The Transmembrane Region Is Responsible for Targeting of Adaptor Protein LAX into “Heavy Rafts”

BACKGROUND: The importance of membrane compartmentalization into specific membrane microdomains has been shown in many biological processes such as immunoreceptor signaling, membrane trafficking, pathogen infection, and tumor progression. Microdomains like lipid rafts, caveolae and tetraspanin enriched microdomains are relatively resistant to solubilization by some detergents. Large detergent-resistant membrane fragments (DRMs) resulting from such membrane solubilization can be conveniently isolated by density gradient ultracentrifugation or gel filtration. Recently, we described a novel type of raft-like membrane microdomains producing, upon detergent Brij98 solubilization, "heavy DRMs" and containing a number of functionally relevant proteins. Transmembrane adaptor protein LAX is a typical "heavy raft" protein. The present study was designed to identify the molecular determinants targeting LAX-derived constructs to heavy rafts. METHODOLOGY/PRINCIPAL FINDINGS: We prepared several constructs encoding chimeric proteins containing various informative segments of the LAX sequence and evaluated their effects on targeting to heavy rafts. Replacement of the polybasic membrane-proximal part of LAX by CD3ε-derived membrane-proximal part had no effect on LAX solubilization. Similarly, the membrane-proximal part of LAX, when introduced into non-raft protein CD25 did not change CD25 detergent solubility. These results indicated that membrane-proximal part of LAX is not important for LAX targeting to heavy rafts. On the other hand, the replacement of transmembrane part of CD25 by the transmembrane part of LAX resulted in targeting into heavy rafts. We also show that LAX is not S-acylated, thus palmitoylation is not involved in LAX targeting to heavy rafts. Also, covalent dimerization was excluded as a cause of targeting into heavy rafts. CONCLUSIONS/SIGNIFICANCE: We identified the transmembrane domain of LAX as a first motif targeting transmembrane protein constructs to detergent-resistant heavy rafts, a novel type of membrane microdomains containing a number of physiologically important proteins

The Csk-binding protein PAG regulates PDGF-induced Src mitogenic signaling via GM1

Spatial regulation is an important feature of signal specificity elicited by cytoplasmic tyrosine kinases of the Src family (SRC family protein tyrosine kinases [SFK]). Cholesterol-enriched membrane domains, such as caveolae, regulate association of SFK with the platelet-derived growth factor receptor (PDGFR), which is needed for kinase activation and mitogenic signaling. PAG, a ubiquitously expressed member of the transmembrane adaptor protein family, is known to negatively regulate SFK signaling though binding to Csk. We report that PAG modulates PDGFR levels in caveolae and SFK mitogenic signaling through a Csk-independent mechanism. Regulation of SFK mitogenic activity by PAG requires the first N-terminal 97 aa (PAG-N), which include the extracellular and transmembrane domains, palmitoylation sites, and a short cytoplasmic sequence. We also show that PAG-N increases ganglioside GM1 levels at the cell surface and, thus, displaces PDGFR from caveolae, a process that requires the ganglioside-specific sialidase Neu-3. In conclusion, PAG regulates PDGFR membrane partitioning and SFK mitogenic signaling by modulating GM1 levels within caveolae independently from Csk

Fab antibody fragment-functionalized liposomes for specific targeting of antigen-positive cells

Liposomes functionalized with monoclonal antibodies or their antigen-binding fragments have attracted much attention as specific drug delivery devices for treatment of various diseases including cancer. The conjugation of antibodies to liposomes is usually achieved by covalent coupling using cross-linkers in a reaction that might adversely affect the characteristics of the final product. Here we present an alternative strategy for liposome functionalization: we created a recombinant Fab antibody fragment genetically fused on its C-terminus to the hydrophobic peptide derived from pulmonary surfactant protein D, which became inserted into the liposomal bilayer during liposomal preparation and anchored the Fab onto the liposome surface. The Fab-conjugated liposomes specifically recognized antigen-positive cells and efficiently delivered their cargo, the Alexa Fluor 647 dye, into target cells in vitro and in vivo. In conclusion, our approach offers the potential for straightforward development of nanomedicines functionalized with an antibody of choice without the need of harmful cross-linkers.This work has received funding from the European Union's Seventh Framework Program (FP7/2007-2013; grant agreement NMP4-LA-2009-228827 NANOFOL) and Horizon 2020 Research and Innovation Program (grant agreement No 683356 - FOLSMART), further from the Portuguese Foundation for Science and Technology under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020.info:eu-repo/semantics/publishedVersio

Rozhovor Britských listů 465. Covid-19: Teď opravdu nerozvolňujte! [Britské listy Interview 465. Covid-19: Do not abolish quarantine measures at this dangerous point!]

Jan Čulík talks to the well-known Czech immunologist Professor Václav Hořejší about the current wave of omicron infection in the Czech Republic. He expresses the hope that the infection wave will subside, not because omicron is less dangerous, but because of a high percentage of Czech who have been vaccinated and a high percentage of Czechs who have already been infected by covid. But the rising omicron wave in the Czech Republic will only have an impact on Czech hospitals in two to three weeks time. So this is NOT the time to cancel quarantine measures as yet. The interview was broadcast by the Czech cable TV station Regionalnitelevize.cz on Friday 4th February 2022

Rozhovor Britských listů 358. Koronavirus: Chování ministra zdravotnictví je skandál [Britské listy Interview 358. Coronavirus: The behaviour of the Czech Health Secretary is scandalous]

In connection with the catastrophic pandemic situation in the Czech Republic (globally, it has the largest numbers of deaths and infections per capita), Jan Čulík again interviews the immunologist Professor Václav Hořejší about the minimum vaccination rate, about the dysfunctionality of the Czech state institutions and about the threat of new coronavirus variants which can avoid vaccinations. The interview was broadcast on the Czech cable TV station Regionalnitelevize.cz from Friday 29th January 2021

Rozhovor Britských listů 325. Koronavirová pandemie v ČR se zhoršuje. Co teď? [Britské listy Interview 325. The coronavirus pandemic is getting worse in the Czech Republic. What now?]

In the first weeks of October 2020, the Czech Republic has recorded the highest number of Covid-19 infections per capita in Europe. Jan Čulík again speaks to top Czech immunologist Václav Hořejší asking him what has caused this and what should the authorities and the inhabitants be doing next. The interview was broadcast by the Czech cable TV Regionalnitelevize.cz from Friday 9th October 2020

Rozhovor Britských listů 343. Nejlépe proti covidu bojuje Německo. Napodobujme to! [Britskí listy Interview 343. Germany is the most successfull country in fighting Covid-19. Let us imitate them!]

The Czech Parliament has extended the anti-coronavirus measures until 23rd December 2020, but it refused to extend it for a full month, which is required. What is the current situation with the pandemic in the Czech Republic, when will vaccination start and should we be afraid of coronavirus mutations? These are the topics of this Britské listy interview in which Jan Čulík talks to the well-know immunologist Professor Václav Hořejší of the Czech Academty of Scientists. The interview was broadcast by the Czech cable TV station Regionalnitelevize.cz from 11th December 2020

Rozhovor Britských listů 479. Covid-19: Urgentně se nechte očkovat třetí dávkou [Britské listy Interview 479. Covid-19: Get vaccinated with the third dose urgently]

We may be in the midst of a Russian military attack against Ukraine, which is keeping the media busy, but covid has not gone away. Jan Čulík talks to immunologist Václav Hořejší about the current pandemic situation in the Czech Republic. The interview was broadcast by the Czech cabler TV station Regionalnitelevize.cz from 25th March 2022

Rozhovor Britských listů 439. Václav Hořejší: Nechte se očkovat, nebo bude katastrofa [Britské listy Interview 439. Václav Hořejší: Get vaccinated, or a catastrophe will follow]

The coronavirus pandemic is getting much worse in the Czech Republic again. Jan Čulík talks to the well-known Czech immunologist Professor Václav Hořejší about what next. The interview was broadcast by the Czech cable TV station Regionalnitelevize.cz from 5th November 2021

Rozhovor Britských listů 329. Bude úspěch, jestli v ČR zemře "jen" 10 000 nebo i 20 000 lidí [Britské listy Interview 329. It will be a success if "only" 10 000 or 20 000 dies in the Czech Republic]

The situation regarding the coronavirus pandemic is catastrophic in the Czech Republic. On Thursday, 22nd October, 15 000 infected individuals were officially recorded, but that is only a fraction of the real number of infections. What will happen next? Jan Čulík talks again to top Czech immunologist Professor Václav Hořejší. The interview was broadcast by the Czech cable TV station Regionalnitelevize.cz from Friday 23rd October 2020