FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2273502465> ?p ?o ?g. }

• W2273502465 endingPage "307" @default.
• W2273502465 startingPage "305" @default.
• W2273502465 abstract "It was a lucky break for me when Ari Helenius decided to join my research group at University of Helsinki where I had started as a principal investigator (PI) after my postdoc at the Rockefeller University in New York. I was interested in lipoproteins and membranes. These were early days – more than 45 years ago – when the bilayer model was being replaced by a membrane model consisting of lipoprotein subunits. Ari decided to use plasma low-density lipoproteins (LDLs) as experimental models to study lipid–protein interactions. We were interested in LDL for two reasons. First, they were abundant and easy to purify. Second, my postdoc friend Kåre Berg had detected an inherited variant of human LDL, the Lp (a) lipoprotein. I was hoping that this serum polymorphism would give us clues to the molecular architecture of LDL. Our first finding was that SDS-gel electrophoresis gave an apparent molecular weight of 230000 for the LDL apoprotein, which was in complete disagreement with earlier observations. We could not understand why previous attempts by the leaders in the field had produced LDL proteins with much smaller molecular weights, down to 35000. But, our estimate turned out to be correct. The others had left their lipoprotein preparation in SDS on the bench, not realizing that proteases contaminating the preparation were SDS-resistant and digested this large protein to pieces. Ari then devised a method to delipidate LDL. He compared four different detergents, SDS, sodium deoxycholate, Nonidet P40 and cetyltrimethylammoniumbromide, and found that they all could remove the lipids from the apoprotein but the state of the protein depended on the detergent employed 1. The lipid-free protein obtained with Nonidet P40 and sodium deoxycholate retained the properties of the native LDL while SDS and cetyltrimethylammoniumbromide denatured the protein. These insights led to the classic paper, showing that lipophilic proteins, lipoproteins and membrane proteins bind mild detergents when delipidated 2. In contrast, hydrophilic proteins bound little or no Triton-X-100 or deoxycholate. The concept that emerged from this work was that the hydrophobic parts of the lipophilic proteins became covered by micellar detergent during the delipidation process, to keep the proteins soluble. Denaturing detergents such as SDS unfolded most proteins and exposed hydrophobic surfaces even in hydrophilic proteins that bound detergent. This simple principle was then employed to devise a charge-shift electrophoresis method to find out whether a membrane protein was integral or peripheral 3. The work on the Lp (a) lipoprotein also progressed. We showed that this lipoprotein contained two subunits, one was a highly sialylated glycoprotein, which carried the Lp (a) immunological epitope, and the other was apo-LDL. We were using enormous amounts of plasma lipoproteins for our biochemical work on LDL and Lp(a) and I was the one who had to donate the blood because I was Lp (a+). I ‘lost’ 800 mL of blood every time I donated. Although the erythrocytes were given back to me by plasmapheresis, I started to rebel. Enough was enough. I decided to shift my lab away from plasma lipoproteins and put the full focus on Semliki Forest virus (SFV), which was our membrane model. I had met Leevi Kääriäinen while I was in New York and he had introduced me to this virus. SFV had the simplest biomembrane that one could envisage. The virus envelope had only one single spike glycoprotein. Ari had already started to work on SFV – in secret. Together with Hans Söderlund, who was a PhD student with Leevi Kääriäinen, they wanted to analyze how a non-ionic detergent, Triton-X-100, solubilized the viral membrane. Ari had asked me what I thought about the project and I had showed little interest. So, Ari and Hans carried out a spectacular study, employing analytical and density gradient centrifugation to stepwise dissociate the SFV membrane with the detergent. The proteins of the virus could be labeled with radioactive lysine and the lipids with radioactive phosphate. Below its critical micellar concentration, Triton-X-100 bound as monomers to the virus membrane. By increasing the detergent concentration, the virus membrane became lysed and the nucleocapsid popped out from the particle. Finally, the membrane was solubilized into small protein–lipid complexes. They followed the stepwise solubilization by negative staining in the electron microscope, obtaining gorgeous and informative micrographs that documented the process 4. When Ari showed me their fantastic data, I wanted to be on the paper but Leevi did not agree. Since we both had done nothing and even been negatively inclined toward the project, we should not be included as authors on the paper. Rightfully so – in retrospect! With Ari as the driver, we continued these studies by expanding them to SDS and deoxycholate and could document how these different detergents solubilized membranes and how the solubilization process mechanistically progressed. Until then researchers had been using detergents more like one does when cooking without understanding the principles. On the basis of our findings, we could then write a review on the solubilization of membranes by detergents and this became a highly cited classic 5. Cell membrane research was picking up speed. The lipoprotein subunit model for membrane organization had been swept away in the 1970s. The bilayer regained its role as the basic structural model for the cell membrane, which developed into the fluid mosaic model characterized by a two-dimensional fluid, in which transmembrane proteins were swimming around. I had received an offer to join the newly founded European Molecular Biology Laboratory (EMBL) in Heidelberg. With SFV packed away in my luggage, my family and I and two other families moved from Helsinki to Heidelberg in 1975. I had managed to convince Ari as well as Henrik Garoff, another PhD student in my lab to form a troika, and continued our studies of the simplest membrane in the world. Now we shifted gears. We began to use SFV as a tool to study the cell biology of the host cell. Henrik teamed up with Bernhard Dobberstein who had joined EMBL after his ground-breaking work together with Günther Blobel to elucidate the mechanism of how secretory proteins become translocated into the endoplasmic reticulum (ER). They used the viral RNA to reconstitute the translocation of the SFV spike glycoprotein into the ER. Ari took a completely novel approach and started to investigate how SFV enters its host cell to initiate infection. He labeled SFV with fluorescent tags and could observe how the virus bound to the BHK-21 cell surface and how the virus then disappeared into the cell. This was of course performed with the microscopes available at the time. But was utterly exciting! Imaging led the way but then a slew of other methods were employed to find out how the virus manages to deliver its RNA into the cytosol. Electron microscopy demonstrated that the surface-bound virus entered the cell by clathrin-coated endocytosis and then sequestered into intracellular vacuoles and lysosomes. Direct penetration from the plasma membrane was never observed. This was the first time that endocytosis as a path of entry for viruses was described 6. The highlight was Ari's demonstration that the SFV membrane can fuse with liposomes at pH 6, or lower. This experiment provided a beautifully simple mechanism, by which the nucleocapsid with the RNA can escape lysosomal destruction by fusing at the low pH prevailing in the lysosomes or in other intracellular vacuoles (later to be named early and late endosomes). The membrane of the virus thereby merged with the membranes of the intracellular vacuoles. The virus had learned from the Greeks and had adopted the Trojan horse trick, as Ari later described this entry pathway that many enveloped viruses employ. The fusion assay that Ari devised consisted of phospholipid-cholesterol liposomes that contained ribonuclease. When the SFV membrane fused with the liposomal membrane at low pH, the nucleocapsid was delivered into the liposome. As a result of content mixing, the P32-labeled viral RNA was digested by the ribonuclease. This was easily assayed by TCA solubility. The first experiment that Ari did was successful; however, the next three to five were not. I cannot exactly remember how many attempts there were before the next positive experiment. But, if it had happened the other way around, this brilliant outcome may never have been realized! In 1981, Ari Helenius moved on to become Professor of Cell Biology at Yale University. When he went to look for a position in the USA, one of the first talks on his seminar tour was at MIT where David Baltimore was his host. When Ari asked if they had a position for him, David looked at his CV and said, your CV is not OK; you have been too long with Kai. Ari went on to his next talk and lost his slides in the plane – pre-powerpoint age – and he had more than 10 seminars ahead of him. But Ari was a fantastic drawer, so he managed brilliantly without slides but with a black board. George Palade at Yale was enthusiastic and offered him a job. At Yale, Ari established a perfect partnership with Ira Mellman. Their first breakthrough was the introduction of endosomes as in-between stations on the route from the plasma membrane to the lysosomes. David indeed missed something. Working together is a strength and not a weakness. This Ari has demonstrated throughout his career as he has continued his brilliant work, employing viruses as tools to understand how cells manage to perform the way they do." @default.
• W2273502465 created "2016-06-24" @default.
• W2273502465 creator A5000708652 @default.
• W2273502465 date "2016-03-23" @default.
• W2273502465 modified "2023-10-16" @default.
• W2273502465 title "My Early Days with Ari Helenius: Detergents and Viruses" @default.
• W2273502465 cites W1599125820 @default.
• W2273502465 cites W1964031596 @default.
• W2273502465 cites W1989675515 @default.
• W2273502465 cites W2023712946 @default.
• W2273502465 cites W2078034671 @default.
• W2273502465 cites W2125545448 @default.
• W2273502465 doi "https://doi.org/10.1111/tra.12377" @default.
• W2273502465 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/26871450" @default.
• W2273502465 hasPublicationYear "2016" @default.
• W2273502465 type Work @default.
• W2273502465 sameAs 2273502465 @default.
• W2273502465 citedByCount "0" @default.
• W2273502465 crossrefType "journal-article" @default.
• W2273502465 hasAuthorship W2273502465A5000708652 @default.
• W2273502465 hasBestOaLocation W22735024651 @default.
• W2273502465 hasConcept C159047783 @default.
• W2273502465 hasConcept C86803240 @default.
• W2273502465 hasConcept C95444343 @default.
• W2273502465 hasConceptScore W2273502465C159047783 @default.
• W2273502465 hasConceptScore W2273502465C86803240 @default.
• W2273502465 hasConceptScore W2273502465C95444343 @default.
• W2273502465 hasIssue "4" @default.
• W2273502465 hasLocation W22735024651 @default.
• W2273502465 hasLocation W22735024652 @default.
• W2273502465 hasOpenAccess W2273502465 @default.
• W2273502465 hasPrimaryLocation W22735024651 @default.
• W2273502465 hasRelatedWork W1641042124 @default.
• W2273502465 hasRelatedWork W1990804418 @default.
• W2273502465 hasRelatedWork W1993764875 @default.
• W2273502465 hasRelatedWork W2013243191 @default.
• W2273502465 hasRelatedWork W2046158694 @default.
• W2273502465 hasRelatedWork W2051339581 @default.
• W2273502465 hasRelatedWork W2082860237 @default.
• W2273502465 hasRelatedWork W2117258802 @default.
• W2273502465 hasRelatedWork W2130076355 @default.
• W2273502465 hasRelatedWork W2151865869 @default.
• W2273502465 hasVolume "17" @default.
• W2273502465 isParatext "false" @default.
• W2273502465 isRetracted "false" @default.
• W2273502465 magId "2273502465" @default.
• W2273502465 workType "article" @default.