Functional screening and molecular characterisation of small proteins
Author: Schlesinger, Dörte
Date: 2023-09-22
Location: Eva & Georg Klein Hall, Biomedicum, Solnavägen 9, Karolinska Institutet, Solna
Time: 09.30
Department: Inst för medicinsk biokemi och biofysik / Dept of Medical Biochemistry and Biophysics
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Thesis (9.967Mb)
Abstract
Proteins are one of the main drivers of cellular function. Most known proteins constitute
various hundred amino acids, but growing evidence suggests that additionally thousands
of short open reading frames (sORFs) are translated, posing the exciting possibility that
many small unknown proteins are yet to be discovered. In Paper I, we designed a knockout
screening pipeline to survey 11,776 curated human sORFs for a role in cancer cell line
growth. We identified 17 candidates and selected the top six hits for further study. We
showed that two of these candidates alleviated their knock-out phenotype when
supplied in trans as GFP-fusion proteins. However, knock-out induced transcriptome
alterations as assessed via RNA-seq could only partially be rescued and illustrated the
difficulties in separating RNA and protein function. We described distinct interaction
partners and subcellular localisations for several of our hits and finally verified translation
of a pseudogene originating sORF. Overall, this study provides insights into novel putative
microproteins.
Microprotein characterisation can be aggravated by the fact that many biochemical tools such as tags are tailored to canonical proteins. In Paper II, we established a minimal singleresidue terminal label (STELLA) strategy. Terminal non-canonical amino acid (ncAA)- tagging was previously not possible, due to inefficient amber suppression. Here, we overcame this issue by generating precursors that are subsequently cleaved, allowing for N- or C-terminal protein labelling with just a single ncAA, which can then be conjugated to fluorescent dyes for in-gel or (live) cell imaging applications. We successfully demonstrated this approach on a range of small proteins with different subcellular localisations. Overall, this paper provides a labelling alternative to larger conventional tags.
Finally, various known small proteins still possess unknown mechanisms of action. For example, it was unclear how viral fusion-associated small transmembrane (FAST) proteins induce cell-cell fusion given the small size of their ectodomains. A previous study showed that the p14 FAST family member exploits actin networks to drive syncytia formation, but it was unknown whether this extends to other FAST proteins. In Paper III, we showed through Co-IP, mutagenic and pharmacological assays, that aquareovirus p22, a FAST protein evolutionary divergent from p14, also exploits N-WASP mediated branched actin networks to induce fusion, albeit by utilising different molecular pathways. A p14/p22 chimera was still functional, even when N-WASP was replaced with the parallel actin nucleator formin. This suggested that the two proteins are modular, and that mechanical pressure coupled to a fusogenic ectodomain is sufficient for syncytia formation. Overall, this study illustrates a more general fusion mechanism for these minimal fusogens.
Taken together, this thesis investigated different aspects of small protein biology, developed microprotein-suitable tools, and contributed to our growing understanding of small protein-mediated function.
Microprotein characterisation can be aggravated by the fact that many biochemical tools such as tags are tailored to canonical proteins. In Paper II, we established a minimal singleresidue terminal label (STELLA) strategy. Terminal non-canonical amino acid (ncAA)- tagging was previously not possible, due to inefficient amber suppression. Here, we overcame this issue by generating precursors that are subsequently cleaved, allowing for N- or C-terminal protein labelling with just a single ncAA, which can then be conjugated to fluorescent dyes for in-gel or (live) cell imaging applications. We successfully demonstrated this approach on a range of small proteins with different subcellular localisations. Overall, this paper provides a labelling alternative to larger conventional tags.
Finally, various known small proteins still possess unknown mechanisms of action. For example, it was unclear how viral fusion-associated small transmembrane (FAST) proteins induce cell-cell fusion given the small size of their ectodomains. A previous study showed that the p14 FAST family member exploits actin networks to drive syncytia formation, but it was unknown whether this extends to other FAST proteins. In Paper III, we showed through Co-IP, mutagenic and pharmacological assays, that aquareovirus p22, a FAST protein evolutionary divergent from p14, also exploits N-WASP mediated branched actin networks to induce fusion, albeit by utilising different molecular pathways. A p14/p22 chimera was still functional, even when N-WASP was replaced with the parallel actin nucleator formin. This suggested that the two proteins are modular, and that mechanical pressure coupled to a fusogenic ectodomain is sufficient for syncytia formation. Overall, this study illustrates a more general fusion mechanism for these minimal fusogens.
Taken together, this thesis investigated different aspects of small protein biology, developed microprotein-suitable tools, and contributed to our growing understanding of small protein-mediated function.
List of papers:
I. Dörte Schlesinger, Christopher Dirks, Carmen Navarro Luzon, Lorenzo Lafranchi, Jürgen Eirich, Simon J Elsässer. A large-scale sORF screen identifies putative microproteins and provides insights into their interaction partners, localisation and function. [Manuscript]
II. Lorenzo Lafranchi, Dörte Schlesinger, Kyle Kimler, Simon J Elsässer. Universal single-residue terminal labels for fluorescent live cell imaging of microproteins. JACS. 2020, 142(47).
Fulltext (DOI)
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III. Ka Man Carmen Chan*, Ashley L. Arthur*, Johannes Morstein*, Meiyan Jin, Abrar Bhat, Dörte Schlesinger, Sungmin Son, Donté A. Stevens, David G. Drubin, Daniel A. Fletcher. Evolutionarily related small viral fusogens hijack distinct but modular actin nucleation pathways to drive cell-cell fusion. PNAS. 2020, 118(1). * These authors contributed equally.
Fulltext (DOI)
Pubmed
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I. Dörte Schlesinger, Christopher Dirks, Carmen Navarro Luzon, Lorenzo Lafranchi, Jürgen Eirich, Simon J Elsässer. A large-scale sORF screen identifies putative microproteins and provides insights into their interaction partners, localisation and function. [Manuscript]
II. Lorenzo Lafranchi, Dörte Schlesinger, Kyle Kimler, Simon J Elsässer. Universal single-residue terminal labels for fluorescent live cell imaging of microproteins. JACS. 2020, 142(47).
Fulltext (DOI)
Pubmed
View record in Web of Science®
III. Ka Man Carmen Chan*, Ashley L. Arthur*, Johannes Morstein*, Meiyan Jin, Abrar Bhat, Dörte Schlesinger, Sungmin Son, Donté A. Stevens, David G. Drubin, Daniel A. Fletcher. Evolutionarily related small viral fusogens hijack distinct but modular actin nucleation pathways to drive cell-cell fusion. PNAS. 2020, 118(1). * These authors contributed equally.
Fulltext (DOI)
Pubmed
View record in Web of Science®
Institution: Karolinska Institutet
Supervisor: Elsässer, Simon
Co-supervisor: Kutter, Claudia; Schmierer, Bernhard; Fernandez Capetillo, Oskar
Issue date: 2023-08-23
Rights:
Publication year: 2023
ISBN: 978-91-8017-093-2
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