Decreased Nuclear Immunoexpression of ING3 is a Frequent Event in Lip Carcinogenesis.

PURPOSE: Evaluate the immunohistochemical expression of the ING3 in actinic cheilitis and squamous cell carcinoma of the lower lip. METHODS: Forty-five specimens of actinic cheilitis and 48 specimens of squamous cell carcinoma of the lower lip were submitted to immunohistochemical detection of ING3. The protein expression in different cellular sublocations was compared between the two groups, and associations with the clinicopathological variables were analyzed. A significance level of 5% was adopted for all tests. RESULTS: Deaths were significantly more frequent in tumors with a high histopathological risk score (p < 0.05). In actinic cheilitis, significant differences were found in the nucleus-cytoplasmic expression of ING3 and expression restricted to the cytoplasm with binary histopathological grading (p < 0.05). In squamous cell carcinoma of the lower lip, there was no statistically significant difference when comparing ING3 expressions with clinical and morphological parameters (p > 0.05). Nucleo-cytoplasmic ING3 expression was significantly lower in squamous cell carcinoma of the lower lip when compared to actinic cheilitis (p < 0.05) and the expression restricted to the cytoplasm was significantly higher in squamous cell carcinoma of the lower lip (p < 0.05). CONCLUSION: The results of this study suggest that there is a marked decrease in the nuclear expression of ING3 as malignant progression occurs, indicating an impaired tumor suppressor function of this protein in actinic cheilitis and squamous cell carcinoma of the lower lip.

Nuclear compensatory evolution driven by mito-nuclear incompatibilities.

Mitochondrial function relies on the coordinated expression of mitochondrial and nuclear genes, exhibiting remarkable resilience despite high mitochondrial mutation rates. The nuclear compensation mechanism suggests deleterious mitochondrial alleles drive compensatory nuclear mutations to preserve mito-nuclear compatibility. However, prevalence and factors conditioning this phenomenon remain debated due to its conflicting evidence. Here, we investigate how mito-nuclear incompatibilities impact substitutions in a model for species radiation. Mating success depends on genetic compatibility (nuclear DNA) and spatial proximity. Populations evolve from partially compatible mito-nuclear states, simulating mitochondrial DNA (mtDNA) introgression. Mutations do not confer advantages nor disadvantages, but individual fecundity declines with increasing incompatibilities, selecting for mito-nuclear coordination. We find that selection for mito-nuclear compatibility affects each genome differently based on their initial state. In compatible gene pairs, selection reduces substitutions in both genomes, while in incompatible nuclear genes, it consistently promotes compensation, facilitated by more mismatches. Interestingly, high mitochondrial mutation rates can reduce nuclear compensation by increasing mtDNA rectification, while substitutions in initially compatible nuclear gene are boosted. Finally, the presence of incompatibilities accelerates species radiation, but equilibrium richness is not directly correlated to substitution rates, revealing the complex dynamics triggered by mitochondrial introgression and mito-nuclear coevolution. Our study provides a perspective on nuclear compensation and the role of mito-nuclear incompatibilities in speciation by exploring extreme scenarios and identifying trends that empirical data alone cannot reveal. We emphasize the challenges in detecting these dynamics and propose analyzing specific genomic signatures could shed light on this evolutionary process.

Alterations of Liver Morphology in Senescent Rats.

Age-related liver changes can have important implications for health and metabolic function. This study aimed to describe the morphoquantitative alterations of the liver in senescent rats compared to adult rats. Twelve male rats were used, divided into 6-month-old adults (group A) and 36-month-old senescent rats (group S). Morphometric and histopathological studies, quantification of collagen types I and III, and stereological analyses were performed to determine the volume density of mononucleated (VvhepM) and binucleated (VvhepB) hepatocyte nuclei, surface area density (SvhepM), and number density (NvhepM) of mononucleated hepatocyte nuclei. The findings reveal an alteration of the normal liver tissue architecture in senescent rats and the presence of inflammatory lesions and fibrosis. In addition, there was a decrease in body and liver mass and volume. Group S showed a significant reduction in VvhepM and NvhepM; however, SvhepM was significantly higher. No significant differences were noted in the percentage of binucleated hepatocytes between the two groups. This study reveals substantial morphological changes in the aging liver, with possible functional implications. More research is needed on the underlying mechanisms and their consequences at older ages.

Dosimetry at cellular level for the alpha-emitting radionuclides actinium-225, astatine-211 and radium-223 for bone metastasis cells from castration resistant prostate cancer.

Objectives.The aim of this work is to evaluate energy deposition in the nucleus and cytoplasm in targeted alpha therapy of metastatic castration-resistant prostate cancer by modeling two cell lines, PC3 (osteolytic) and LNCaP C4-2 (osteoblastic), for actinium-225, astatine-211, and radium-223 and their progeny, using Monte Carlo simulations with the GATE/Geant4 code.Approach.We developed single cell and cell clusters models to Monte Carlo simulations, performed on the GATE platform version 9.3, with the GEANT4-DNA physics list emstandard_opt3_mixed_dna for At-211, Ac-225 and Ra-223 progenies. We considered three radionuclide distributions as a sources: the nucleus, the cytoplasm and the whole cell.Main results.When the nucleus was considered as a target, theS-values (N←N) calculated for At-211, Ac-225 and Ra-223 progenies were significantly higher, within 60%-90%, thanS-values (N←Cy), demonstrating less influence of cytoplasm only internalization. When the cytoplasm was considering as a target, theS-values (Cy←Cy) calculated for At-211, Ac-225 and Ra-223 progeny were significantly higher, within 30%-90%, than theS-values (Cy←N). When no progeny migration occurs and for target nucleus , the cumulativeS-values (N←N) calculated for At-211, Ac-225 and Ra-223 were significantly higher, within 50%-70%, than theS-values (N←N) computed for At-211, Ac-225, and Ra-223. Comparing the cumulativeS-values, Ac-225 and Ra-223 therapies is more effective, in terms of deposited energy in a target, than that with At-211.Significance.The data presented in this research indicates that Ac-225 therapy may be the optimum choice due to the energy deposited in the nucleus, as long as the recoil effects and redistribution of progeny are understood. In contrast, At-211 is an alternative to avoid progeny migration. However, to completely analyze the efficacy of radionuclide therapy, other parameters must be considered, such as biological half-life, stability of the transport molecule, progeny migration, excretion pathways, and uptake in different organs.

Lysosomes accumulate at the perinuclear region of muscle cells during chick myogenesis.

Lysosomes are involved in a myriad of cellular functions, such as degradation of macromolecules, endocytosis and exocytosis, modulation of several signaling pathways, and regulation of cell metabolism. To fulfill these diverse functions, lysosomes can undergo several dynamic changes in their content, size, pH, and location within cells. Here, we studied some of these parameters during embryonic chick skeletal muscle cells. We used an anti-lysosome-associated membrane protein 2 (LAMP2) antibody to specifically determine the intracellular localization of lysosomes in these cells. Our data shows that lysosomes are highly enriched in the perinuclear region of chick embryonic muscle cells. We also showed that the wingless signaling pathway (Wnt)/ß-catenin signaling pathway can modulate the location of LAMP2 in chick myogenic cells. Our results highlight the role of lysosomes during muscle differentiation and particularly the presence of a subcellular population of lysosomes that are concentrated in the perinuclear region of muscle cells.

Upregulated Nuclear Expression of Soluble Epoxide Hydrolase Predicts Poor Outcome in Breast Cancer Patients: Importance of the Digital Pathology Approach.

Breast cancer (BC) is the most common cancer in women, with incidence rates increasing globally in recent years. Therefore, it is important to find new molecules with prognostic and therapeutic value to improve therapeutic response and quality of life. The polyunsaturated fatty acids (PUFAs) metabolic pathway participates in various physiological processes, as well as in the development of malignancies. Although aberrancies in the PUFAs metabolic pathway have been implicated in carcinogenesis, the functional and clinical relevance of this pathway has not been well explored in BC. To evaluate the clinical significance of soluble epoxide hydrolase (EPHX2) expression in Mexican patients with BC using tissue microarrays (TMAs) and digital pathology (DP). Immunohistochemical analyses were performed on 11 TMAs with 267 BC samples to quantify this enzyme. Using DP, EPHX2 protein expression was evaluated solely in tumor areas. The association of EPHX2 with overall survival (OS) was detected through bioinformatic analysis in public databases and confirmed in our cohort via Cox regression analysis. Clear nuclear expression of EPHX2 was identified. Receiver operating characteristics (ROC) curves revealed the optimal cutoff point at 2.847062 × 10-3 pixels, with sensitivity of 69.2% and specificity of 67%. Stratification based on this cutoff value showed elevated EPHX2 expression in multiple clinicopathological features, including older age and nuclear grade, human epidermal growth factor receptor 2 (HER2) and triple negative breast cancer (TNBC) subtypes, and recurrence. Kaplan-Meier curves demonstrated how higher nuclear expression of EPHX2 predicts shorter OS. Consistently, multivariate analysis confirmed EPHX2 as an independent predictor of OS, with a hazard ratio (HR) of 3.483 and a 95% confidence interval of 1.804-6.724 (p < 0.001). Our study demonstrates for the first time that nuclear overexpression of EPHX2 is a predictor of poor prognosis in BC patients. The DP approach was instrumental in identifying this significant association. Our study provides valuable insights into the potential clinical utility of EPHX2 as a prognostic biomarker and therapeutic target in BC.

Dynamic changes in mRNA nucleocytoplasmic localization in the nitrate response of Arabidopsis roots.

Nitrate is a nutrient and signal that regulates gene expression. The nitrate response has been extensively characterized at the organism, organ, and cell-type-specific levels, but intracellular mRNA dynamics remain unexplored. To characterize nuclear and cytoplasmic transcriptome dynamics in response to nitrate, we performed a time-course expression analysis after nitrate treatment in isolated nuclei, cytoplasm, and whole roots. We identified 402 differentially localized transcripts (DLTs) in response to nitrate treatment. Induced DLT genes showed rapid and transient recruitment of the RNA polymerase II, together with an increase in the mRNA turnover rates. DLTs code for genes involved in metabolic processes, localization, and response to stimulus indicating DLTs include genes with relevant functions for the nitrate response that have not been previously identified. Using single-molecule RNA FISH, we observed early nuclear accumulation of the NITRATE REDUCTASE 1 (NIA1) transcripts in their transcription sites. We found that transcription of NIA1, a gene showing delayed cytoplasmic accumulation, is rapidly and transiently activated; however, its transcripts become unstable when they reach the cytoplasm. Our study reveals the dynamic localization of mRNAs between the nucleus and cytoplasm as an emerging feature in the temporal control of gene expression in response to nitrate treatment in Arabidopsis roots.

Microscopic Analysis of Nuclear Speckles in a Viviparous Reptile.

Nuclear speckles are compartments enriched in splicing factors present in the nucleoplasm of eucaryote cells. Speckles have been studied in mammalian culture and tissue cells, as well as in some non-mammalian vertebrate cells and invertebrate oocytes. In mammals, their morphology is linked to the transcriptional and splicing activities of the cell through a recruitment mechanism. In rats, speckle morphology depends on the hormonal cycle. In the present work, we explore whether a similar situation is also present in non-mammalian cells during the reproductive cycle. We studied the speckled pattern in several tissues of a viviparous reptile, the lizard Sceloporus torquatus, during two different stages of reproduction. We used immunofluorescence staining against splicing factors in hepatocytes and oviduct epithelium cells and fluorescence and confocal microscopy, as well as ultrastructural immunolocalization and EDTA contrast in Transmission Electron Microscopy. The distribution of splicing factors in the nucleoplasm of oviductal cells and hepatocytes coincides with the nuclear-speckled pattern described in mammals. Ultrastructurally, those cell types display Interchromatin Granule Clusters and Perichromatin Fibers. In addition, the morphology of speckles varies in oviduct cells at the two stages of the reproductive cycle analyzed, paralleling the phenomenon observed in the rat. The results show that the morphology of speckles in reptile cells depends upon the reproductive stage as it occurs in mammals.

Decoding how receptor tyrosine kinases (RTKs) mediate nuclear calcium signaling.

Calcium (Ca2+) is a highly versatile intracellular messenger that regulates several cellular processes. Although it is unclear how a single-second messenger coordinates various effects within a cell, there is growing evidence that spatial patterns of Ca2+ signals play an essential role in determining their specificity. Ca2+ signaling patterns can differ in various cell regions, and Ca2+ signals in the nuclear and cytoplasmic compartments have been observed to occur independently. The initiation and function of Ca2+ signaling within the nucleus are not yet fully understood. Receptor tyrosine kinases (RTKs) induce Ca2+ signaling resulting from phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and inositol 1,4,5-trisphosphate (InsP3) formation within the nucleus. This signaling mechanism may be responsible for the effects of specific growth factors on cell proliferation and gene transcription. This review highlights the recent advances in RTK trafficking to the nucleus and explains how these receptors initiate nuclear calcium signaling.

The basement membrane regulates the cellular localization and the cytoplasmic interactome of Yes-Associated Protein (YAP) in mammary epithelial cells.

The Hippo pathway, a signaling cascade involved in the regulation of organ size and several other processes, acts as a conduit between extracellular matrix (ECM) cues and cellular responses. We asked whether the basement membrane (BM), a specialized ECM component known to induce quiescence and differentiation in mammary epithelial cells, would regulate the localization, activity, and interactome of YAP, a Hippo pathway effector. To address this question, we used a broad range of experimental approaches, including 2D and 3D cultures of both mouse and human mammary epithelial cells, as well as the developing mouse mammary gland. In contrast to malignant cells, nontumoral cells cultured with a reconstituted BM (rBM) displayed higher concentrations of YAP in the cytoplasm. Incidentally, when in the nucleus of rBM-treated cells, YAP resided preferentially at the nuclear periphery. In agreement with our cell culture experiments, YAP exhibited cytoplasmic predominance in ductal cells of developing mammary epithelia, where a denser BM is found. Conversely, terminal end bud (TEB) cells with a thinner BM displayed higher nucleus-to-cytoplasm ratios of YAP. Bioinformatic analysis revealed that genes regulated by YAP were overrepresented in the transcriptomes of microdissected TEBs. Consistently, mouse epithelial cells exposed to the rBM expressed lower levels of YAP-regulated genes, although the protein level of YAP and Hippo components were slightly altered by the treatment. Mass spectrometry analysis identified a differential set of proteins interacting with YAP in cytoplasmic fractions of mouse epithelial cells in the absence or presence of rBM. In untreated cells, YAP interactants were enriched in processes related to ubiquitin-mediated proteolysis, whereas in cells exposed to rBM YAP interactants were mainly key proteins related to amino acid, amino sugar, and carbohydrate metabolism. Collectively, we unraveled that the BM induces YAP translocation or retention in the cytoplasm of nontumoral epithelial cells and that in the cytoplasm YAP seems to undertake novel functions in metabolic pathways.

Translation initiation factor eIF1A rescues hygromycin B sensitivity caused by deleting the carboxy-terminal tail in the GPN-loop GTPase Npa3.

The essential yeast protein GPN-loop GTPase 1 (Npa3) plays a critical role in RNA polymerase II (RNAPII) assembly and subsequent nuclear import. We previously identified a synthetic lethal interaction between a mutant lacking the carboxy-terminal 106-amino acid tail of Npa3 (npa3ΔC) and a bud27Δ mutant. As the prefoldin-like Bud27 protein participates in ribosome biogenesis and translation, we hypothesized that Npa3 may also regulate these biological processes. We investigated this proposal by using Saccharomyces cerevisiae strains episomally expressing either wild-type Npa3 or hypomorphic mutants (Npa3ΔC, Npa3K16R, and Npa3G70A). The Npa3ΔC mutant fully supports RNAPII nuclear localization and activity. However, the Npa3K16R and Npa3G70A mutants only partially mediate RNAPII nuclear targeting and exhibit a higher reduction in Npa3 function. Cell proliferation in these strains displayed an increased sensitivity to protein synthesis inhibitors hygromycin B and geneticin/G418 (npa3G70A > npa3K16R > npa3ΔC > NPA3 cells) but not to transcriptional elongation inhibitors 6-azauracil, mycophenolic acid or 1,10-phenanthroline. In all three mutant strains, the increase in sensitivity to both aminoglycoside antibiotics was totally rescued by expressing NPA3. Protein synthesis, visualized by quantifying puromycin incorporation into nascent-polypeptide chains, was markedly more sensitive to hygromycin B inhibition in npa3ΔC, npa3K16R, and npa3G70A than NPA3 cells. Notably, high-copy expression of the TIF11 gene, that encodes the eukaryotic translation initiation factor 1A (eIF1A) protein, completely suppressed both phenotypes (of reduced basal cell growth and increased sensitivity to hygromycin B) in npa3ΔC cells but not npa3K16R or npa3G70A cells. We conclude that Npa3 plays a critical RNAPII-independent and previously unrecognized role in translation initiation.

Structural basis of nuclear transport for NEIL DNA glycosylases mediated by importin-alpha.

NEIL glycosylases, including NEIL1, NEIL2, and NEIL3, play a crucial role in the base excision DNA repair pathway (BER). The classical importin pathway mediated by importin α/ß and cargo proteins containing nuclear localization sequences (NLS) is the most common transport mechanism of DNA repair proteins to the nucleus. Previous studies have identified putative NLSs located at the C-terminus of NEIL3 and NEIL1. Crystallographic, bioinformatics, calorimetric (ITC), and fluorescence assays were used to investigate the interaction between NEIL1 and NEIL3 putative NLSs and importin-α (Impα). Our findings showed that NEIL3 contains a typical cNLS, with medium affinity for the major binding site of Impα. In contrast, crystallographic analysis of NEIL1 NLS revealed its binding to Impα, but with high B-factors and a lack of electron density at the linker region. ITC and fluorescence assays indicated no detectable affinity between NEIL1 NLS and Impα. These data suggest that NEIL1 NLS is a non-classical NLS with low affinity to Impα. Additionally, we compared the binding mode of NEIL3 and NEIL1 with Mus musculus Impα to human isoforms HsImpα1 and HsImpα3, which revealed interesting binding differences for HsImpα3 variant. NEIL3 is a classical medium affinity monopartite NLS, while NEIL1 is likely to be an unclassical low-affinity bipartite NLS. The base excision repair pathway is one of the primary systems involved in repairing DNA. Thus, understanding the mechanisms of nuclear transport of NEIL proteins is crucial for comprehending the role of these proteins in DNA repair and disease development.

CTCF and Its Multi-Partner Network for Chromatin Regulation.

Architectural proteins are essential epigenetic regulators that play a critical role in organizing chromatin and controlling gene expression. CTCF (CCCTC-binding factor) is a key architectural protein responsible for maintaining the intricate 3D structure of chromatin. Because of its multivalent properties and plasticity to bind various sequences, CTCF is similar to a Swiss knife for genome organization. Despite the importance of this protein, its mechanisms of action are not fully elucidated. It has been hypothesized that its versatility is achieved through interaction with multiple partners, forming a complex network that regulates chromatin folding within the nucleus. In this review, we delve into CTCF's interactions with other molecules involved in epigenetic processes, particularly histone and DNA demethylases, as well as several long non-coding RNAs (lncRNAs) that are able to recruit CTCF. Our review highlights the importance of CTCF partners to shed light on chromatin regulation and pave the way for future exploration of the mechanisms that enable the finely-tuned role of CTCF as a master regulator of chromatin.

The paralogues MAGOH and MAGOHB are oncogenic factors in high-grade gliomas and safeguard the splicing of cell division and cell cycle genes.

The exon junction complex (EJC) plays key roles throughout the lifespan of RNA and is particularly relevant in the nervous system. We investigated the roles of two EJC members, the paralogs MAGOH and MAGOHB, with respect to brain tumour development. High MAGOH/MAGOHB expression was observed in 14 tumour types; glioblastoma (GBM) showed the greatest difference compared to normal tissue. Increased MAGOH/MAGOHB expression was associated with poor prognosis in glioma patients, while knockdown of MAGOH/MAGOHB affected different cancer phenotypes. Reduced MAGOH/MAGOHB expression in GBM cells caused alterations in the splicing profile, including re-splicing and skipping of multiple exons. The binding profiles of EJC proteins indicated that exons affected by MAGOH/MAGOHB knockdown accumulated fewer complexes on average, providing a possible explanation for their sensitivity to MAGOH/MAGOHB knockdown. Transcripts (genes) showing alterations in the splicing profile are mainly implicated in cell division, cell cycle, splicing, and translation. We propose that high MAGOH/MAGOHB levels are required to safeguard the splicing of genes in high demand in scenarios requiring increased cell proliferation (brain development and GBM growth), ensuring efficient cell division, cell cycle regulation, and gene expression (splicing and translation). Since differentiated neuronal cells do not require increased MAGOH/MAGOHB expression, targeting these paralogs is a potential option for treating GBM.

Nfinder: automatic inference of cell neighborhood in 2D and 3D using nuclear markers.

BACKGROUND: In tissues and organisms, the coordination of neighboring cells is essential to maintain their properties and functions. Therefore, knowing which cells are adjacent is crucial to understand biological processes that involve physical interactions among them, e.g. cell migration and proliferation. In addition, some signaling pathways, such as Notch or extrinsic apoptosis, are highly dependent on cell-cell communication. While this is straightforward to obtain from membrane images, nuclei labelling is much more ubiquitous for technical reasons. However, there are no automatic and robust methods to find neighboring cells based only on nuclear markers. RESULTS: In this work, we describe Nfinder, a method to assess the cell's local neighborhood from images with nuclei labeling. To achieve this goal, we approximate the cell-cell interaction graph by the Delaunay triangulation of nuclei centroids. Then, links are filtered by automatic thresholding in cell-cell distance (pairwise interaction) and the maximum angle that a pair of cells subtends with shared neighbors (non-pairwise interaction). We systematically characterized the detection performance by applying Nfinder to publicly available datasets from Drosophila melanogaster, Tribolium castaneum, Arabidopsis thaliana and C. elegans. In each case, the result of the algorithm was compared to a cell neighbor graph generated by manually annotating the original dataset. On average, our method detected 95% of true neighbors, with only 6% of false discoveries. Remarkably, our findings indicate that taking into account non-pairwise interactions might increase the Positive Predictive Value up to + 11.5%. CONCLUSION: Nfinder is the first robust and automatic method for estimating neighboring cells in 2D and 3D based only on nuclear markers and without any free parameters. Using this tool, we found that taking non-pairwise interactions into account improves the detection performance significantly. We believe that using our method might improve the effectiveness of other workflows to study cell-cell interactions from microscopy images. Finally, we also provide a reference implementation in Python and an easy-to-use napari plugin.

Deciphering Divergent Trypanosomatid Nuclear Complexes by Analyzing Interactomic Datasets with AlphaFold2 and Genetic Approaches.

Acetylation signaling pathways in trypanosomatids, a group of early branching organisms, are poorly understood due to highly divergent protein sequences. To overcome this challenge, we used interactomic datasets and AlphaFold2 (AF2)-multimer to predict direct interactions and validated them using yeast two and three-hybrid assays. We focused on MORF4 related gene (MRG) domain-containing proteins and their interactions, typically found in histone acetyltransferase/deacetylase complexes. The results identified a structurally conserved complex, TcTINTIN, which is orthologous to human and yeast trimer independent of NuA4 for transcription interaction (TINTIN) complexes; and another trimeric complex involving an MRG domain, only seen in trypanosomatids. The identification of a key component of TcTINTIN, TcMRGBP, would not have been possible through traditional homology-based methods. We also conducted molecular dynamics simulations, revealing a conformational change that potentially affects its affinity for TcBDF6. The study also revealed a novel way in which an MRG domain participates in simultaneous interactions with two MRG binding proteins binding two different surfaces, a phenomenon not previously reported. Overall, this study demonstrates the potential of using AF2-processed interactomic datasets to identify protein complexes in deeply branched eukaryotes, which can be challenging to study based on sequence similarity. The findings provide new insights into the acetylation signaling pathways in trypanosomatids, specifically highlighting the importance of MRG domain-containing proteins in forming complexes, which may have important implications for understanding the biology of these organisms and developing new therapeutics. On the other hand, our validation of AF2 models for the determination of multiprotein complexes illuminates the power of using such artificial intelligence-derived tools in the future development of biology.

Molecular Mechanisms of Nuclear Transport of the Neuronal Voltage-gated Ca2+ Channel ß3 Auxiliary Subunit.

Previous studies have shown that in addition to its role within the voltage-gated calcium channel complex in the plasma membrane, the neuronal CaVß subunit can translocate to the cell nucleus. However, little is known regarding the role this protein could play in the nucleus, nor the molecular mechanism used by CaVß to enter this cell compartment. This report shows evidence that CaVß3 has nuclear localization signals (NLS) that are not functional, suggesting that the protein does not use a classical nuclear import pathway. Instead, its entry into the nucleus could be associated with another protein that would function as a carrier, using a mechanism known as a piggyback. Mass spectrometry assays and bioinformatic analysis allowed the identification of proteins that could be participating in the entry of CaVß3 into the nucleus. Likewise, through proximity ligation assays (PLA), it was found that members of the heterogeneous nuclear ribonucleoproteins (hnRNPs) and B56δ, a regulatory subunit of the protein phosphatase 2A (PP2A), could function as proteins that regulate this piggyback mechanism. On the other hand, bioinformatics and site-directed mutagenesis assays allowed the identification of a functional nuclear export signal (NES) that controls the exit of CaVß3 from the nucleus, which would allow the completion of the nuclear transport cycle of the protein. These results reveal a novel mechanism for the nuclear transport cycle of the neuronal CaVß3 subunit.

Elucidation of the mechanism of nuclear localization of Mexican tetra Neu4 via bipartite nuclear localization signal and less conserved regions.

Nuclear sialoglycans are minor components in the nucleus, and their biological significance was not well understood. Recently, Nile tilapia Neu4 sialidase (OnNeu4) was identified and reported as the first nuclear sialidase in vertebrates. Although OnNeu4 possesses the nuclear localization signal (NLS) required for nuclear localization, other fish Neu4 sialidases, such as zebrafish and Japanese medaka, also possess NLS, but their subcellular localizations are not nucleus. To understand the nuclear localization mechanism of fish Neu4, we focused on Mexican tetra Neu4 (AmNeu4), which, unlike Neu4 in other fishes, has a bipartite NLS. AmNeu4 exhibited a wide range of optimal pH and substrate specificity, and its gene expression was specifically detected in the liver, spleen, and gut in adult fish. AmNeu4, like OnNeu4, exhibited nuclear localization, which was attenuated by importin inhibitor, and deletion of the bipartite NLS completely reduced the nuclear localization. In addition, the conjugation of the bipartite NLS of AmNeu4 made GFP show nuclear localization. To understand the mechanism of nuclear localization of AmNeu4 and OnNeu4, we compared fish Neu4 amino acid sequences and focused on the less conserved region of Neu4 sialidase (LCR). LCR-deletion mutants of AmNeu4 and OnNeu4 showed significantly reduced the nuclear localization. The LCR region in AmNeu4 and OnNeu4 possessed consecutive Ser/Thr. The Neu4 mutants in which consecutive Ser/Thr in LCR were changed to Ala or deleted significantly suppressed the nuclear localization. These results suggest that the nuclear localization of Neu4 in Nile tilapia and Mexican tetra may be regulated by NLS and LCR.

Binding of Venezuelan Equine Encephalitis Virus Inhibitors to Importin-α Receptors Explored with All-Atom Replica Exchange Molecular Dynamics.

Although Venezuelan equine encephalitis virus (VEEV) is a life-threatening pathogen with a capacity for epidemic outbreaks, there are no FDA-approved VEEV antivirals for humans. VEEV cytotoxicity is partially attributed to the formation of a tetrameric complex between the VEEV capsid protein, the nuclear import proteins importin-α and importin-ß, and the nuclear export protein CRM1, which together block trafficking through the nuclear pore complex. Experimental studies have identified small molecules from the CL6662 scaffold as potential inhibitors of the viral nuclear localization signal (NLS) sequence binding to importin-α. However, little is known about the molecular mechanism of CL6662 inhibition. To address this issue, we employed all-atom replica exchange molecular dynamics simulations to probe, in atomistic detail, the binding mechanism of CL6662 ligands to importin-α. Three ligands, including G281-1485 and two congeners with varying hydrophobicities, were considered. We investigated the distribution of ligand binding poses, their locations, and ligand specificities measured by the strength of binding interactions. We found that G281-1485 binds nonspecifically without forming well-defined binding poses throughout the NLS binding site. Binding of the less hydrophobic congener becomes strongly on-target with respect to the NLS binding site but remains nonspecific. However, a more hydrophobic congener is a strongly specific binder and the only ligand out of three to form a well-defined binding pose, while partially overlapping with the NLS binding site. On the basis of free energy estimates, we argue that all three ligands weakly compete with the viral NLS sequence for binding to importin-α in an apparent compromise to preserve host NLS binding. We further show that all-atom replica exchange binding simulations are a viable tool for studying ligands binding nonspecifically without forming well-defined binding poses.

Rescue of Mitochondrial Function in Hutchinson-Gilford Progeria Syndrome by the Pharmacological Modulation of Exportin CRM1.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging disorder caused by the expression of progerin, a mutant variant of Lamin A. Recently, HGPS studies have gained relevance because unraveling its underlying mechanism would help to understand physiological aging. We previously reported that the CRM1-mediated nuclear protein export pathway is exacerbated in HGPS cells, provoking the mislocalization of numerous protein targets of CRM1. We showed that normalization of this mechanism by pharmacologically inhibiting CRM1 with LMB (specific CRM1 inhibitor), mitigates the senescent phenotype of HGPS cells. Since mitochondrial dysfunction is a hallmark of HGPS, in this study we analyze the effect of LMB on mitochondrial function. Remarkably, LMB treatment induced the recovery of mitochondrial function in HGPS cells, as shown by the improvement in mitochondrial morphology, mitochondrial membrane potential, and ATP levels, which consequently impeded the accumulation of ROS but not mitochondrial superoxide. We provide evidence that the beneficial effect of LMB is mechanistically based on a combinatory effect on mitochondrial biogenesis via upregulation of PGC-1α expression (master transcription cofactor of mitochondrial genes), and mitophagy through the recovery of lysosomal content. The use of exportin CRM1 inhibitors constitutes a promising strategy to treat HGPS and other diseases characterized by mitochondrial impairment.