Multiplexed phospholipid membrane platform for curvature sensitive protein screening

The curvature of lipid membranes plays a key role in many relevant biological processes such as membrane trafficking, vesicular budding or host-virus interactions. In-vitro studies on membrane curvature of simplified...

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STUDIES ON HOST-VIRUS INTERACTIONS IN THE CHICK EMBRYO-INFLUENZA VIRUS SYSTEM

Journal of Experimental Medicine ◽

10.1084/jem.101.5.493 ◽

1955 ◽

Vol 101 (5) ◽

pp. 493-506 ◽

Cited By ~ 7

Author(s):

Kurt Paucker ◽

Werner Henle

Keyword(s):

Influenza Virus ◽

Infectious Virus ◽

Allantoic Fluid ◽

Progressive Reduction ◽

Virus Particles ◽

Host Virus Interactions ◽

Virus Interactions ◽

Available Information ◽

Virus System

An experimental analysis is here presented of the conditions that lead to the appearance of non-infectious hemagglutinins (NIHA) in the allantoic fluid of chick embryos injected with standard influenza virus (PR8 strain) which had been exposed to 37°C. in vitro for various periods of time. On progressive reduction of the infectivity of the undiluted inocula from about 109 to 103 ID50 (103.2 HA units) the yields of infectious virus in 24 hours decreased in straight correspondence 1 millionfold, but those of hemagglutinins only by a factor of 10. Thus the proportions of NIHA in the yields increased sharply but the total quantity obtained decreased gradually. The quantities of infectious virus produced per ID50 injected were the same throughout this range; i.e., between 50 and 100 ID50, regardless of increasing proportions of heat-inactivated virus in the seeds. This value agrees with previous estimates of yields under other conditions. Thus, initiation and completion of first cycles by the infectious virus remaining in the inocula were not, or at most, slightly inhibited. The inactivated virus, therefore, failed to establish immediate interference. It was capable, however, of holding the infectious process to one cycle. Upon 10-fold dilution of the seeds essentially similar results were obtained except that a slight loss in interfering activity could now be detected with an increase in exposure to 37°C. With further dilutions little or no interference was noted. The capacity to yield NIHA decreased slowly during exposure of the seeds to 37°C. over a period of 5 days, thereafter more rapidly. It could not be restored by addition of infectious virus. Furthermore, since NIHA was obtained when the seeds contained as little as 102 or 103 ID50, it is unlikely that it was derived from those cells which had adsorbed both infectious and inactivated seed virus. It is suggestive that multiple adsorption of inactivated virus particles per se will yield NIHA. The available information, as discussed, favors the view that the NIHA does not represent seed virus in some form but is newly produced.

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Discrete Determinants in ArfGAP2/3 Conferring Golgi Localization and Regulation by the COPI Coat

Molecular Biology of the Cell ◽

10.1091/mbc.e08-10-1010 ◽

2009 ◽

Vol 20 (3) ◽

pp. 859-869 ◽

Cited By ~ 32

Author(s):

Lena Kliouchnikov ◽

Joëlle Bigay ◽

Bruno Mesmin ◽

Anna Parnis ◽

Moran Rawet ◽

...

Keyword(s):

Lipid Membranes ◽

Membrane Curvature ◽

In Vitro Assays ◽

Minor Role ◽

Gtp Hydrolysis ◽

Gtpase Activating Proteins ◽

Golgi Localization ◽

Adp Ribosylation Factor ◽

Fusion Approach

From yeast to mammals, two types of GTPase-activating proteins, ArfGAP1 and ArfGAP2/3, control guanosine triphosphate (GTP) hydrolysis on the small G protein ADP-ribosylation factor (Arf) 1 at the Golgi apparatus. Although functionally interchangeable, they display little similarity outside the catalytic GTPase-activating protein (GAP) domain, suggesting differential regulation. ArfGAP1 is controlled by membrane curvature through its amphipathic lipid packing sensor motifs, whereas Golgi targeting of ArfGAP2 depends on coatomer, the building block of the COPI coat. Using a reporter fusion approach and in vitro assays, we identified several functional elements in ArfGAP2/3. We show that the Golgi localization of ArfGAP3 depends on both a central basic stretch and a carboxy-amphipathic motif. The basic stretch interacts directly with coatomer, which we found essential for the catalytic activity of ArfGAP3 on Arf1-GTP, whereas the carboxy-amphipathic motif interacts directly with lipid membranes but has minor role in the regulation of ArfGAP3 activity. Our findings indicate that the two types of ArfGAP proteins that reside at the Golgi use a different combination of protein–protein and protein–lipid interactions to promote GTP hydrolysis in Arf1-GTP.

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Coding variants identified in diabetic patients alter PICK1 BAR domain function in insulin granule biogenesis

10.1101/2020.10.05.325951 ◽

2020 ◽

Author(s):

Rita C. Andersen ◽

Matthew D. Lycas ◽

Jan H. Schmidt ◽

Nikolaj R. Christensen ◽

Viktor K. Lund ◽

...

Keyword(s):

Membrane Trafficking ◽

Lipid Membranes ◽

Binding Capacity ◽

Secretory Granules ◽

Dominant Negative ◽

Diabetic Patients ◽

Bar Domain ◽

Coding Variants ◽

Positively Charged

SummaryBin/amphiphysin/Rvs (BAR) domains are positively charged crescent-shaped modules that shape negatively charged curved lipid membranes during membrane remodeling processes. The BAR domain proteins ICA69, PICK1 and arfaptins have recently been demonstrated to coordinate the budding and formation of immature secretory granules (ISGs) at the trans-Golgi network. Here, we identify four coding variants in the PICK1 gene from a Danish whole-exome screening of diabetic patients, that all involve change of positively charged residues in the PICK1 BAR domain. All four coding variants failed to rescue the insulin content in INS-1E cells upon KD of endogenous PICK1. Moreover, two variants showed dominant negative properties. Interestingly, in vitro assays addressing the BAR domain function suggest that the coding variants compromised membrane binding capacity but increased capacity to cause fission of liposomes.Live confocal microscopy and super-resolution microscopy further revealed that PICK1 resides transiently on ISGs before egress via vesicular budding events. Interestingly, this egress of PICK1 was accelerated in the coding variants. We propose that PICK1 assists or complements the removal of excess membrane and generic membrane trafficking proteins, and possibly also insulin from ISGs during the maturation process and that the coding variants may cause premature budding possibly explaining their dominant negative function.

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Comparative study of curvature sensing mediated by F-BAR domain and an intrinsically disordered region of FBP17

10.1101/2020.07.30.230037 ◽

2020 ◽

Author(s):

Maohan Su ◽

Yinyin Zhuang ◽

Xinwen Miao ◽

Yongpeng Zeng ◽

Weibo Gao ◽

...

Keyword(s):

Lipid Bilayer ◽

Membrane Trafficking ◽

Membrane Curvature ◽

Wave System ◽

Common Belief ◽

Bar Domain ◽

Curvature Sensing ◽

Intrinsically Disordered ◽

Intrinsically Disordered Region

Membrane curvature has emerged as an intriguing physical organization principle underlying biological signaling and membrane trafficking. FBP17 of the CIP4/FBP17/Toca-1 F-BAR family is unique in the BAR family because its structurally folded F-BAR domain does not contain any hydrophobic motifs that insert into lipid bilayer. While it has been widely assumed so, whether the banana-shaped F-BAR domain alone can sense curvature has never been experimentally demonstrated. Using a nanopillar-supported lipid bilayer system, we found that the F-BAR domain of FBP17 displayed minimal curvature sensing in vitro. We further identified an alternatively spliced intrinsically disordered region (IDR) of FBP17 next to its F-BAR domain that is conserved in sequence across species. The IDR senses membrane curvature and its sensing ability greatly exceeds that of F-BAR domain alone. In living cells, presence of the IDR domain changed the dynamics of FBP17 recruitment in a curvature-coupled cortical wave system. Collectively, we propose that FBP17 does sense curvature but contrary to the common belief, its curvature sensing capability largely originates from its disordered region, not F-BAR domain itself.

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Reversible ADP-ribosylation of RNA

Nucleic Acids Research ◽

10.1093/nar/gkz305 ◽

2019 ◽

Vol 47 (11) ◽

pp. 5658-5669 ◽

Cited By ~ 33

Author(s):

Deeksha Munnur ◽

Edward Bartlett ◽

Petra Mikolčević ◽

Ilsa T Kirby ◽

Johannes Gregor Matthias Rack ◽

...

Keyword(s):

Dna Repair ◽

Stress Response ◽

Nicotinamide Adenine Dinucleotide ◽

Cellular Stress Response ◽

Biological Processes ◽

Adp Ribosylation ◽

Human Proteins ◽

Novel Target ◽

Host Virus Interactions ◽

Virus Interactions

Abstract ADP-ribosylation is a reversible chemical modification catalysed by ADP-ribosyltransferases such as PARPs that utilize nicotinamide adenine dinucleotide (NAD+) as a cofactor to transfer monomer or polymers of ADP-ribose nucleotide onto macromolecular targets such as proteins and DNA. ADP-ribosylation plays an important role in several biological processes such as DNA repair, transcription, chromatin remodelling, host-virus interactions, cellular stress response and many more. Using biochemical methods we identify RNA as a novel target of reversible mono-ADP-ribosylation. We demonstrate that the human PARPs - PARP10, PARP11 and PARP15 as well as a highly diverged PARP homologue TRPT1, ADP-ribosylate phosphorylated ends of RNA. We further reveal that ADP-ribosylation of RNA mediated by PARP10 and TRPT1 can be efficiently reversed by several cellular ADP-ribosylhydrolases (PARG, TARG1, MACROD1, MACROD2 and ARH3), as well as by MACROD-like hydrolases from VEEV and SARS viruses. Finally, we show that TRPT1 and MACROD homologues in bacteria possess activities equivalent to the human proteins. Our data suggest that RNA ADP-ribosylation may represent a widespread and physiologically relevant form of reversible ADP-ribosylation signalling.

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STUDIES ON HOST-VIRUS INTERACTIONS IN THE CHICK EMBRYO-INFLUENZA VIRUS SYSTEM

Journal of Experimental Medicine ◽

10.1084/jem.94.4.269 ◽

1951 ◽

Vol 94 (4) ◽

pp. 269-289 ◽

Cited By ~ 11

Author(s):

Oscar C. Liu ◽

Werner Henle

Keyword(s):

Influenza Virus ◽

Growth Curve ◽

High Speed ◽

Allantoic Fluid ◽

In Vitro Tests ◽

Host Virus Interactions ◽

Virus Interactions ◽

Virus System

The role of inhibitors of hemagglutination in the evaluation of host-virus interactions in the chick embryo-influenza virus system has been analyzed. Comparisons were made between materials (allantoic fluids and membrane suspensions) derived from in vivo (growth curve) experiments at hourly intervals after inoculation, and from in vitro tests in which normal allantoic fluids and membrane suspensions were incubated with virus at 37°C. for various periods of time. In both instances large amounts of virus were added to the systems, resulting in comparable concentrations of the agent. The seeds employed were either fully active or irradiated by ultraviolet light to the extent that the virus lost its capacity to increase but kept its interfering and hemagglutinating properties. The various materials were assayed for (a) the hemagglutinating titers of the virus present in the systems before and after heating to 56°C.; (b) the concentration of inhibitor in the materials at various stages of incubation after heating to 70°C. for 30 minutes as measured by the hemagglutination-inhibition reaction with native or heated test virus (30 minutes 56°C.); and (c) the degree of adsorption of the hemagglutinins present in the materials onto chicken red cells at 0°C. and their subsequent elution at 37°C. The effects of receptor-destroying enzyme (RDE), treatment with sodium periodate, or high speed centrifugation on the inhibitory activities were studied in some of the tests. The essential results which indicate certain sources of error in the evaluation of host-virus interactions as well as means for studying virus activity at the early stages of the infectious process, were as follows: 1. Though some inhibitory effects on hemagglutination were noticeable in the allantoic fluid during the 1st hour after inoculation they were, as a rule, no longer apparent after this interval, and treatment with RDE did not increase the hemagglutinin titers. Thus, the interpretation of growth curve data concerning allantoic fluids hardly seems to be affected by inhibitor. On the other hand, striking effects were noted with the membrane suspensions of growth curve experiments in that RDE shortened the latent period to 2 hours and the titers in the first few positive samples (4 to 5 hours) increased) whereas in later harvests no such effect was noted. Under these conditions complement-fixation antigens and hemagglutinins made their appearance in the tissues simultaneously and not as previously reported the former prior to the latter. However, the infectivity showed increments only several hours after these two activities had become measurable. Thus the hypothesis of the stagewise development of influenza virus is still supported by these data. 2. Using the inhibition of hemagglutination technic it was found that the inhibitor in allantoic fluid rapidly decreased as a result of the action of active and irradiated virus, but destruction was never complete. In the membranes of the in vivo series only active seed led to loss of inhibitor, again without complete destruction, beginning at the time complement-fixing antigen and hemagglutinins became measurable. Irradiated seed was without effect in vivo whereas, in the in vitro tests it equalled the activity of the active virus. The implications of this difference in the effectiveness of active and irradiated seed in vivo with regard to the understanding of the mode of viral multiplication are discussed. 3. Although many factors may influence the shape of adsorption-elution curves it is felt that at 0°C. the extent of adsorption is directly related to the amount of inhibitor present in the systems. In the early hours after inoculation the degree of adsorption was relatively small but it increased gradually with the time of incubation. The inhibitor of adsorption was destroyed by RDE and NaIO4 and was only partially sedimentable by high speed centrifugation. In every respect studied its properties corresponded with the findings obtained with inhibitors in the hemagglutination-inhibition technic. Although the difference in the rapidity of inhibitor destruction as measured by the various technics might suggest a multiplicity of inhibitors it is felt that it rather denotes a greater sensitivity of the adsorption technic as compared to the others.

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PI(3,5)P2 controls endosomal branched actin dynamics by regulating cortactin–actin interactions

The Journal of Cell Biology ◽

10.1083/jcb.201412127 ◽

2015 ◽

Vol 210 (5) ◽

pp. 753-769 ◽

Cited By ~ 49

Author(s):

Nan Hyung Hong ◽

Aidong Qi ◽

Alissa M. Weaver

Keyword(s):

Membrane Trafficking ◽

Membrane Curvature ◽

Actin Dynamics ◽

Complex Activity ◽

Actin Networks ◽

Binding Partner ◽

Late Endosomes ◽

Actin Turnover ◽

Direct Binding

Branched actin critically contributes to membrane trafficking by regulating membrane curvature, dynamics, fission, and transport. However, how actin dynamics are controlled at membranes is poorly understood. Here, we identify the branched actin regulator cortactin as a direct binding partner of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) and demonstrate that their interaction promotes turnover of late endosomal actin. In vitro biochemical studies indicated that cortactin binds PI(3,5)P2 via its actin filament-binding region. Furthermore, PI(3,5)P2 competed with actin filaments for binding to cortactin, thereby antagonizing cortactin activity. These findings suggest that PI(3,5)P2 formation on endosomes may remove cortactin from endosome-associated branched actin. Indeed, inhibition of PI(3,5)P2 production led to cortactin accumulation and actin stabilization on Rab7+ endosomes. Conversely, inhibition of Arp2/3 complex activity greatly reduced cortactin localization to late endosomes. Knockdown of cortactin reversed PI(3,5)P2-inhibitor–induced actin accumulation and stabilization on endosomes. These data suggest a model in which PI(3,5)P2 binding removes cortactin from late endosomal branched actin networks and thereby promotes net actin turnover.

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Glycosyltransferases encoded by viruses

Journal of General Virology ◽

10.1099/vir.0.80320-0 ◽

2004 ◽

Vol 85 (10) ◽

pp. 2741-2754 ◽

Cited By ~ 62

Author(s):

Nicolas Markine-Goriaynoff ◽

Laurent Gillet ◽

James L. Van Etten ◽

Haralambos Korres ◽

Naresh Verma ◽

...

Keyword(s):

Evolution Process ◽

Cellular Biology ◽

Biological Processes ◽

Virally Encoded ◽

Host Virus Interactions ◽

Virus Interactions

Studies of cellular biology in recent decades have highlighted the crucial roles of glycans in numerous important biological processes, raising the concept of glycomics that is now considered as important as genomics, transcriptomics and proteomics. For millions of years, viruses have been co-evolving with their hosts. Consequently, during this co-evolution process, viruses have acquired mechanisms to mimic, hijack or sabotage host processes that favour their replication, including mechanisms to modify the glycome. The importance of the glycome in the regulation of host–virus interactions has recently led to a new concept called ‘glycovirology’. One fascinating aspect of glycovirology is the study of how viruses affect the glycome. Viruses reach that goal either by regulating expression of host glycosyltransferases or by expressing their own glycosyltransferases. This review describes all virally encoded glycosyltransferases and discusses their established or putative functions. The description of these enzymes illustrates several intriguing aspects of virology and provides further support for the importance of glycomics in biological processes.

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Structure and Dynamics of GPCRs in Lipid Membranes: Physical Principles and Experimental Approaches

Molecules ◽

10.3390/molecules25204729 ◽

2020 ◽

Vol 25 (20) ◽

pp. 4729

Author(s):

Andrew J. Y. Jones ◽

Florian Gabriel ◽

Aditi Tandale ◽

Daniel Nietlispach

Keyword(s):

Lipid Bilayer ◽

Lipid Membranes ◽

In Vitro Studies ◽

Specific Effects ◽

Biophysical Studies ◽

Experimental Approaches ◽

Specific Lipid ◽

Physical Principles ◽

Insight Into

Over the past decade, the vast amount of information generated through structural and biophysical studies of GPCRs has provided unprecedented mechanistic insight into the complex signalling behaviour of these receptors. With this recent information surge, it has also become increasingly apparent that in order to reproduce the various effects that lipids and membranes exert on the biological function for these allosteric receptors, in vitro studies of GPCRs need to be conducted under conditions that adequately approximate the native lipid bilayer environment. In the first part of this review, we assess some of the more general effects that a membrane environment exerts on lipid bilayer-embedded proteins such as GPCRs. This is then followed by the consideration of more specific effects, including stoichiometric interactions with specific lipid subtypes. In the final section, we survey a range of different membrane mimetics that are currently used for in vitro studies, with a focus on NMR applications.

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