ◽  
Specific Lipid

Association of proteins with cellular membranes is critical for signaling and membrane trafficking processes. Many peripheral lipid-binding domains have been identified in the last few decades and have been investigated for their specific lipid-sensing properties using traditional in vivo and in vitro studies. However, several knowledge gaps remain owing to intrinsic limitations of these methodologies. Thus, novel approaches are necessary to further our understanding in lipid–protein biology. This review briefly discusses lipid-binding domains that act as specific lipid biosensors and provides a broad perspective on the computational approaches such as molecular dynamics (MD) simulations and machine learning (ML)-based techniques that can be used to study protein–membrane interactions. We also highlight the need for de novo design of proteins that elicit specific lipid-binding properties.

scholarly journals Endosomal trafficking of yeast membrane proteins

2018 ◽  
Vol 46 (6) ◽  
pp. 1551-1558 ◽  
Author(s):  
Kamilla M. E. Laidlaw ◽  
Chris MacDonald
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Membrane Trafficking ◽  
Surface Membrane ◽  
Multivesicular Body ◽  
Endosomal Trafficking ◽  
Yeast Saccharomyces Cerevisiae ◽  
Endosomal Membrane ◽  
Recent Developments ◽  
Intracellular Compartments

Various membrane trafficking pathways transport molecules through the endosomal system of eukaryotic cells, where trafficking decisions control the localisation and activity of a diverse repertoire of membrane protein cargoes. The budding yeast Saccharomyces cerevisiae has been used to discover and define many mechanisms that regulate conserved features of endosomal trafficking. Internalised surface membrane proteins first localise to endosomes before sorting to other compartments. Ubiquitination of endosomal membrane proteins is a signal for their degradation. Ubiquitinated cargoes are recognised by the endosomal sorting complex required for transport (ESCRT) apparatus, which mediate sorting through the multivesicular body pathway to the lysosome for degradation. Proteins that are not destined for degradation can be recycled to other intracellular compartments, such as the Golgi and the plasma membrane. In this review, we discuss recent developments elucidating the mechanisms that drive membrane protein degradation and recycling pathways in yeast.

scholarly journals Desmin Aggregate Formation by R120G αB-Crystallin Is Caused by Altered Filament Interactions and Is Dependent upon Network Status in Cells

2004 ◽  
Vol 15 (5) ◽  
pp. 2335-2346 ◽  
Author(s):  
Ming Der Perng ◽  
Shu Fang Wen ◽  
Paul van den IJssel ◽  
Alan R. Prescott ◽  
Roy A. Quinlan
Keyword(s):  
De Novo ◽  
Late Onset ◽  
Binding Capacity ◽  
Disease Process ◽  
Apparent Dissociation Constant ◽  
Binding Characteristics ◽  
Αb Crystallin ◽  
Network Status ◽  
Filament Networks

The R120G mutation in αB-crystallin causes desmin-related myopathy. There have been a number of mechanisms proposed to explain the disease process, from altered protein processing to loss of chaperone function. Here, we show that the mutation alters the in vitro binding characteristics of αB-crystallin for desmin filaments. The apparent dissociation constant of R120G αB-crystallin was decreased while the binding capacity was increased significantly and as a result, desmin filaments aggregated. These data suggest that the characteristic desmin aggregates seen as part of the disease histopathology can be caused by a direct, but altered interaction of R120G αB-crystallin with desmin filaments. Transfection studies show that desmin networks in different cell backgrounds are not equally affected. Desmin networks are most vulnerable when they are being made de novo and not when they are already established. Our data also clearly demonstrate the beneficial role of wild-type αB-crystallin in the formation of desmin filament networks. Collectively, our data suggest that R120G αB-crystallin directly promotes desmin filament aggregation, although this gain of a function can be repressed by some cell situations. Such circumstances in muscle could explain the late onset characteristic of the myopathies caused by mutations in αB-crystallin.

scholarly journals Lysophosphatidic acid acyltransferase 3 regulates Golgi complex structure and function

2009 ◽  
Vol 186 (2) ◽  
pp. 211-218 ◽  
Author(s):  
John A. Schmidt ◽  
William J. Brown
Keyword(s):  
Lysophosphatidic Acid ◽  
Golgi Complex ◽  
Membrane Trafficking ◽  
Functional Organization ◽  
Complex Structure ◽  
Golgi Membrane ◽  
Direct Role ◽  
Tubule Formation ◽  
In Cells

Recent studies have suggested that the functional organization of the Golgi complex is dependent on phospholipid remodeling enzymes. Here, we report the identification of an integral membrane lysophosphatidic acid–specific acyltransferase, LPAAT3, which regulates Golgi membrane tubule formation, trafficking, and structure by altering phospholipids and lysophospholipids. Overexpression of LPAAT3 significantly inhibited the formation of Golgi membrane tubules in vivo and in vitro. Anterograde and retrograde protein trafficking was slower in cells overexpressing LPAAT3 and accelerated in cells with reduced expression (by siRNA). Golgi morphology was also dependent on LPAAT3 because its knockdown caused the Golgi to become fragmented. These data are the first to show a direct role for a specific phospholipid acyltransferase in regulating membrane trafficking and organelle structure.

scholarly journals Acaps Are Arf6 Gtpase-Activating Proteins That Function in the Cell Periphery

2000 ◽  
Vol 151 (3) ◽  
pp. 627-638 ◽  
Author(s):  
Trevor R. Jackson ◽  
Fraser D. Brown ◽  
Zhongzhen Nie ◽  
Koichi Miura ◽  
Letizia Foroni ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Coiled Coil ◽  
Bound State ◽  
Cell Periphery ◽  
Endosomal Membrane ◽  
3T3 Fibroblasts ◽  
Gtpase Activating Proteins ◽  
Nih 3T3 ◽  
Tubular Membranes

The GTP-binding protein ADP-ribosylation factor 6 (Arf6) regulates endosomal membrane trafficking and the actin cytoskeleton in the cell periphery. GTPase-activating proteins (GAPs) are critical regulators of Arf function, controlling the return of Arf to the inactive GDP-bound state. Here, we report the identification and characterization of two Arf6 GAPs, ACAP1 and ACAP2. Together with two previously described Arf GAPs, ASAP1 and PAP, they can be grouped into a protein family defined by several common structural motifs including coiled coil, pleckstrin homology, Arf GAP, and three complete ankyrin-repeat domains. All contain phosphoinositide-dependent GAP activity. ACAP1 and ACAP2 are widely expressed and occur together in the various cultured cell lines we examined. Similar to ASAP1, ACAP1 and ACAP2 were recruited to and, when overexpressed, inhibited the formation of platelet-derived growth factor (PDGF)–induced dorsal membrane ruffles in NIH 3T3 fibroblasts. However, in contrast with ASAP1, ACAP1 and ACAP2 functioned as Arf6 GAPs. In vitro, ACAP1 and ACAP2 preferred Arf6 as a substrate, rather than Arf1 and Arf5, more so than did ASAP1. In HeLa cells, overexpression of either ACAP blocked the formation of Arf6-dependent protrusions. In addition, ACAP1 and ACAP2 were recruited to peripheral, tubular membranes, where activation of Arf6 occurs to allow membrane recycling back to the plasma membrane. ASAP1 did not inhibit Arf6-dependent protrusions and was not recruited by Arf6 to tubular membranes. The additional effects of ASAP1 on PDGF-induced ruffling in fibroblasts suggest that multiple Arf GAPs function coordinately in the cell periphery.

scholarly journals WIF-B cells: an in vitro model for studies of hepatocyte polarity.

1993 ◽  
Vol 123 (6) ◽  
pp. 1761-1775 ◽  
Author(s):  
G Ihrke ◽  
E B Neufeld ◽  
T Meads ◽  
M R Shanks ◽  
D Cassio ◽  
...  
Keyword(s):  
B Cells ◽  
Membrane Trafficking ◽  
Incubation Temperature ◽  
De Novo ◽  
Hybrid Cell ◽  
Rat Hepatocytes ◽  
Membrane Lipid ◽  
Suitable Model

We have evaluated the utility of the hepatoma-derived hybrid cell line, WIF-B, for in vitro studies of polarized hepatocyte functions. The majority (&gt; 70%) of cells in confluent culture formed closed spaces with adjacent cells. These bile canalicular-like spaces (BC) accumulated fluorescein, a property of bile canaliculi in vivo. By indirect immunofluorescence, six plasma membrane (PM) proteins showed polarized distributions similar to rat hepatocytes in situ. Four apical PM proteins were concentrated in the BC membrane of WIF-B cells. Microtubules radiated from the BC (apical) membrane, and actin and foci of gamma-tubulin were concentrated in this region. The tight junction-associated protein ZO-1 was present in belts marking the boundary between apical and basolateral PM domains. We explored the functional properties of this boundary in living cells using fluorescent membrane lipid analogs and soluble tracers. When cells were incubated at 4 degrees C with a fluorescent analog of sphingomyelin, only the basolateral PM was labeled. In contrast, when both PM domains were labeled by de novo synthesis of fluorescent sphingomyelin from ceramide, fluorescent lipid could only be removed from the basolateral domain. These data demonstrate the presence of a barrier to the lateral diffusion of lipids between the PM domains. However, small soluble FITC-dextrans (4,400 mol wt) were able to diffuse into BC, while larger FITC-dextrans were restricted to various degrees depending on their size and incubation temperature. At 4 degrees C, the surface labeling reagent sNHS-LC-biotin (557 mol wt) had access to the entire PM, but streptavidin (60,000 mol wt), which binds to biotinylated molecules, was restricted to only the basolateral domain. Such differential accessibility of well-characterized probes can be used to mark each membrane domain separately. These results show that WIF-B cells are a suitable model to study membrane trafficking and targeting in hepatocytes in vitro.

scholarly journals Connected function of PRAF/RLD and GNOM in membrane trafficking controls intrinsic cell polarity in plants

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Lu Wang ◽  
Dongmeng Li ◽  
Kezhen Yang ◽  
Xiaoyu Guo ◽  
Chao Bian ◽  
...  
Keyword(s):  
Cell Polarity ◽  
Membrane Trafficking ◽  
Asymmetric Cell Division ◽  
Endosomal Trafficking ◽  
Dependent Manner ◽  
Developmental Defects ◽  
Organismal Development ◽  
Stomatal Lineage

AbstractCell polarity is a fundamental feature underlying cell morphogenesis and organismal development. In the Arabidopsis stomatal lineage, the polarity protein BASL controls stomatal asymmetric cell division. However, the cellular machinery by which this intrinsic polarity site is established remains unknown. Here, we identify the PRAF/RLD proteins as BASL physical partners and mutating four PRAF members leads to defects in BASL polarization. Members of PRAF proteins are polarized in stomatal lineage cells in a BASL-dependent manner. Developmental defects of the praf mutants phenocopy those of the gnom mutants. GNOM is an activator of the conserved Arf GTPases and plays important roles in membrane trafficking. We further find PRAF physically interacts with GNOM in vitro and in vivo. Thus, we propose that the positive feedback of BASL and PRAF at the plasma membrane and the connected function of PRAF and GNOM in endosomal trafficking establish intrinsic cell polarity in the Arabidopsis stomatal lineage.

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