An efficient auxin-inducible degron system with low basal degradation in human cells

Auxin-inducible degron technology allows rapid and controlled protein depletion. However, basal degradation without auxin and inefficient auxin-inducible depletion have limited its utility. We have identified a potent auxin-inducible degron system composed of auxin receptor F-box protein AtAFB2 and short degron minilAA7. The system showed minimal basal degradation and enabled rapid auxin-inducible depletion of endogenous human transmembrane, cytoplasmic and nuclear proteins in 1 h with robust functional phenotypes.Peer reviewe

Autophagy Genes for Wet Age-Related Macular Degeneration in a Finnish Case-Control Study

Age-related macular degeneration is an eye disease that is the main cause of legal blindness in the elderly in developed countries. Despite this, its pathogenesis is not completely known, and many genetic, epigenetic, environmental and lifestyle factors may be involved. Vision loss in age-related macular degeneration (AMD) is usually consequence of the occurrence of its wet (neovascular) form that is targeted in the clinic by anti-VEGF (vascular endothelial growth factor) treatment. The wet form of AMD is associated with the accumulation of cellular waste in the retinal pigment epithelium, which is removed by autophagy and the proteosomal degradation system. In the present work, we searched for the association between genotypes and alleles of single nucleotide polymorphisms (SNPs) of autophagy-related genes and wet AMD occurrence in a cohort of Finnish patients undergoing anti-VEGF therapy and controls. Additionally, the correlation between treatment efficacy and genotypes was investigated. Overall, 225 wet AMD patients and 161 controls were enrolled in this study. Ten SNPs (rs2295080, rs11121704, rs1057079, rs1064261, rs573775, rs11246867, rs3088051, rs10902469, rs73105013, rs10277) in the mTOR (Mechanistic Target of Rapamycin), ATG5 (Autophagy Related 5), ULK1 (Unc-51-Like Autophagy Activating Kinase 1), MAP1LC3A (Microtubule Associated Protein 1 Light Chain 3 α), SQSTM1 (Sequestosome 1) were analyzed with RT-PCR-based genotyping. The genotype/alleles rs2295080-G, rs11121704-C, rs1057079-C and rs73105013-T associated with an increased, whereas rs2295080-TT, rs2295080-T, rs11121704-TT, rs1057079-TT, rs1057079-T, rs573775-AA and rs73105013-C with a decreased occurrence of wet AMD. In addition, the rs2295080-GG, rs2295080-GT, rs1057079-TT, rs11246867-AG, rs3088051-CC and rs10277-CC genotypes were a positively correlated cumulative number of anti-VEGF injections in 2 years. Therefore, variability in autophagy genes may have an impact on the risk of wet AMD occurrence and the efficacy of anti-VEGF treatment

Inhibition of prolyl oligopeptidase : A promising pathway to prevent the progression of age-related macular degeneration

Dry age-related macular degeneration (AMD) is a currently untreatable vision threatening disease. Impaired proteasomal clearance and autophagy in the retinal pigment epithelium (RPE) and subsequent photoreceptor damage are connected with dry AMD, but detailed pathophysiology is still unclear. In this paper, we discover inhibition of cytosolic protease, prolyl oligopeptidase (PREP), as a potential pathway to treat dry AMD. We showed that PREP inhibitor exposure induced autophagy in the RPE cells, shown by increased LC3-II levels and decreased p62 levels. PREP inhibitor treatment increased total levels of autophagic vacuoles in the RPE cells. Global proteomics was used to examine the phenotype of a commonly used cell model displaying AMD characteristics, oxidative stress and altered protein metabolism, in vitro. These RPE cells displayed induced protein aggregation and clear alterations in macromolecule metabolism, confirming the relevance of the cell model. Differences in intracellular target engagement of PREP inhibitors were observed with cellular thermal shift assay (CETSA). These differences were explained by intracellular drug exposure (the unbound cellular partition coefficient, Kpuu). Importantly, our data is in line with previous observations regarding the discrepancy between PREP's cleaving activity and outcomes in autophagy. This highlights the need to further explore PREP's role in autophagy so that more effective compounds can be designed to battle diseases in which autophagy induction is needed. The present work is the first report investigating the PREP pathway in the RPE and we predict that the PREP inhibitors can be further optimized for treatment of dry AMD.Peer reviewe

Enzymatic Oxidation of Cholesterol : Properties and Functional Effects of Cholestenone in Cell Membranes

Peer reviewe

An efficient auxin-inducible degron system with low basal degradation in human cells

Auxin-inducible degron technology allows rapid and controlled protein depletion. However, basal degradation without auxin and inefficient auxin-inducible depletion have limited its utility. We have identified a potent auxin-inducible degron system composed of auxin receptor F-box protein AtAFB2 and short degron minilAA7. The system showed minimal basal degradation and enabled rapid auxin-inducible depletion of endogenous human transmembrane, cytoplasmic and nuclear proteins in 1 h with robust functional phenotypes.Peer reviewe

Concerted regulation of NPC2 binding to endosomal/lysosomal membranes by bis(monoacylglycero)phosphate and sphingomyelin

Niemann-Pick Protein C2 (NPC2) is a small soluble protein critical for cholesterol transport within and from the lysosome and the late endosome. Intriguingly, NPC2-mediated cholesterol transport has been shown to be modulated by lipids, yet the molecular mechanism of NPC2-membrane interactions has remained elusive. Here, based on an extensive set of atomistic simulations and free energy calculations, we clarify the mechanism and energetics of NPC2-membrane binding and characterize the roles of physiologically relevant key lipids associated with the binding process. Our results capture in atomistic detail two competitively favorable membrane binding orientations of NPC2 with a low interconversion barrier. The first binding mode (Prone) places the cholesterol binding pocket in direct contact with the membrane and is characterized by membrane insertion of a loop (V59-M60-G61-I62-P63-V64P65). This mode is associated with cholesterol uptake and release. On the other hand, the second mode (Supine) places the cholesterol binding pocket away from the membrane surface, but has overall higher membrane binding affinity. We determined that bis(monoacylglycero) phosphate (BMP) is specifically required for strong membrane binding in Prone mode, and that it cannot be substituted by other anionic lipids. Meanwhile, sphingomyelin counteracts BMP by hindering Prone mode without affecting Supine mode. Our results provide concrete evidence that lipids modulate NPC2-mediated cholesterol transport either by favoring or disfavoring Prone mode and that they impose this by modulating the accessibility of BMP for interacting with NPC2. Overall, we provide a mechanism by which NPC2-mediated cholesterol transport is controlled by the membrane composition and how NPC2-lipid interactions can regulate the transport rate.Peer reviewe

Seipin traps triacylglycerols to facilitate their nanoscale clustering in the endoplasmic reticulum membrane

Seipin is a disk-like oligomeric endoplasmic reticulum (ER) protein important for lipid droplet (LD) biogenesis and triacylglycerol (TAG) delivery to growing LDs. Here we show through biomolecular simulations bridged to experiments that seipin can trap TAGs in the ER bilayer via the luminal hydrophobic helices of the protomers delineating the inner opening of the seipin disk. This promotes the nanoscale sequestration of TAGs at a concentration that by itself is insufficient to induce TAG clustering in a lipid membrane. We identify Ser166 in the alpha 3 helix as a favored TAG occupancy site and show that mutating it compromises the ability of seipin complexes to sequester TAG in silico and to promote TAG transfer to LDs in cells. While the S166D-seipin mutant colocalizes poorly with promethin, the association of nascent wild-type seipin complexes with promethin is promoted by TAGs. Together, these results suggest that seipin traps TAGs via its luminal hydrophobic helices, serving as a catalyst for seeding the TAG cluster from dissolved monomers inside the seipin ring, thereby generating a favorable promethin binding interface.Peer reviewe

High‐content imaging and structure‐based predictions reveal functional differences between Niemann‐Pick C1 variants

LDL-peräisen kolesterolin solunsisäinen kuljetus on riippuvaista NPC1-nimisen proteiinin toiminnasta lyso- somien kalvorakenteissa. Kyseistä proteiinia koodaavassa geenissä esiintyvät NPC1-proteiinin normaalin toi- minnan estävät geenimutaatiot aiheuttavat hankalan kolesterolikertymäsairauden, joka ilmenee tyypillisesti hermosolujen rappeumana johtaen ennenaikaiseen kuolemaan lapsuusiässä. Ihmisen NPC1-proteiinia koodaava geeni on hyvin heterogeeninen. Osa tämän geenin sisältämistä geenimuunnoksista tiedetään haitattomiksi poly- morfioiksi, mutta osalla NPC1-taudin kantajista ja NPC1-potilaista on geenissään muutoksia, joiden toiminnal- linen merkitys on epäselvä. Eri tutkimuslaboratorioiden välillä jaettu NPC1 cDNA-konstrukti, joka tunnetaan nimellä NPC1 Wild-Type Variant (WT-V) ja, jota on käytetty villityyppi-NPC1 vertailunäytteenä monissa tut- kimuksissa, sisältää myös tiettyjen aminohappojen muutoksia verrattuna Genbankissa julkaistuun NPC1 viite- sekvenssiin. Havainnoidaksemme paremmin pieniä toiminnallisia eroavaisuuksia NPC1-varianttien välillä, ge- neroimme stabiilisti NPC1-poistogeenisessä taustassa AAVS1 safe-harbor-lokuksesta NPC1-variantteja eks- pressoivia ihmissoluja käyttäen CRISPR/Cas9-tekniikkaa. Tämän jälkeen hyödynsimme automaattista kuvanta- mista ja automaattista kuva-analyysia arvioidaksemme kvantitatiivisesti LDL:n indusoimaa muutosta lyso- somien kolesterolimäärässä ja rasvapisaroiden muodostumisessa. Tuloksemme osoittavat, että NPC1 WT-V konstruktin sisältämä L472P-aminohappomuutos heikentää NPC1-proteiinin toimintaa lysosomaalisen koleste- rolin kuljetuksessa. Atomistiset simulaatiot vihjaavat, että L472P-mutaatio kiertää NPC1-proteiinin kahden lu- minaalisen toiminnallisen osan asentoa toistensa suhteen ja hajottaa hiljattain karakterisoidun kolesterolinsiirto- tunnelin. Nämä tulokset paljastavat toiminnallisen vian NPC1 WT-V proteiinissa ja korostavat simulaatioiden, kvantitatiivisen kuvantamisen ja stabiilin proteiiniekspression merkitystä proteiinien toiminnallisten muutosten osoittamisessa.The human Niemann-Pick C1 (NPC1) gene encoding a 1278 amino acid protein is very heterogeneous. While some variants represent benign polymorphisms, NPC disease carriers and patients may possess rare variants, whose functional importance remains unknown. An NPC1 cDNA construct known as NPC1 wild-type variant (WT-V), distributed between laboratories and used as a WT control in several studies, also contains changes regarding specific amino acids compared to the NPC1 Genbank reference sequence. To improve the dissection of subtle functional differences, we generated human cells stably expressing NPC1 variants from the AAVS1 safe-harbor locus on an NPC1-null background engineered by CRISPR/Cas9 editing. We then employed high-content imaging with automated image analysis to quantitatively assess LDL-induced, time-dependent changes in lysosomal cholesterol content and lipid droplet formation. Our results indicate that the L472P change present in NPC1 WT-V compromises NPC1 functionality in lysosomal cholesterol export. All-atom molecular dynamics simulations suggest that the L472P change alters the relative position of the NPC1 middle and the C-terminal luminal domains, disrupting the recently characterized cholesterol efflux tunnel. These results reveal functional defects in NPC1 WT-V and highlight the strength of simulations and quantitative imaging upon stable protein expression in elucidating subtle differences in protein function

A Ceramide-Regulated Element in the Late Endosomal Protein LAPTM4B Controls Amino Acid Transporter Interaction

Membrane proteins are functionally regulated by the composition of the surrounding lipid bilayer. The late endosomal compartment is a central site for the generation of ceramide, a bioactive sphingolipid, which regulates responses to cell stress. The molecular interactions between ceramide and late endosomal transmembrane proteins are unknown. Here, we uncover in atomistic detail the ceramide interaction of Lysosome Associated Protein Transmembrane 4B (LAPTM4B), implicated in ceramide-dependent cell death and autophagy, and its functional relevance in lysosomal nutrient signaling. The ceramide-mediated regulation of LAPTM4B depends on a sphingolipid interaction motif and an adjacent aspartate residue in the protein's third transmembrane (TM3) helix. The interaction motif provides the preferred contact points for ceramide while the neighboring membrane-embedded acidic residue confers flexibility that is subject to ceramide-induced conformational changes, reducing TM3 bending. This facilitates the interaction between LAPTM4B and the amino acid transporter heavy chain 4F2hc, thereby controlling mTORC signaling. These findings provide mechanistic insights into how transmembrane proteins sense and respond to ceramide.Peer reviewe