TY - JOUR

T1 - New Evidence of the Importance of Weak Interactions in the Formation of PML-Bodies

AU - Fonin, Alexander V.

AU - Silonov, Sergey A.

AU - Fefilova, Anna S.

AU - Stepanenko, Olesya V.

AU - Gavrilova, Anastasia A.

AU - Petukhov, Alexey V.

AU - Romanovich, Anna E.

AU - Modina, Anna L.

AU - Zueva, Tatiana S.

AU - Nedelyaev, Evgeniy M.

AU - Pleskach, Nadejda M.

AU - Kuranova, Mirya L.

AU - Kuznetsova, Irina M.

AU - Uversky, Vladimir N.

AU - Turoverov, Konstantin K.

N1 - Publisher Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

PY - 2022/1/30

Y1 - 2022/1/30

N2 - In this work, we performed a comparative study of the formation of PML bodies by full-length PML isoforms and their C-terminal domains in the presence and absence of endogenous PML. Based on the analysis of the distribution of intrinsic disorder predisposition in the amino acid sequences of PML isoforms, regions starting from the amino acid residue 395 (i.e., sequences encoded by exons 4–6) were assigned as the C-terminal domains of these proteins. We demonstrate that each of the full-sized nuclear isoforms of PML is capable of forming nuclear liquid-droplet compartments in the absence of other PML isoforms. These droplets possess dynamic characteristics of the exchange with the nucleoplasm close to those observed in the wild-type cells. Only the C-terminal domains of the PML-II and PML-V isoforms are able to be included in the composition of the endogenous PML bodies, while being partially distributed in the nucleoplasm. The bodies formed by the C-terminal domain of the PML-II isoform are dynamic liquid droplet compartments, regardless of the presence or absence of endogenous PML. The C-terminal domain of PML-V forms dynamic liquid droplet compartments in the knockout cells (PML−/− ), but when the C-terminus of the PML-V isoform is inserted into the existing endogenous PML bodies, the molecules of this protein cease to exchange with the nucleoplasm. It was demonstrated that the K490R substitution, which disrupts the PML sumoylation, promotes diffuse distribution of the C-terminal domains of PML-II and PML-V isoforms in endogenous PML knockout HeLa cells, but not in the wild-type cells. These data indicate the ability of the C-terminal domains of the PML-II and PML-V isoforms to form dynamic liquid droplet-like compartments, regardless of the ordered N-terminal RBCC motifs of the PML. This indicates a significant role of the non-specific interactions between the mostly disordered C-terminal domains of PML isoforms for the initiation of liquid–liquid phase separation (LLPS) leading to the formation of PML bodies.

AB - In this work, we performed a comparative study of the formation of PML bodies by full-length PML isoforms and their C-terminal domains in the presence and absence of endogenous PML. Based on the analysis of the distribution of intrinsic disorder predisposition in the amino acid sequences of PML isoforms, regions starting from the amino acid residue 395 (i.e., sequences encoded by exons 4–6) were assigned as the C-terminal domains of these proteins. We demonstrate that each of the full-sized nuclear isoforms of PML is capable of forming nuclear liquid-droplet compartments in the absence of other PML isoforms. These droplets possess dynamic characteristics of the exchange with the nucleoplasm close to those observed in the wild-type cells. Only the C-terminal domains of the PML-II and PML-V isoforms are able to be included in the composition of the endogenous PML bodies, while being partially distributed in the nucleoplasm. The bodies formed by the C-terminal domain of the PML-II isoform are dynamic liquid droplet compartments, regardless of the presence or absence of endogenous PML. The C-terminal domain of PML-V forms dynamic liquid droplet compartments in the knockout cells (PML−/− ), but when the C-terminus of the PML-V isoform is inserted into the existing endogenous PML bodies, the molecules of this protein cease to exchange with the nucleoplasm. It was demonstrated that the K490R substitution, which disrupts the PML sumoylation, promotes diffuse distribution of the C-terminal domains of PML-II and PML-V isoforms in endogenous PML knockout HeLa cells, but not in the wild-type cells. These data indicate the ability of the C-terminal domains of the PML-II and PML-V isoforms to form dynamic liquid droplet-like compartments, regardless of the ordered N-terminal RBCC motifs of the PML. This indicates a significant role of the non-specific interactions between the mostly disordered C-terminal domains of PML isoforms for the initiation of liquid–liquid phase separation (LLPS) leading to the formation of PML bodies.

KW - Acute hydrogen peroxide-induced oxidative stress

KW - Fluorescence recovery after photobleaching (FRAP)

KW - Liquid–liquid phase separation (LLPS)

KW - Membrane-less organelles (MLOs)

KW - PML-bodies

KW - Promyelocytic leukemia protein (PML) isoforms

KW - Amino Acid Sequence

KW - Promyelocytic Leukemia Nuclear Bodies/metabolism

KW - Protein Isoforms/chemistry

KW - Cell Nucleus/metabolism

KW - Humans

KW - Cytoplasm/metabolism

KW - Gene Knockout Techniques

KW - Protein Domains

KW - Sumoylation

KW - Promyelocytic Leukemia Protein/chemistry

KW - HeLa Cells

KW - Amino Acid Substitution

UR - http://www.scopus.com/inward/record.url?scp=85123548551&partnerID=8YFLogxK

UR - https://www.mendeley.com/catalogue/7f2c2e7d-5536-36bd-8436-3c029a197541/

U2 - 10.3390/ijms23031613

DO - 10.3390/ijms23031613

M3 - Article

C2 - 35163537

AN - SCOPUS:85123548551

VL - 23

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

SN - 1422-0067

IS - 3

M1 - 1613

ER -