Publications - Icosagen

2025

A conserved phosphorylation mechanism for regulating the interaction between the CMG replicative helicase and its forked DNA substrate.

Sandra Koit, Nele Tamberg, Allan Reinapae, Lauri Peil, Arnold Kristjuhan, Ivar Ilves

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Abstract

The CMG helicase is a crucial enzyme complex that plays a vital role in the replication of genomic DNA in eukaryotes. Besides unwinding the DNA template and coordinating the replisome’s structure, it is also a key target for signaling pathways that regulate the replication process. We show that a specific serine/threonine residue in the MCM3 subunit of CMG, which has been previously linked to phosphorylation-dependent control mechanisms of genomic DNA replication in human cells, is a conserved phosphorylation site for Chk1 and potentially other protein kinases. This suggests a conserved regulatory mechanism associated with it in metazoans and several other eukaryotes, including budding yeast. Our in vitro analysis links this mechanism directly to the modulation of the CMG helicase activity by impacting its interactions with the forked DNA substrate. Further supporting its conserved role in regulation, we found that phosphomimetic substitution with aspartic acid and alanine knock-out of this conserved residue lead to opposite phenotypic defects in the growth of budding yeast cells. These findings outline a candidate conserved phosphorylation pathway for regulating genomic DNA replication in eukaryotes, which adjusts the interactions between the replicative helicase complex and its DNA substrate according to the specific needs of various physiological conditions.

Keywords: Eukaryote, DNA replication, DNA helicase, checkpoint control, protein phosphorylation.

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2024

Anti-SARS-CoV-2 antibodies in a nasal spray efficiently block viral transmission between ferrets.

Gildemann K, Tsernant ML, Liivand L, Ennomäe R, Poikalainen V, Lepasalu L, Rom S, Kavak A, Cox RM, Wolf JD, Lieber CM, Plemper RK, Männik A, Ustav M Jr, Ustav M, Gerhold JM.

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Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread in the population. We recently reported the production of bovine colostrum-derived antibodies that can neutralize the virus. These have been formulated into a nasal spray. The immunoglobulin preparation is capable of blocking interaction of the trimeric spike protein (Tri S) of SARS-CoV-2 with the cellular receptor angiotensin-converting enzyme 2 (ACE2), entry of a pseudovirus carrying the Tri S into ACE2 over-expressing human embryonic kidney (HEK) cells, and entry of the virus into live Vero E6 cells. Using an ELISA assay, we demonstrate here that this holds true for different SARS-CoV-2 variants of concern. Using the ferret transmission model, we show that the nasal spray formulation of anti-SARS-CoV-2 immunoglobulins efficiently blocks transmission of SARS-CoV-2 from infected to uninfected ferrets. The results indicate that the use of the nasal spray in humans can add an effective additional layer of protection against the virus, and might be applicable for other viruses of the upper respiratory tract.

Keywords: Immunology; Virology.

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Artesunate treats obesity in male mice and non-human primates through GDF15/GFRAL signalling axis.

Guo X, Asthana P, Zhai L, Cheng KW, Gurung S, Huang J, Wu J, Zhang Y, Mahato AK, Saarma M, Ustav M, Kwan HY, Lyu A, Chan KM, Xu P, Bian ZX, Wong HLX.

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Abstract

Obesity, a global health challenge, is a major risk factor for multiple life-threatening diseases, including diabetes, fatty liver, and cancer. There is an ongoing need to identify safe and tolerable therapeutics for obesity management. Herein, we show that treatment with artesunate, an artemisinin derivative approved by the FDA for the treatment of severe malaria, effectively reduces body weight and improves metabolic profiles in preclinical models of obesity, including male mice with overnutrition-induced obesity and male cynomolgus macaques with spontaneous obesity, without inducing nausea and malaise. Artesunate promotes weight loss and reduces food intake in obese mice and cynomolgus macaques by increasing circulating levels of Growth Differentiation Factor 15 (GDF15), an appetite-regulating hormone with a brainstem-restricted receptor, the GDNF family receptor α-like (GFRAL). Mechanistically, artesunate induces the expression of GDF15 in multiple organs, especially the liver, in mice through a C/EBP homologous protein (CHOP)-directed integrated stress response. Inhibition of GDF15/GFRAL signalling by genetic ablation of GFRAL or tissue-specific knockdown of GDF15 abrogates the anti-obesity effect of artesunate in mice with diet-induced obesity, suggesting that artesunate controls bodyweight and appetite in a GDF15/GFRAL signalling-dependent manner. These data highlight the therapeutic benefits of artesunate in the treatment of obesity and related comorbidities.

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Structural and functional validation of a highly specific Smurf2 inhibitor.

Tessier TM, Chowdhury A, Stekel Z, Fux J, Sartori MA, Teyra J, Jarvik N, Chung J, Kurinov I, Sicheri F, Sidhu SS, Singer AU, Zhang W.

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Abstract

Smurf1 and Smurf2 are two closely related member of the HECT (homologous to E6AP carboxy terminus) E3 ubiquitin ligase family and play important roles in the regulation of various cellular processes. Both were initially identified to regulate transforming growth factor-β and bone morphogenetic protein signaling pathways through regulating Smad protein stability and are now implicated in various pathological processes. Generally, E3 ligases, of which over 800 exist in humans, are ideal targets for inhibition as they determine substrate specificity; however, there are few inhibitors with the ability to precisely target a particular E3 ligase of interest. In this work, we explored a panel of ubiquitin variants (UbVs) that were previously identified to bind Smurf1 or Smurf2. In vitro binding and ubiquitination assays identified a highly specific Smurf2 inhibitor, UbV S2.4, which was able to inhibit ligase activity with high potency in the low nanomolar range. Orthologous cellular assays further demonstrated high specificity of UbV S2.4 toward Smurf2 and no cross-reactivity toward Smurf1. Structural analysis of UbV S2.4 in complex with Smurf2 revealed its mechanism of inhibition was through targeting the E2 binding site. In summary, we investigated several protein-based inhibitors of Smurf1 and Smurf2 and identified a highly specific Smurf2 inhibitor that disrupts the E2-E3 protein interaction interface.

Keywords: E3 ligases; HECT domain; crystal structure; inhibitor; phage display; protein engineering; ubiquitin variants.

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2023

Tolle I, Oehm S, Hoesl MG, Treiber-Kleinke C, Peil L, Bozukova M, Albers S, Adamu Bukari A-R, Semmler T, Rappsilber J, Ignatova Z, Gerstein AC and Budisa N. Evolving a mitigation of the stress response pathway to change the basic chemistry of life. Front. Synth. Biol. 2023 Aug 24; 1:1248065. Read the paper here.

Hermet P, Delache B, Herate C, Wolf E, Kivi G, et al. (2023) Broadly neutralizing humanized SARS-CoV-2 antibody binds to a conserved epitope on Spike and provides antiviral protection through inhalation-based delivery in non-human primates. PLOS Pathogens. 2023 Aug 2; 19(8): e1011532. Read the paper here.
Kovaleva V, Yu LY, Ivanova L, Shpironok O, Nam J, Eesmaa A, Kumpula EP, Sakson S, Toots U, Ustav M, Huiskonen JT, Voutilainen MH, Lindholm P, Karelson M, Saarma M. MANF regulates neuronal survival and UPR through its ER-located receptor IRE1α. Cell Rep. 2023 Feb 3;42(2):112066. Read the paper here.

2022

Porosk L, Nebogatova J, Härk HH, Vunk B, Arukuusk P, Toots U, Ustav M, Langel Ü, Kurrikoff K. Predicting Transiently Expressed Protein Yields: Comparison of Transfection Methods in CHO and HEK293. Pharmaceutics. 2022 Sep 14;14(9):1949. Read the paper here.
Naaber P, Tserel L, Kangro K, Punapart M, Sepp E, Jürjenson V, Kärner J, Haljasmägi L, Haljasorg U, Kuusk M, Sankovski E, Planken A, Ustav M, Žusinaite E, Gerhold JM, Kisand K, Peterson P. Protective antibodies and T cell responses to Omicron variant after the booster dose of BNT162b2 vaccine. Cell Rep Med. 2022 Aug 16;3(8):100716. Read the paper here.
Kangro K, Roose E, Dekimpe C, Vandenbulcke A, Graça NAG, Voorberg J, Ustav M, Männik A, Vanhoorelbeke K. Improvement of recombinant ADAMTS13 production through a more optimal signal peptide or an N-terminal fusion protein. J Thromb Haemost. 2022 Oct;20(10):2379-2385. Read the paper here.
Nacer A, Kivi G, Pert R, Juronen E, Holenya P, Aliprandini E, Amino R, Silvie O, Quinkert D, Le Duff Y, Hurley M, Reimer U, Tover A, Draper SJ, Gilbert S, Ho MM, Bowyer PW. Expanding the Malaria Antibody Toolkit: Development and Characterisation of Plasmodium falciparum RH5, CyRPA, and CSP Recombinant Human Monoclonal Antibodies. Front Cell Infect Microbiol. 2022 Jun 16;12:901253. Read the paper here.
Kangro K, Kurašin M, Gildemann K, Sankovski E, Žusinaite E, Lello LS, Pert R, Kavak A, Poikalainen V, Lepasalu L, Kuusk M, Pau R, Piiskop S, Rom S, Oltjer R, Tiirik K, Kogermann K, Plaas M, Tiirats T, Aasmäe B, Plaas M, Mumm K, Krinka D, Talpsep E, Kadaja M, Gerhold JM, Planken A, Tover A, Merits A, Männik A, Ustav M Jr, Ustav M. Bovine colostrum-derived antibodies against SARS-CoV-2 show great potential to serve as prophylactic agents. PLoS One. 2022 Jun 10;17(6):e0268806. Read the paper here.
Uusküla A, Keis A, Toompere K, Planken A, Rebrov K. Cluster randomised, controlled, triple-blind trial assessing the efficacy of intranasally administered virus-neutralising bovine colostrum supplement in preventing SARS-CoV-2 infection in household contacts of SARS-CoV-2-positive individuals: a study protocol. Trials. 2022 Jan 31;23(1):92. Read the paper here.

2021

Kuznetsov A, Arukuusk P, Härk H, Juronen E, Ustav M, Langel Ü, Järv J. ACE2 Peptide Fragment Interaction with Different S1 Protein Sites. Int J Pept Res Ther. 2022;28(1):7. Read the paper here.
Kasemaa K, Talts K, Täht E, Toots U, Ustav M. DNA Transfer into Animal Cells Using Stearylated CPP Based Transfection Reagent. Methods Mol Biol. 2022;2383:569-578. Read the paper here.
De Waele L, Curie A, Kangro K, Tellier E, Kaplanski G, Männik A, Tersteeg C, Joly BS, Coppo P, Veyradier A, De Meyer SF, Roose E, Vanhoorelbeke K. Anti-cysteine/spacer antibodies that open ADAMTS13 are a common feature in iTTP. Blood Adv. 2021 Nov 9;5(21):4480-4484. Read the paper here.
Naaber P, Tserel L, Kangro K, Sepp E, Jürjenson V, Adamson A, Haljasmägi L, Rumm AP, Maruste R, Kärner J, Gerhold JM, Planken A, Ustav M, Kisand K, Peterson P. Dynamics of antibody response to BNT162b2 vaccine after six months: a longitudinal prospective study. Lancet Reg Health Eur. 2021 Nov;10:100208. Read the paper here.
Kangro K, Roose E, Joly BS, Sinkovits G, Falter T, von Auer C, Rossmann H, Reti M, Voorberg J, Prohászka Z, Lämmle B, Coppo P, Veyradier A, De Meyer SF, Männik A, Vanhoorelbeke K. Anti-ADAMTS13 autoantibody profiling in patients with immune-mediated thrombotic thrombocytopenic purpura. Blood Adv. 2021 Sep 14;5(17):3427-3435. Read the paper here.
Miersch S, Li Z, Saberianfar R, Ustav M, Brett Case J, Blazer L, Chen C, Ye W, Pavlenco A, Gorelik M, Garcia Perez J, Subramania S, Singh S, Ploder L, Ganaie S, Chen RE, Leung DW, Pandolfi PP, Novelli G, Matusali G, Colavita F, Capobianchi MR, Jain S, Gupta JB, Amarasinghe GK, Diamond MS, Rini J, Sidhu SS. Tetravalent SARS-CoV-2 Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations. J Mol Biol. 2021 Sep 17;433(19):167177. Read the paper here.
Horta S, Neumann F, Yeh SH, Langseth CM, Kangro K, Breukers J, Madaboosi N, Geukens N, Vanhoorelbeke K, Nilsson M, Lammertyn J. Evaluation of Immuno-Rolling Circle Amplification for Multiplex Detection and Profiling of Antigen-Specific Antibody Isotypes. Anal Chem. 2021 Apr 20;93(15):6169-6177. Read the paper here.
Dekimpe C, Roose E, Kangro K, Bonnez Q, Vandenbulcke A, Tellier E, Kaplanski G, Feys HB, Tersteeg C, Männik A, De Meyer SF, Vanhoorelbeke K. Determination of anti-ADAMTS-13 autoantibody titers in ELISA: Influence of ADAMTS-13 presentation and autoantibody detection. J Thromb Haemost. 2021 Sep;19(9):2248-2255. Read the paper here.
Hrdinová J, D’Angelo S, Graça NAG, Ercig B, Vanhoorelbeke K, Veyradier A, Voorberg J, Coppo P. Dissecting the pathophysiology of immune thrombotic thrombocytopenic purpura: interplay between genes and environmental triggers. Haematologica. 2021 Mar 1;106(3):924. Read the paper here.
Ercig B, Graça NAG, Kangro K, Arfman T, Wichapong K, Hrdinová J, Kaijen P, van Alphen FPJ, van den Biggelaar M, Vanhoorelbeke K, Veyradier A, Coppo P, Reutelingsperger C, Nicolaes GAF, Männik A, Voorberg J. N-glycan-mediated shielding of ADAMTS13 prevents binding of pathogenic autoantibodies in immune-mediated TTP. Blood. 2021 May 13;137(19):2694-2698. Read the paper here.

2020

Tegel H, Dannemeyer M, Kanje S, Sivertsson Å, Berling A, Svensson A-S, Hober A, Enstedt H, Volk A-L, Lundqvist M, Moradi M, Afshari D, Ekblad S, Xu L, Westin M, Bidad F, Holmberg Schiavone L, Davies R, Mayr LM, Knight S, Göpel SO, Voldborg BG, Edfors F, Forsström B, von Feilitzen K, Zwahlen M, Rockberg J, Ottosson Takanen J, Uhlén M, Hober S. High throughput generation of a resource of the human secretome in mammalian cells. New Biotechnology. 2020 Jun;58:45–54. Read the paper here.

Velásquez Pereira LC, Roose E, Graça NAG, Sinkovits G, Kangro K, Joly BS, Tellier E, Kaplanski G, Falter T, Von Auer C, Rossmann H, Feys HB, Reti M, Prohászka Z, Lämmle B, Voorberg J, Coppo P, Veyradier A, De Meyer SF, Männik A, Vanhoorelbeke K. Immunogenic hotspots in the spacer domain of ADAMTS13 in immune-mediated thrombotic thrombocytopenic purpura. J Thromb Haemost. 2021 Feb;19(2):478-488. Read the paper here.
Graça NAG, Ercig B, Carolina Velásquez Pereira L, Kangro K, Kaijen P, Nicolaes GAF, Veyradier A, Coppo P, Vanhoorelbeke K, Männik A, Voorberg J. Modifying ADAMTS13 to modulate binding of pathogenic autoantibodies of patients with acquired thrombotic thrombocytopenic purpura. Haematologica. 2020 Nov 1;105(11):2619-2630. Read the paper here.
Ianevski A, Yao R, Biza S, Zusinaite E, Mannik A, Kivi G, Planken A, Kurg K, Tombak EM, Ustav M Jr, Shtaida N, Kulesskiy E, Jo E, Yang J, Lysvand H, Løseth K, Oksenych V, Aas PA, Tenson T, Vitkauskienė A, Windisch MP, Fenstad MH, Nordbø SA, Ustav M, Bjørås M, Kainov DE. Identification and Tracking of Antiviral Drug Combinations. Viruses. 2020 Oct 18;12(10):1178. Read the paper here.
Kangro K, Roose E, Schelpe AS, Tellier E, Kaplanski G, Voorberg J, De Meyer SF, Männik A, Vanhoorelbeke K. Generation and validation of small ADAMTS13 fragments for epitope mapping of anti-ADAMTS13 autoantibodies in immune-mediated thrombotic thrombocytopenic purpura. Res Pract Thromb Haemost. 2020 Jun 25;4(5):918-930. doi: 10.1002/rth2.12379. Read the paper here.

2019

Laaneväli A, Ustav M, Ustav E, Piirsoo M. E2 protein is the major determinant of specificity at the human papillomavirus origin of replication. PLoS One. 2019 Oct 23;14(10):e0224334. Read the paper here.
Pereira Aguilar P, Schneider TA, Wetter V, Maresch D, Ling WL, Tover A, Steppert P, Jungbauer A. Polymer-grafted chromatography media for the purification of enveloped virus-like particles, exemplified with HIV-1 gag VLP. Vaccine. 2019 Nov 8;37(47):7070-7080. Read the paper here.
Reiter K, Aguilar PP, Wetter V, Steppert P, Tover A, Jungbauer A. Separation of virus-like particles and extracellular vesicles by flow-through and heparin affinity chromatography. J Chromatogr A. 2019 Mar 15;1588:77-84. Read the paper here.

2018

Öhlund P, García-Arriaza J, Zusinaite E, Szurgot I, Männik A, Kraus A, Ustav M, Merits A, Esteban M, Liljeström P, Ljungberg K. DNA-launched RNA replicon vaccines induce potent anti-Ebolavirus immune responses that can be further improved by a recombinant MVA boost. Sci Rep. 2018 Aug 20;8(1):12459. Read the paper here.
Podgórska M, Ołdak M, Marthaler A, Fingerle A, Walch-Rückheim B, Lohse S, Müller CSL, Vogt T, Ustav M, Wnorowski A, Malejczyk M, Majewski S, Smola S. Chronic Inflammatory Microenvironment in Epidermodysplasia Verruciformis Skin Lesions: Role of the Synergism Between HPV8 E2 and C/EBPβ to Induce Pro-Inflammatory S100A8/A9 Proteins. Front Microbiol. 2018 Mar 7;9:392. Read the paper here.
Lázaro-Frías A, Gómez-Medina S, Sánchez-Sampedro L, Ljungberg K, Ustav M, Liljeström P, Muñoz-Fontela C, Esteban M, García-Arriaza J. Distinct Immunogenicity and Efficacy of Poxvirus-Based Vaccine Candidates against Ebola Virus Expressing GP and VP40 Proteins. J Virol. 2018 May 14;92(11):e00363-18. Read the paper here.
Sankovski E, Abroi A, Ustav M Jr, Ustav M. Nuclear myosin 1 associates with papillomavirus E2 regulatory protein and influences viral replication. Virology. 2018 Jan 15;514:142-155. Read the paper here.

2017

Toots M, Ustav M Jr, Männik A, Mumm K, Tämm K, Tamm T, Ustav E, Ustav M. Identification of several high-risk HPV inhibitors and drug targets with a novel high-throughput screening assay. PLoS Pathog. 2017 Feb 9;13(2):e1006168. Read the paper here.
Steppert P, Burgstaller D, Klausberger M, Tover A, Berger E, Jungbauer A. Quantification and characterization of virus-like particles by size-exclusion chromatography and nanoparticle tracking analysis. J Chromatogr A. 2017 Mar 3;1487:89-99. Read the paper here.
Steppert P, Burgstaller D, Klausberger M, Kramberger P, Tover A, Berger E, Nöbauer K, Razzazi-Fazeli E, Jungbauer A. Separation of HIV-1 gag virus-like particles from vesicular particles impurities by hydroxyl-functionalized monoliths. J Sep Sci. 2017 Feb;40(4):979-990. Read the paper here.

2016

Galli E, Härkönen T, Sainio MT, Ustav M, Toots U, Urtti A, Yliperttula M, Lindahl M, Knip M, Saarma M, Lindholm P. Increased circulating concentrations of mesencephalic astrocyte-derived neurotrophic factor in children with type 1 diabetes. Sci Rep. 2016 Jun 30;6:29058. Read the paper here.
Steppert P, Burgstaller D, Klausberger M, Berger E, Aguilar PP, Schneider TA, Kramberger P, Tover A, Nöbauer K, Razzazi-Fazeli E, Jungbauer A. Purification of HIV-1 gag virus-like particles and separation of other extracellular particles. J Chromatogr A. 2016 Jul 15;1455:93-101. Read the paper here.
Kivi G, Teesalu K, Parik J, Kontkar E, Ustav M Jr, Noodla L, Ustav M, Männik A. HybriFree: a robust and rapid method for the development of monoclonal antibodies from different host species. BMC Biotechnol. 2016 Jan 8;16:2. Read the paper here.

2015

Orav M, Geimanen J, Sepp EM, Henno L, Ustav E, Ustav M. Initial amplification of the HPV18 genome proceeds via two distinct replication mechanisms. Sci Rep. 2015 Nov 2;5:15952. Read the paper here.
Saare M, Hämarik U, Venta R, Panarina M, Zucchelli C, Pihlap M, Remm A, Kisand K, Toots U, Möll K, Salupere R, Musco G, Uibo R, Peterson P. SP140L, an Evolutionarily Recent Member of the SP100 Family, Is an Autoantigen in Primary Biliary Cirrhosis. J Immunol Res. 2015;2015:526518. Read the paper here.
Ustav M Jr, Castaneda FR, Reinson T, Männik A, Ustav M. Human Papillomavirus Type 18 cis-Elements Crucial for Segregation and Latency. PLoS One. 2015 Aug 19;10(8):e0135770. Read the paper here.
Karro K, Männik T, Männik A, Ustav M. DNA Transfer into Animal Cells Using Stearylated CPP Based Transfection Reagent. Methods Mol Biol. 2015;1324:435-45. Read the paper here.
Reinson T, Henno L, Toots M, Ustav M Jr, Ustav M. The Cell Cycle Timing of Human Papillomavirus DNA Replication. PLoS One. 2015 Jul 1;10(7):e0131675. Read the paper here.
Isok-Paas H, Männik A, Ustav E, Ustav M. The transcription map of HPV11 in U2OS cells adequately reflects the initial and stable replication phases of the viral genome. Virol J. 2015 Apr 14;12:59. Read the paper here.

2014

Toots M, Männik A, Kivi G, Ustav M Jr, Ustav E, Ustav M. The transcription map of human papillomavirus type 18 during genome replication in U2OS cells. PLoS One. 2014 Dec 30;9(12):e116151. Read the paper here.
dos Santos R, Rosa SA, Aires-Barros MR, Tover A, Azevedo AM. Phenylboronic acid as a multi-modal ligand for the capture of monoclonal antibodies: development and optimization of a washing step. J Chromatogr A. 2014 Aug 15;1355:115-24. Read the paper here.