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The interaction between SARS-CoV-2 non-structural protein Nsp9 and the nanobody 2NSP90 was investigated by NMR spectroscopy using the paramagnetic perturbation methodology PENELOP (Paramagnetic Equilibrium vs Nonequilibrium magnetization Enhancement or LOss Perturbation). The Nsp9 monomer is an essential component of the replication and transcription complex (RTC) that reproduces the viral gRNA for subsequent propagation. Therefore preventing Nsp9 recruitment in RTC would represent an efficient antiviral strategy that could be applied to different coronaviruses, given the Nsp9 relative invariance. The NMR results were consistent with a previous characterization suggesting a 4:4 Nsp9-to-nanobody stoichiometry with the occurrence of two epitope pairs on each of the Nsp9 units that establish the inter-dimer contacts of Nsp9 tetramer. The oligomerization state of Nsp9 was also analyzed by molecular dynamics simulations and both dimers and tetramers resulted plausible. A different distribution of the mapped epitopes on the tetramer surface with respect to the former 4:4 complex could also be possible, as well as different stoichiometries of the Nsp9-nanobody assemblies such as the 2:2 stoichiometry suggested by the recent crystal structure of the Nsp9 complex with 2NSP23 (PDB ID: 8dqu), a nanobody exhibiting essentially the same affinity as 2NSP90. The experimental NMR evidence, however, ruled out the occurrence in liquid state of the relevant Nsp9 conformational change observed in the same crystal structure. [ABSTRACT FROM AUTHOR]

Following the entry into the host cell, SARS‐CoV‐2 replication is mediated by the replication transcription complex (RTC) assembled through a number of nonstructural proteins (Nsps). A monomeric form of Nsp9 is particularly important for RTC assembly and function. In the present study, 136 unique nanobodies targeting Nsp9 are generated. Several nanobodies belonging to different B‐cell lineages are expressed, purified, and characterized. Results from immunoassays applied to purified Nsp9 and neat saliva from coronavirus disease (COVID‐19) patients show that these nanobodies effectively and specifically recognize both recombinant and endogenous Nsp9. Nuclear magnetic resonance analyses supported by molecular dynamics reveal a composite Nsp9 oligomerization pattern and demonstrate that both nanobodies stabilize the tetrameric form of wild‐type Nsp9 also identifying the epitopes on the tetrameric assembly. These results can have important implications in the potential use of these nanobodies to combat viral replication. [ABSTRACT FROM AUTHOR]

Actin is an essential regulator of cellular functions. In the eukaryotic cell nucleus, actin regulates chromatin as a bona fide component of chromatin remodelling complexes, it associates with nuclear RNA polymerases to regulate transcription and is involved in co-transcriptional assembly of nascent RNAs into ribonucleoprotein complexes. Actin dynamics are, therefore, emerging as a major regulatory factor affecting diverse cellular processes. Importantly, the involvement of actin dynamics in nuclear functions is redefining the concept of nucleoskeleton from a rigid scaffold to a dynamic entity that is likely linked to the three-dimensional organization of the nuclear genome. In this review, we discuss how nuclear actin, by regulating chromatin structure through phase separation may contribute to the architecture of the nuclear genome during cell differentiation and facilitate the expression of specific gene programs. We focus specifically on mitochondrial genes and how their dysregulation in the absence of actin raises important questions about the role of cytoskeletal proteins in regulating chromatin structure. The discovery of a novel pool of mitochondrial actin that serves as 'mitoskeleton' to facilitate organization of mtDNA supports a general role for actin in genome architecture and a possible function of distinct actin pools in the communication between nucleus and mitochondria. [ABSTRACT FROM AUTHOR]

Cell-penetrating peptides (CPPs) are short peptides that are able to efficiently penetrate cellular lipid bilayers. Although CPPs have been used as carriers in conjugation with certain cargos to target specific genes and pathways, how rationally designed CPPs per se affect global gene expression has not been investigated. Therefore, following time course treatments with 4 CPPs-penetratin, PepFect14, mtCPP1 and TP10, HeLa cells were transcriptionally profiled by RNA sequencing. Results from these analyses showed a time-dependent response to different CPPs, with specific sets of genes related to ribosome biogenesis, microtubule dynamics and long-noncoding RNAs being differentially expressed compared to untreated controls. By using an image-based high content phenotypic profiling platform we confirmed that differential gene expression in CPP-treated HeLa cells strongly correlates with changes in cellular phenotypes such as increased nucleolar size and dispersed microtubules, compatible with altered ribosome biogenesis and cell growth. Altogether these results suggest that cells respond to different cell penetrating peptides by alteration of specific sets of genes, which are possibly part of the common response to such stimulus. [ABSTRACT FROM AUTHOR]

Actin cytoskeletal dynamics drive cellular shape changes, linking numerous cell functions to physiological and pathological cues. Mutations in actin regulators that are differentially expressed or enriched in immune cells cause severe human diseases known as primary immunodeficiencies underscoring the importance of efficienct actin remodeling in immune cell homeostasis. Here we discuss recent findings on how immune cells sense the mechanical properties of their environement. Moreover, while the organization and biochemical regulation of cytoplasmic actin have been extensively studied, nuclear actin reorganization is a rapidly emerging field that has only begun to be explored in immune cells. Based on the critical and multifaceted contributions of cytoplasmic actin in immune cell functionality, nuclear actin regulation is anticipated to have a large impact on our understanding of immune cell development and functionality., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Record, Saeed, Venit, Percipalle and Westerberg.)

Cytoskeletal proteins are beginning to be considered as key regulators of nuclear function. Among them, actin and myosin have been implicated in numerous tasks, including chromatin regulation, transcription and assembly of nascent ribonucleoprotein complexes. We also know from work performed by several labs that influx of actin and myosin into the nucleus and out of the nucleus is tightly regulated. In particular, in the case of actin, its nucleocytoplasmic import/export cycle is controlled by the importin/exportin system and it correlates with the transcriptional state of the cell. These basic molecular functions of both actin and myosin seem to impact key cellular functions, including development and differentiation as well as the cellular response to DNA damage by directly affecting transcriptional reprograming. These observations are beginning to suggest that actin and myosin could play an important role in consolidating the organization of the mammalian genome and that loss of actin and myosin likely leads to a general instability of the genome. In this chapter, we provide a general background on evidence that actin and myosin are important in key nuclear functions. Following this, we will focus on evidence supporting of a role in genome organization and finally we will discuss increasingly striking results on the role of actin and myosin in the maintenance of genome integrity., (© 2020 Elsevier Inc. All rights reserved.)