bims-nucpor 2022-11-20 papers

Issue of 2022‒11‒20
two papers selected by
Sara Mingu
Johannes Gutenberg University

Comput Struct Biotechnol J. 2022 ;20 5952-5961

Recognition motifs for importin 4 [(L)PPRS(G/P)P] and importin 5 [KP(K/Y)LV] binding, identified by bio-informatic simulation and experimental in vitro validation.

Athanasios A Panagiotopoulos, Konstantina Kalyvianaki, Paraskevi K Tsodoulou, Maria N Darivianaki, Dimitris Dellis, George Notas, Vangelis Daskalakis, Panayiotis A Theodoropoulos, Christos Α Panagiotidis, Elias Castanas, Marilena Kampa.

Nuclear translocation of large proteins is mediated through karyopherins, carrier proteins recognizing specific motifs of cargo proteins, known as nuclear localization signals (NLS). However, only few NLS signals have been reported until now. In the present work, NLS signals for Importins 4 and 5 were identified through an unsupervised in silico approach, followed by experimental in vitro validation. The sequences LPPRS(G/P)P and KP(K/Y)LV were identified and are proposed as recognition motifs for Importins 4 and 5 binding, respectively. They are involved in the trafficking of important proteins into the nucleus. These sequences were validated in the breast cancer cell line T47D, which expresses both Importins 4 and 5. Elucidating the complex relationships of the nuclear transporters and their cargo proteins is very important in better understanding the mechanism of nuclear transport of proteins and laying the foundation for the development of novel therapeutics, targeting specific importins.

Keywords: IPO4, Importin 4; IPO5, Importin 5; IPO7, Importin 7; IPOα, Importin α; IPOβ, Importin β; Importin 4; Importin 5; Karyopherins; Nuclear localization signal (NLS)

DOI: https://doi.org/10.1016/j.csbj.2022.10.015

STAR Protoc. 2022 Dec 16. 3(4): 101813

Protocol to image and quantify nucleocytoplasmic transport in cultured cells using fluorescent in situ hybridization and a dual reporter system.

Haochen Cui, Masood Sepehrimanesh, Casey A Coutee, Masuma Akter, Md Abir Hosain, Baojin Ding.

Nucleocytoplasmic transport (NCT) plays critical roles in maintaining cellular homeostasis. Here, we present a protocol to measure NCT for both transcript and protein cargos in cultured cells. We first describe the fluorescent in situ hybridization (FISH) assay to measure the nuclear mRNA export. We then detail a dual reporter system to measure the protein NCT. This protocol also includes image analysis and data output using CellProfiler™. The combined approach can be used to unbiasedly analyze NCT activities in cultured cells. For complete details on the use and execution of this protocol, please refer to Ding et al. (2020, 2021).

Keywords: Cell Biology; Cell culture; Cell-based Assays; Gene Expression; In Situ Hybridization; Microscopy; Molecular Biology; Neuroscience; Single Cell

DOI: https://doi.org/10.1016/j.xpro.2022.101813