WW domains: A globular NLS recognized by importin-α.

In this issue, the discovery by Yang et al. (https://doi.org/10.1083/jcb.202308013) that folded WW domains of YAP1 and other proteins bind to Impα introduces a new class of globular NLS, contrasting with the extensively studied linear NLS motifs. This finding underscores the versatility of importins in recognizing their cargo proteins.

The Identification of a Novel Nucleomodulin MbovP467 of Mycoplasmopsis bovis and Its Potential Contribution in Pathogenesis.

Mycoplasmopsis bovis is a causative agent of crucial diseases in both dairy and beef cattle leading to substantial economic losses. However, limited control measures for M. bovis-related diseases exist due to a lack of understanding about the virulence factors of this pathogen, a common challenge in mycoplasma research. Consequently, this study aimed to characterize a novel nucleomodulin as a virulence-related factor of M. bovis. Employing bioinformatic tools, we initially predicted MbovP467 to be a secreted protein with a nuclear localization signal based on SignalP scores and the cNLS (Nuclear Localization Signal) Mapper, respectively. Subsequently, the MbovP467 gene was synthesized and cloned into a pEGFP plasmid with EGFP labeling to obtain a recombinant plasmid (rpEGFP-MbovP467) and then was also cloned in pET-30a with a consideration for an Escherichia coli codon bias and expressed and purified for the production of polyclonal antibodies against the recombinant MbovP467 protein. Confocal microscopy and a Western blotting assay confirmed the nuclear location of MbovP467 in bovine macrophages (BoMacs). RNA-seq data revealed 220 up-regulated and 20 down-regulated genes in the rpEGFP-MbovP467-treated BoMac group compared to the control group (pEGFP). A GO- and KEGG-enrichment analysis identified associations with inflammatory responses, G protein-coupled receptor signaling pathways, nuclear receptor activity, sequence-specific DNA binding, the regulation of cell proliferation, IL-8, apoptotic processes, cell growth and death, the TNF signaling pathway, the NF-κB signaling pathway, pathways in cancer, and protein families of signaling and cellular processes among the differentially expressed up-regulated mRNAs. Further experiments, investigating cell viability and the inflammatory response, demonstrated that MbovP467 reduces BoMac cell viability and induces the mRNA expression of IL-1ß, IL-6, IL-8, TNF-α, and apoptosis in BoMac cells. Further, MbovP467 increased the promoter activity of TNF-α. In conclusion, this study identified a new nucleomodulin, MbovP467, for M. bovis, which might have an important role in M. bovis pathogenesis.

STMND1 is a phylogenetically ancient stathmin which localizes to motile cilia and exhibits nuclear translocation that is inhibited when soluble tubulin concentration increases.

Stathmins are small, unstructured proteins that bind tubulin dimers and are implicated in several human diseases, but whose function remains unknown. We characterized a new stathmin, STMND1 (Stathmin Domain Containing 1) as the human representative of an ancient subfamily. STMND1 features a N-terminal myristoylated and palmitoylated motif which directs it to membranes and a tubulin-binding stathmin-like domain (SLD) that contains an internal nuclear localization signal. Biochemistry and proximity labeling showed that STMND1 binds tubulin, and live imaging showed that tubulin binding inhibits translocation from cellular membranes to the nucleus. STMND1 is highly expressed in multiciliated epithelial cells, where it localizes to motile cilia. Overexpression in a model system increased the length of primary cilia. Our study suggests that the most ancient stathmins have cilium-related functions that involve sensing soluble tubulin.

Myospreader improves gene editing in skeletal muscle by myonuclear propagation.

Successful CRISPR/Cas9-based gene editing in skeletal muscle is dependent on efficient propagation of Cas9 to all myonuclei in the myofiber. However, nuclear-targeted gene therapy cargos are strongly restricted to their myonuclear domain of origin. By screening nuclear localization signals and nuclear export signals, we identify "Myospreader," a combination of short peptide sequences that promotes myonuclear propagation. Appending Myospreader to Cas9 enhances protein stability and myonuclear propagation in myoblasts and myofibers. AAV-delivered Myospreader dCas9 better inhibits transcription of toxic RNA in a myotonic dystrophy mouse model. Furthermore, Myospreader Cas9 achieves higher rates of gene editing in CRISPR reporter and Duchenne muscular dystrophy mouse models. Myospreader reveals design principles relevant to all nuclear-targeted gene therapies and highlights the importance of the spatial dimension in therapeutic development.

Structural basis for nuclear import of bat adeno-associated virus capsid protein.

Adeno-associated viruses (AAV) are one of the world's most promising gene therapy vectors and as a result, are one of the most intensively studied viral vectors. Despite a wealth of research into these vectors, the precise characterisation of AAVs to translocate into the host cell nucleus remains unclear. Recently we identified the nuclear localization signals of an AAV porcine strain and determined its mechanism of binding to host importin proteins. To expand our understanding of diverse AAV import mechanisms we sought to determine the mechanism in which the Cap protein from a bat-infecting AAV can interact with transport receptor importins for translocation into the nucleus. Using a high-resolution crystal structure and quantitative assays, we were able to not only determine the exact region and residues of the N-terminal domain of the Cap protein which constitute the functional NLS for binding with the importin alpha two protein, but also reveal the differences in binding affinity across the importin-alpha isoforms. Collectively our results allow for a detailed molecular view of the way AAV Cap proteins interact with host proteins for localization into the cell nucleus.

Nuclear Localization Signal Enhances the Targeting and Therapeutic Efficacy of a Porphyrin-Based Molecular Cargo: A Systemic In Vitro and Ex Vivo Evaluation.

The objective of the present work was to evaluate the potential of a nuclear localization signal (NLS) toward facilitating intracellular delivery and enhancement in the therapeutic efficacy of the molecular cargo. Toward this, an in-house synthesized porphyrin derivative, namely, 5-carboxymethyelene-oxyphenyl-10,15,20-tris(4-methoxyphenyl) porphyrin (UTriMA), was utilized for conjugation with the NLS sequence [PKKKRKV]. The three compounds synthesized during the course of the present work, namely DOTA-Lys-NLS, DOTA-UTriMA-Lys-NLS, and DOTA-Lys-UTriMA, were evaluated for cellular toxicity in cancer cell lines (HT1080), wherein all exhibited minimal dark toxicity. However, during photocytotoxicity studies with DOTA-Lys-UTriMA and DOTA-UTriMA-Lys-NLS conjugates in the same cell line, the latter exhibited significantly higher light-dependent toxicity compared to the former. Furthermore, the photocytotoxicity for DOTA-UTriMA-Lys-NLS in a healthy cell line (WI26VA4) was found to be significantly lower than that observed in the cancer cells. Fluorescence cell imaging studies carried out in HT1080 cancer cells revealed intracellular accumulation for the NLS-conjugated porphyrin (DOTA-UTriMA-Lys-NLS), whereas unconjugated porphyrin (DOTA-Lys-UTriMA) failed to do so. To evaluate the radiotherapeutic effects of the synthesized conjugates, all three compounds were radiolabeled with 177Lu, a well-known therapeutic radionuclide with high radiochemical purity (>95%). During in vitro studies, the [177Lu]Lu-DOTA-UTriMA-Lys-NLS complex exhibited the highest cell binding as well as internalization among the three radiolabeled complexes. Biological distribution studies for the radiolabeled compounds were performed in a fibrosarcoma-bearing small animal model, wherein significantly higher accumulation and prolonged retention of [177Lu]Lu-DOTA-UTriMA-Lys-NLS (9.32 ± 1.27% IA/g at 24 h p.i.) in the tumorous lesion compared to [177Lu]Lu-UTriMA-Lys-DOTA (2.3 ± 0.13% IA/g at 24 h p.i.) and [177Lu]Lu-DOTA-Lys-NLS complexes (0.26 ± 0.17% IA/g at 24 h p.i.) were observed. The results of the biodistribution studies were further corroborated by recording serial SPECT-CT images of fibrosarcoma-bearing Swiss mice administered with [177Lu]Lu-DOTA-UTriMA-Lys-NLS at different time points. Tumor regression studies performed with [177Lu]Lu-DOTA-UTriMA-Lys-NLS in the same animal model with two different doses [250 µCi (9.25 MBq) and 500 µCi (18.5 MBq)] resulted in a significant reduction in tumor mass in the treated group of animals. The above results revealed a definite enhancement in the targeting ability of molecular cargo upon conjugation with NLS and hence indicated that this strategy may be helpful for the preparation of drug-NLS conjugates as multimodal agents.

WW domains form a folded type of nuclear localization signal to guide YAP1 nuclear import.

The nuclear translocation of YAP1 is significantly implicated in the proliferation, stemness, and metastasis of cancer cells. Although the molecular basis underlying YAP1 subcellular distribution has been extensively explored, it remains to be elucidated how the nuclear localization signal guides YAP1 to pass through the nuclear pore complex. Here, we define a globular type of nuclear localization signal composed of folded WW domains, named as WW-NLS. It directs YAP1 nuclear import through the heterodimeric nuclear transport receptors KPNA-KPNB1, bypassing the canonical nuclear localization signal that has been well documented in KPNA/KPNB1-mediated nuclear import. Strikingly, competitive interference with the function of the WW-NLS significantly attenuates YAP1 nuclear translocation and damages stemness gene activation and sphere formation in malignant breast cancer cells. Our findings elucidate a novel globular type of nuclear localization signal to facilitate nuclear entry of WW-containing proteins including YAP1.

C9orf72 polyPR directly binds to various nuclear transport components.

The disruption of nucleocytoplasmic transport (NCT) is an important mechanism in neurodegenerative diseases. In the case of C9orf72-ALS, trafficking of macromolecules through the nuclear pore complex (NPC) might get frustrated by the binding of C9orf72-translated arginine-containing dipeptide repeat proteins (R-DPRs) to the Kapß family of nuclear transport receptors. Besides Kapßs, several other types of transport components have been linked to NCT impairments in R-DPR-expressed cells, but the molecular origin of these observations has not been clarified. Here, we adopt a coarse-grained molecular dynamics model at amino acid resolution to study the direct interaction between polyPR, the most toxic DPR, and various nuclear transport components to elucidate the binding mechanisms and provide a complete picture of potential polyPR-mediated NCT defects. We found polyPR to directly bind to several isoforms of the Impα family, CAS (the specific exporter of Impα) and RanGAP. We observe no binding between polyPR and Ran. Longer polyPRs at lower salt concentrations also make contact with RanGEF and NTF2. Analyzing the polyPR contact sites on the transport components reveals that polyPR potentially interferes with RanGTP/RanGDP binding, with nuclear localization signal (NLS)-containing cargoes (cargo-NLS) binding to Impα, with cargo-NLS release from Impα, and with Impα export from the nucleus. The abundance of polyPR-binding sites on multiple transport components combined with the inherent polyPR length dependence makes direct polyPR interference of NCT a potential mechanistic pathway of C9orf72 toxicity.

The Nuclear Localization Signal of Porcine Circovirus Type 4 Affects the Subcellular Localization of the Virus Capsid and the Production of Virus-like Particles.

Porcine circovirus 4 (PCV4) is a newly identified virus belonging to PCV of the Circoviridae family, the Circovirus genus. We previously found that PCV4 is pathogenic in vitro, while the virus's replication in cells is still unknown. In this study, we evaluated the N-terminal of the PCV4 capsid (Cap) and identified an NLS at amino acid residues 4-37 of the N-terminus of the PCV4 Cap, 4RSRYSRRRRNRRNQRRRGLWPRASRRRYRWRRKN37. The NLS was further divided into two fragments (NLS-A and NLS-B) based on the predicted structure, including two α-helixes, which were located at 4RSRYSRRRRNRRNQRR19 and 24PRASRRRYRWRRK36, respectively. Further studies showed that the NLS, especially the first α-helixes formed by the NLS-A fragment, determined the nuclear localization of the Cap protein, and the amino acid 4RSRY7 in the NLS of the PCV4 Cap was the critical motif affecting the VLP packaging. These results will provide a theoretical basis for elucidating the infection mechanism of PCV4 and developing subunit vaccines based on VLPs.

Identification and characterization of nuclear localization signals in the circumsporozoite protein of Plasmodium falciparum.

Secretory proteins of Plasmodium exhibit differential spatial and functional activity within the host cell nucleus. However, the nuclear localization signals (NLSs) for these proteins remain largely uncharacterized. In this study, we have identified and characterized two NLSs in the circumsporozoite protein of Plasmodium falciparum (Pf-CSP). Both NLSs in the Pf-CSP contain clusters of lysine and arginine residues essential for specific interactions with the conserved tryptophan and asparagine residues of importin-α, facilitating nuclear translocation of Pf-CSP. While the two NLSs of Pf-CSP function independently and are both crucial for nuclear localization, a single NLS of Pf-CSP leads to weak nuclear localization. These findings shed light on the mechanism of nuclear penetrability of secretory proteins of Plasmodium proteins.

Hitchhiking of Cas9 with nucleus-localized proteins impairs its controllability and leads to efficient genome editing of NLS-free Cas9.

CRISPR-Cas9 is the most commonly used genome-editing tool in eukaryotic cells. To modulate Cas9 entry into the nucleus to enable control of genome editing, we constructed a light-controlled CRISPR-Cas9 system to control exposure of the Cas9 protein nuclear localization signal (NLS). Although blue-light irradiation was found to effectively control the entry of Cas9 protein into the nucleus with confocal microscopy observation, effective gene editing occurred in controls with next-generation sequencing analysis. To further clarify this phenomenon, a CRISPR-Cas9 editing system without the NLS and a CRISPR-Cas9 editing system containing a nuclear export signal were also constructed. Interestingly, both Cas9 proteins could achieve effective editing of target sites with significantly reduced off-target effects. Thus, we speculated that other factors might mediate Cas9 entry into the nucleus. However, NLS-free Cas9 was found to produce effective target gene editing even following inhibition of cell mitosis to prevent nuclear import caused by nuclear membrane disassembly. Furthermore, multiple nucleus-localized proteins were found to interact with Cas9, which could mediate the "hitchhiking" of NLS-free Cas9 into the nucleus. These findings will inform future attempts to construct controllable gene-editing systems and provide new insights into the evolution of the nucleus and compatible protein functions.

PTHrP intracrine actions divergently influence breast cancer growth through p27 and LIFR.

The role of parathyroid hormone (PTH)-related protein (PTHrP) in breast cancer remains controversial, with reports of PTHrP inhibiting or promoting primary tumor growth in preclinical studies. Here, we provide insight into these conflicting findings by assessing the role of specific biological domains of PTHrP in tumor progression through stable expression of PTHrP (-36-139aa) or truncated forms with deletion of the nuclear localization sequence (NLS) alone or in combination with the C-terminus. Although the full-length PTHrP molecule (-36-139aa) did not alter tumorigenesis, PTHrP lacking the NLS alone accelerated primary tumor growth by downregulating p27, while PTHrP lacking the NLS and C-terminus repressed tumor growth through p27 induction driven by the tumor suppressor leukemia inhibitory factor receptor (LIFR). Induction of p27 by PTHrP lacking the NLS and C-terminus persisted in bone disseminated cells, but did not prevent metastatic outgrowth, in contrast to the primary tumor site. These data suggest that the PTHrP NLS functions as a tumor suppressor, while the PTHrP C-terminus may act as an oncogenic switch to promote tumor progression through differential regulation of p27 signaling.

Nucleocytoplasmic shuttling of BEFV M protein-modulated by lamin A/C and chromosome maintenance region 1 through a transcription-, carrier- and energy-dependent pathway.

This study demonstrates for the first time that the matrix (M) protein of BEFV is a nuclear targeting protein that shuttles between the nucleus and the cytoplasm in a transcription-, carrier-, and energy-dependent manner. Experiments performed in both intact cells and digitonin-permeabilized cells revealed that M protein targets the nucleolus and requires carrier, cytosolic factors or energy input. By employing sequence and mutagenesis analyses, we have determined both nuclear localization signal (NLS) 6KKGKSK11 and nuclear export signal (NES) 98LIITSYL TI106 of M protein that are important for the nucleocytoplasmic shuttling of M protein. Furthermore, we found that both lamin A/C and chromosome maintenance region 1 (CRM-1) proteins could be coimmunoprecipitated and colocalized with the BEFV M protein. Knockdown of lamin A/C by shRNA and inhibition of CRM-1 by leptomycin B significantly reduced virus yield. Collectively, this study provides novel insights into nucleocytoplasmic shuttling of the BEFV M protein modulated by lamin A/C and CRM-1 and by a transcription- and carrier- and energy-dependent pathway.

Should I stay or should I go? Trafficking of plant extra-nuclear transcription factors.

At the heart of all biological processes lies the control of nuclear gene expression, which is primarily achieved through the action of transcription factors (TFs) that generally contain a nuclear localization signal (NLS) to facilitate their transport into the nucleus. However, some TFs reside in the cytoplasm in a transcriptionally inactive state and only enter the nucleus in response to specific signals, which in plants include biotic or abiotic stresses. These extra-nuclear TFs can be found in the cytosol or associated with various membrane systems, including the endoplasmic reticulum and plasma membrane. They may be integral proteins with transmembrane domains or associate peripherally with the lipid bilayer via acylation or membrane-binding domains. Although over 30 plant TFs, most of them involved in stress responses, have been experimentally shown to reside outside the nucleus, computational predictions suggest that this number is much larger. Understanding how extra-nuclear TFs are trafficked into the nucleus is essential for reconstructing transcriptional regulatory networks that govern major cellular pathways in response to biotic and abiotic signals. Here, we provide a perspective on what is known on plant extranuclear-nuclear TF retention, nuclear trafficking, and the post-translational modifications that ultimately enable them to regulate gene expression upon entering the nucleus.

Analysis of the effects of importin α1 on the nuclear translocation of IL-1α in HeLa cells.

Interleukin-1α (IL-1α), a cytokine released by necrotic cells, causes sterile inflammation. On the other hand, IL-1α is present in the nucleus and also regulates the expression of many proteins. A protein substrate containing a classical nuclear localization signal (cNLS) typically forms a substrate/importin α/ß complex, which is subsequently transported to the nucleus. To the best of our knowledge, no study has directly investigated whether IL-1α-which includes cNLS-is imported into the nucleus in an importin α/ß-dependent manner. In this study, we noted that all detected importin α subtypes interacted with IL-1α. In HeLa cells, importin α1-mediated nuclear translocation of IL-1α occurred at steady state and was independent of importin ß1. Importin α1 not only was engaged in IL-1α nuclear transport but also concurrently functioned as a molecule that regulated IL-1α protein level in the cell. Furthermore, we discussed the underlying mechanism of IL-1α nuclear translocation by importin α1 based on our findings.

Signal-regulated Unmasking of Nuclear Localization Motif in the PAS Domain Regulates the Nuclear Translocation of PASK.

The ligand-regulated PAS domains are one of the most diverse signal-integrating domains found in proteins from prokaryotes to humans. By biochemically connecting cellular processes with their environment, PAS domains facilitate an appropriate cellular response. PAS domain-containing Kinase (PASK) is an evolutionarily conserved protein kinase that plays important signaling roles in mammalian stem cells to establish stem cell fate. We have shown that the nuclear translocation of PASK is stimulated by differentiation signaling cues in muscle stem cells. However, the mechanistic basis of the regulation of PASK nucleo-cytoplasmic translocation remains unknown. Here, we show that the PAS-A domain of PASK contains a putative monopartite nuclear localization sequence (NLS) motif. This NLS is inhibited in cells through intramolecular association with a short linear motif, termed the PAS Interacting Motif (PIM), found upstream of the kinase domain. This interaction serves to retain PASK in the cytosol in the absence of signaling cues. Consistent with that, we show that metabolic inputs induce PASK nuclear import, likely by disrupting this association. We suggest that a route for such linkage may occur through the PAS-A ligand binding cavity. We show that PIM recruitment and artificial ligand binding to the PAS-A domain occur at neighboring locations that could facilitate metabolic control of the PAS-PIM interaction. Thus, the intramolecular interaction in PASK integrates metabolic signaling cues for nuclear translocation and could be targeted to control the balance between self-renewal and differentiation in stem cells.

LAIR1-mediated resistance of hepatocellular carcinoma cells to T cells through a GSK-3ß/ß-catenin/MYC/PD-L1 pathway.

BACKGROUND: An increasing number of studies have reported the involvement of oncogenes in the regulation of the immune system. LAIR1 is an immunosuppressive molecule and its role in immune-related diseases has been mainly reported. To date, it is unclear whether LAIR1 in tumor cells is involved in immune regulation. Therefore, the aim of this study was to investigate the role of LAIR1 in the immune microenvironment of hepatocellular carcinoma (HCC) to seek the novel therapeutic discoveries. METHODS: Tumor Immune Dysfunction and Exclusion database was used to predict the response of LAIR1 expression to immune checkpoint blockade. CD8+ T cells were co-cultured with HCC cells, and the killing efficiency of leukocytes on HCC cells was detected by flow cytometry. Flow cytometry was also used to detect the expression of inhibitory receptors. In addition, Western blot, immunofluorescence, and nucleus/cytoplasm fractionation experiments were performed to explore the molecular mechanisms by which LAIR1 created a suppressive tumor microenvironment. RESULTS: LAIR1 expression in HCC was associated with worse immune prognosis and T-cell dysfunction. HCC cells overexpressing LAIR1 co-cultured with CD8+ T cells induced exhaustion of latter. Mechanism studies indicated that LAIR1 in HCC cells up-regulated the phosphorylation of ß-catenin by inducing the phosphorylation of GSK-3ß, leading to the impairment of the expression and the nuclear localization signal of ß-catenin. Low ß-catenin expression and nuclear localization signal inhibited MYC-mediated PD-L1 expression. Therefore, PD-L1 up-regulated by LAIR1 caused the exhaustion of infiltrating CD8+ T cells in HCC, which aggravated the malignant progression of HCC. CONCLUSION: LAIR1 increased PD-L1 expression through the GSK-3ß/ß-catenin/MYC/PD-L1 pathway and promoted immune evasion of HCC cells. Targeted inhibition of LAIR1 helped to enhance the immune killing effect of CD8+ T cells in HCC.

Structural and functional characterization of siadenovirus core protein VII nuclear localization demonstrates the existence of multiple nuclear transport pathways.

Adenovirus protein VII (pVII) plays a crucial role in the nuclear localization of genomic DNA following viral infection and contains nuclear localization signal (NLS) sequences for the importin (IMP)-mediated nuclear import pathway. However, functional analysis of pVII in adenoviruses to date has failed to fully determine the underlying mechanisms responsible for nuclear import of pVII. Therefore, in the present study, we extended our analysis by examining the nuclear trafficking of adenovirus pVII from a non-human species, psittacine siadenovirus F (PsSiAdV). We identified a putative classical (c)NLS at pVII residues 120-128 (120PGGFKRRRL128). Fluorescence polarization and electrophoretic mobility shift assays demonstrated direct, high-affinity interaction with both IMPα2 and IMPα3 but not IMPß. Structural analysis of the pVII-NLS/IMPα2 complex confirmed a classical interaction, with the major binding site of IMPα occupied by K124 of pVII-NLS. Quantitative confocal laser scanning microscopy showed that PsSiAdV pVII-NLS can confer IMPα/ß-dependent nuclear localization to GFP. PsSiAdV pVII also localized in the nucleus when expressed in the absence of other viral proteins. Importantly, in contrast to what has been reported for HAdV pVII, PsSiAdV pVII does not localize to the nucleolus. In addition, our study demonstrated that inhibition of the IMPα/ß nuclear import pathway did not prevent PsSiAdV pVII nuclear targeting, indicating the existence of alternative pathways for nuclear localization, similar to what has been previously shown for human adenovirus pVII. Further examination of other potential NLS signals, characterization of alternative nuclear import pathways, and investigation of pVII nuclear targeting across different adenovirus species is recommended to fully elucidate the role of varying nuclear import pathways in the nuclear localization of pVII.

Nuclear localization signal-tagged systems: relevant nuclear import principles in the context of current therapeutic design.

Nuclear targeting of therapeutics provides a strategy for enhancing efficacy of molecules active in the nucleus and minimizing off-target effects. 'Active' nuclear-directed transport and efficient translocations across nuclear pore complexes provide the most effective means of maximizing nuclear localization. Nuclear-targeting systems based on nuclear localization signal (NLS) motifs have progressed significantly since the beginning of the current millennium. Here, we offer a roadmap for understanding the basic mechanisms of nuclear import in the context of actionable therapeutic design for developing NLS-therapeutics with improved treatment efficacy.