Nuclear Protein Import Research Articles

Abstract 890: Calmodulin governs nuclear entry of fusion PAX3/FOXO1 oncoprotein, a target in alveolar rhabdomyosarcoma

Abstract Reciprocal chromosomal translocations resulting in the fusion of the DNA binding domain of the transcription factor PAX3 and the transactivational domain of FOXO1 underlie most cases of alveolar rhabdomyosarcoma (aRMS). Testing of compound MTPF63 against a panel of sarcoma cell lines showed that it had an inhibitory effect on proliferation of PAX3-FOXO1 translocation-positive cells. Further evaluation of its activity in Rh4 cells transduced with a PAX3-FOXO1 luciferase reporter showed that it attenuated reporter activity. Additional confirmation for the compound's inhibitory impact on PAX3-FOXO1 activity was obtained by detecting significant attenuation in the levels of Alk1 and MyoD1 proteins whose expression is driven by PAX3-FOXO1 in aRMS cells. Cell viability assays performed under 2D and 3D culturing conditions demonstrated that MTPF63 inhibits cell growth more effectively in translocation-positive alveolar RMS than in translocation-negative embryonal RMS. A hypothesis for MTPF63's mode of action was generated using the complete pool of barcoded essential heterozygous (~1150 strains) and homozygous (~4800 strains) diploid gene deletion mutants of Saccharomyces cerevisiae to identify gene deletions that confer sensitivity to the compound. Gene ontology enrichment analysis of 255 sensitive mutants whose growth rates were significantly inhibited in the presence of the compound compared to control, not only revealed calmodulin as main target of the compound but also illustrated the compound's interference with calmodulin-dependent nuclear protein import pathway, suggesting that it might impact PAX3-FOXO1 nuclear localization. Immunostaining of nuclear-localized PAX3-FOXO1 oncoprotein in two aRMS cell lines revealed cytoplasmic localization. Immunoblot analysis of fractionated cytosolic and nuclear protein lysates for PAX3-FOXO1 in MTPF63 treated cells also showed increased amounts in the cytosolic fractions. Transient overexpression of calmodulin was shown to partially rescue the inhibitory effect of the compound on PAX3-FOXO1-driven luciferase expression in RH4 reporter cells. Taken together, the data demonstrate the involvement of calmodulin in the nuclear import of PAX3-FOXO1 and introduce calmodulin antagonists as potential therapeutics against translocation-positive childhood alveolar rhabdomyosarcoma. Citation Format: Emad Darvishi, Cheryl Lynn Thomas, Berkley Eric Gryder, John Frederick Shern, Javed Khan, Girma M Woldemichael. Calmodulin governs nuclear entry of fusion PAX3/FOXO1 oncoprotein, a target in alveolar rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 890.

Mitochondrial proteome disruption in the diabetic heart through targeted epigenetic regulation at the mitochondrial heat shock protein 70 (mtHsp70) nuclear locus

>99% of the mitochondrial proteome is nuclear-encoded. The mitochondrion relies on a coordinated multi-complex process for nuclear genome-encoded mitochondrial protein import. Mitochondrial heat shock protein 70 (mtHsp70) is a key component of this process and a central constituent of the protein import motor. Type 2 diabetes mellitus (T2DM) disrupts mitochondrial proteomic signature which is associated with decreased protein import efficiency. The goal of this study was to manipulate the mitochondrial protein import process through targeted restoration of mtHsp70, in an effort to restore proteomic signature and mitochondrial function in the T2DM heart. A novel line of cardiac-specific mtHsp70 transgenic mice on the db/db background were generated and cardiac mitochondrial subpopulations were isolated with proteomic evaluation and mitochondrial function assessed. MicroRNA and epigenetic regulation of the mtHsp70 gene during T2DM were also evaluated. MtHsp70 overexpression restored cardiac function and nuclear-encoded mitochondrial protein import, contributing to a beneficial impact on proteome signature and enhanced mitochondrial function during T2DM. Further, transcriptional repression at the mtHsp70 genomic locus through increased localization of H3K27me3 during T2DM insult was observed. Our results suggest that restoration of a key protein import constituent, mtHsp70, provides therapeutic benefit through attenuation of mitochondrial and contractile dysfunction in T2DM.

Importin α5 negatively regulates importin β1-mediated nuclear import of Newcastle disease virus matrix protein and viral replication and pathogenicity in chicken fibroblasts

ABSTRACTThe matrix (M) protein of Newcastle disease virus (NDV) is demonstrated to localize in the nucleus via intrinsic nuclear localization signal (NLS), but cellular proteins involved in the nuclear import of NDV M protein and the role of M's nuclear localization in the replication and pathogenicity of NDV remain unclear. In this study, importin β1 was screened to interact with NDV M protein by yeast two-hybrid screening. This interaction was subsequently confirmed by co-immunoprecipitation and pull-down assays. In vitro binding studies indicated that the NLS region of M protein and the amino acids 336–433 of importin β1 that belonged to the RanGTP binding region were important for binding. Importantly, a recombinant virus with M/NLS mutation resulted in a pathotype change of NDV and attenuated viral replication and pathogenicity in chicken fibroblasts and SPF chickens. In agreement with the binding data, nuclear import of NDV M protein in digitonin-permeabilized HeLa cells required both importin β1 and RanGTP. Interestingly, importin α5 was verified to interact with M protein through binding importin β1. However, importin β1 or importin α5 depletion by siRNA resulted in different results, which showed the obviously cytoplasmic or nuclear accumulation of M protein and the remarkably decreased or increased replication ability and pathogenicity of NDV in chicken fibroblasts, respectively. Our findings therefore demonstrate for the first time the nuclear import mechanism of NDV M protein and the negative regulation role of importin α5 in importin β1-mediated nuclear import of M protein and the replication and pathogenicity of a paramyxovirus.

Beyond Histones: New Substrate Proteins of Lysine Deacetylases in Arabidopsis Nuclei

The reversible acetylation of lysine residues is catalyzed by the antagonistic action of lysine acetyltransferases and deacetylases, which can be considered as master regulators of their substrate proteins. Lysine deacetylases, historically referred to as histone deacetylases, have profound functions in regulating stress defenses and development in plants. Lysine acetylation of the N-terminal histone tails promotes gene transcription and decondensation of chromatin, rendering the DNA more accessible to the transcription machinery. In plants, the classical lysine deacetylases from the RPD3/HDA1-family have thus far mainly been studied in the context of their deacetylating activities on histones, and their versatility in molecular activities is still largely unexplored. Here we discuss the potential impact of lysine acetylation on the recently identified nuclear substrate proteins of lysine deacetylases from the Arabidopsis RPD3/HDA1-family. Among the deacetylase substrate proteins, many interesting candidates involved in nuclear protein import, transcriptional regulation, and chromatin remodeling have been identified. These candidate proteins represent key starting points for unraveling new molecular functions of the Arabidopsis lysine deacetylases. Site-directed engineering of lysine acetylation sites on these target proteins might even represent a new approach for optimizing plant growth under climate change conditions.

Roles of the CSE1L-mediated nuclear import pathway in epigenetic silencing

Epigenetic silencing can be mediated by various mechanisms, and many regulators remain to be identified. Here, we report a genome-wide siRNA screening to identify regulators essential for maintaining gene repression of a CMV promoter silenced by DNA methylation. We identified CSE1L (chromosome segregation 1 like) as an essential factor for the silencing of the reporter gene and many endogenous methylated genes. CSE1L depletion did not cause DNA demethylation. On the other hand, the methylated genes derepressed by CSE1L depletion largely overlapped with methylated genes that were also reactivated by treatment with histone deacetylase inhibitors (HDACi). Gene silencing defects observed upon CSE1L depletion were linked to its nuclear import function for certain protein cargos because depletion of other factors involved in the same nuclear import pathway, including KPNAs and KPNB1 proteins, displayed similar derepression profiles at the genome-wide level. Therefore, CSE1L appears to be critical for the nuclear import of certain key repressive proteins. Indeed, NOVA1, HDAC1, HDAC2, and HDAC8, genes known as silencing factors, became delocalized into cytosol upon CSE1L depletion. This study suggests that the cargo specificity of the protein nuclear import system may impact the selectivity of gene silencing.

MOS6 and TN13 in plant immunity

ABSTRACTThe Arabidopsis nuclear transport receptor IMPORTIN-α3/MOS6 (MODIFIER OF SNC1, 6) is required for constitutive defense responses of the auto-immune mutant snc1 (suppressor of npr1-1, constitutive 1) and contributes to basal disease resistance, suggesting a role in nuclear import of defense-regulatory cargo proteins. We recently showed that MOS6 selectively interacts with TN13, a TIR-NBS protein involved in basal resistance to Pseudomonas syringae pv. tomato (Pst) DC3000 lacking the effectors AvrPto and AvrPtoB. Consistent with a predicted N-terminal transmembrane domain, TN13 localizes to the endoplasmic reticulum (ER) and the nuclear envelope (NE) where it interacts with MOS6 in a transient expression assay. Here, we propose a model that summarizes the subcellular localization, association and function of TN13 and MOS6 in plant defense signaling.

Depletion of nuclear import protein karyopherin alpha 7 (KPNA7) induces mitotic defects and deformation of nuclei in cancer cells

BackgroundNucleocytoplasmic transport is a tightly regulated process carried out by specific transport machinery, the defects of which may lead to a number of diseases including cancer. Karyopherin alpha 7 (KPNA7), the newest member of the karyopherin alpha nuclear importer family, is expressed at a high level during embryogenesis, reduced to very low or absent levels in most adult tissues but re-expressed in cancer cells.MethodsWe used siRNA-based knock-down of KPNA7 in cancer cell lines, followed by functional assays (proliferation and cell cycle) and immunofluorescent stainings to determine the role of KPNA7 in regulation of cancer cell growth, proper mitosis and nuclear morphology.ResultsIn the present study, we show that the silencing of KPNA7 results in a dramatic reduction in pancreatic and breast cancer cell growth, irrespective of the endogenous KPNA7 expression level. This growth inhibition is accompanied by a decrease in the fraction of S-phase cells as well as aberrant number of centrosomes and severe distortion of the mitotic spindles. In addition, KPNA7 depletion leads to reorganization of lamin A/C and B1, the main nuclear lamina proteins, and drastic alterations in nuclear morphology with lobulated and elongated nuclei.ConclusionsTaken together, our data provide new important evidence on the contribution of KPNA7 to the regulation of cancer cell growth and the maintenance of nuclear envelope environment, and thus deepens our understanding on the impact of nuclear transfer proteins in cancer pathogenesis.

Heat shock protein 60 involvement in vascular smooth muscle cell proliferation

AimHeat shock protein 60 (Hsp60) is a mediator of stress-induced vascular smooth muscle cell (VSMC) proliferation. This study will determine, first, if the mitochondrial or cytoplasmic localization of Hsp60 is critical to VSMC proliferation and, second, the mechanism of Hsp60 induction of VSMC proliferation with a focus on modification of nucleocytoplasmic trafficking. Methods and resultsHsp60 was overexpressed in primary rabbit VSMCs with or without a mitochondrial targeting sequence (AdHsp60mito-). Both interventions induced an increase in VSMC PCNA expression and proliferation. The increase in VSMC PCNA expression and growth was not observed after siRNA-mediated knockdown of Hsp60 expression. Nuclear protein import in VSMC was measured by fluorescent microscopy using a microinjected fluorescent import substrate. Nuclear protein import was stimulated by both AdHsp60 and AdHsp60mito- treatments. AdHsp60 treatment also induced increases in nucleoporin (Nup) 62, Nup153, importin-α, importin-β and Ran expression as well as cellular ATP levels compared to control. AdHsp60mito- treatment induced an up-regulation in importin-α, importin-β and Ran expression compared to control. Hsp60 knockdown did not change nuclear protein import nor the expression of any nuclear transport receptors or nucleoporins. Both heat shock treatment and Hsp60 overexpression promoted the interaction of Ran with Hsp60. ConclusionsVSMC proliferation can be modulated via an Hsp60 dependent, cytosol localized mechanism that in part involves a stimulation of nuclear protein import through an interaction with Ran. This novel cellular signaling role for Hsp60 may be important in growth-based vascular pathologies like atherosclerosis and hypertension.

KPNB1 inhibition disrupts proteostasis and triggers unfolded protein response-mediated apoptosis in glioblastoma cells

The nuclear import receptor karyopherin β1 (KPNB1) is involved in the nuclear import of most proteins and in the regulation of multiple mitotic events. Upregulation of KPNB1 has been observed in cancers including glioblastoma. Depletion of KPNB1 induces mitotic arrest and apoptosis in cancer cells, but the underlying mechanism is not clearly elucidated. Here, we found that downregulation and functional inhibition of KPNB1 in glioblastoma cells induced growth arrest and apoptosis without apparent mitotic arrest. KPNB1 inhibition upregulated Puma and Noxa and freed Mcl-1-sequestered Bax and Bak, leading to mitochondrial outer membrane permeabilization (MOMP) and apoptosis. Moreover, combination of Bcl-xL inhibitors and KPNB1 inhibition enhanced apoptosis in glioblastoma cells. KPNB1 inhibition promoted cytosolic retention of its cargo and impaired cellular proteostasis, resulting in elevated polyubiquitination, formation of aggresome-like-induced structure (ALIS), and unfolded protein response (UPR). Ubiquitination elevation and UPR activation in KPNB1-deficient cells were reversed by KPNB1 overexpression or inhibitors of protein synthesis but aggravated by inhibitors of autophagy-lysosome or proteasome, indicating that rebalance of cytosolic/nuclear protein distribution and alleviation of protein overload favor proteostasis and cell survival. Chronic activation of eIF2α/ATF4 cascade of UPR was responsible for the upregulation of Puma and Noxa, apoptosis and ABT-263 sensitivity. Taken together, our findings demonstrate that KPNB1 is required for proteostasis maintenance and its inhibition induces apoptosis in glioblastoma cells through UPR-mediated deregulation of Bcl-2 family members.

Non-equivalence of nuclear import among nuclei in multinucleated skeletal muscle cells.

Skeletal muscle is primarily composed of large myofibers containing thousands of post-mitotic nuclei distributed throughout a common cytoplasm. Protein production and localization in specialized myofiber regions is crucial for muscle function. Myonuclei differ in transcriptional activity and protein accumulation, but how these differences among nuclei sharing a cytoplasm are achieved is unknown. Regulated nuclear import of proteins is one potential mechanism for regulating transcription spatially and temporally in individual myonuclei. The best-characterized nuclear localization signal (NLS) in proteins is the classical NLS (cNLS), but many other NLS motifs exist. We examined cNLS and non-cNLS reporter protein import using multinucleated muscle cells generated in vitro, revealing that cNLS and non-cNLS nuclear import differs among nuclei in the same cell. Investigation of cNLS nuclear import rates in isolated myofibers ex vivo confirmed differences in nuclear import rates among myonuclei. Analyzing nuclear import throughout myogenesis revealed that cNLS and non-cNLS import varies during differentiation. Taken together, our results suggest that both spatial and temporal regulation of nuclear import pathways are important in muscle cell differentiation and protein regionalization in myofibers.

Visualization of human karyopherin beta-1/importin beta-1 interactions with protein partners in mitotic cells by co-immunoprecipitation and proximity ligation assays

Karyopherin beta-1/Importin beta-1 is a conserved nuclear transport receptor, acting in protein nuclear import in interphase and as a global regulator of mitosis. These pleiotropic functions reflect its ability to interact with, and regulate, different pathways during the cell cycle, operating as a major effector of the GTPase RAN. Importin beta-1 is overexpressed in cancers characterized by high genetic instability, an observation that highlights the importance of identifying its partners in mitosis. Here we present the first comprehensive profile of importin beta-1 interactors from human mitotic cells. By combining co-immunoprecipitation and proteome-wide mass spectrometry analysis of synchronized cell extracts, we identified expected (e.g., RAN and SUMO pathway factors) and novel mitotic interactors of importin beta-1, many with RNA-binding ability, that had not been previously associated with importin beta-1. These data complement interactomic studies of interphase transport pathways. We further developed automated proximity ligation assay (PLA) protocols to validate selected interactors. We succeeded in obtaining spatial and temporal resolution of genuine importin beta-1 interactions, which were visualized and localized in situ in intact mitotic cells. Further developments of PLA protocols will be helpful to dissect importin beta-1-orchestrated pathways during mitosis.

Myosin‐1C uses a novel phosphoinositide‐dependent pathway for nuclear localization

Accurate control of macromolecule transport between nucleus and cytoplasm underlines several essential biological processes, including gene expression. According to the canonical model, nuclear import of soluble proteins is based on nuclear localization signals and transport factors. We challenge this view by showing that nuclear localization of the actin‐dependent motor protein Myosin‐1C (Myo1C) resembles the diffusion–retention mechanism utilized by inner nuclear membrane proteins. We show that Myo1C constantly shuttles in and out of the nucleus and that its nuclear localization does not require soluble factors, but is dependent on phosphoinositide binding. Nuclear import of Myo1C is preceded by its interaction with the endoplasmic reticulum, and phosphoinositide binding is specifically required for nuclear import, but not nuclear retention, of Myo1C. Our results therefore demonstrate, for the first time, that membrane association and binding to nuclear partners is sufficient to drive nuclear localization of also soluble proteins, opening new perspectives to evolution of cellular protein sorting mechanisms.

In Vitro Modeling of Congenital Heart Defects Associated with an NKX2-5 Mutation Revealed a Dysregulation in BMP/Notch-Mediated Signaling.

The Nkx2-5 gene codes for a transcription factor that plays a critical role in heart development. Heterozygous mutations in NKX2-5 in both human and mice result in congenital heart defects (CHDs). However, the molecular mechanisms by which these mutations cause the disease are still unknown. Recently, we have generated the heterozygous mouse model of the human CHDs associated mutation NKX2-5 R142C (Nkx2-5R141C/+ mouse ortholog of human NKX2-5 R142C variant) that developed septal and conduction defects. This study generated a heterozygous Nkx2-5 R141C mouse embryonic stem cell line (Nkx2-5R141C/+ mESCs) to model CHDs in vitro. We observed that Nkx2-5R141C/+ mESCs display an alteration in the expression of genes that are essential for normal heart development. Furthermore, the reduced cardiomyogenesis is paralleled by a reduction in nuclear import of Nkx2-5 protein. Examination of the Nkx2-5R141C/+ embryos at E8.5 revealed a transient loss of cardiomyogenesis, which is consistent with the phenotype observed in vitro. Moreover, gene expression profiling of Nkx2-5R141C/+ cells at an early stage of cardiac differentiation revealed pronounced deregulation of several cardiac differentiation and function genes. Collectively, our data showed that heterozygosity for the R141C mutation results in disruption of the cellular distribution of Nkx2-5 protein, a transient reduction in cardiomyogenesis that may disrupt the early patterning of the heart, and this, in turn, affects the intricate orchestration of signaling pathways leading to downregulation of Bone morphogenetic protein (BMP) and Notch signaling. Therefore, we have developed mESCs model of a human CHD, providing an in vitro system to examine early stages of heart development, which are otherwise difficult to study in vivo. Stem Cells 2018;36:514-526.

TDP-43 pathology disrupts nuclear pore complexes and nucleocytoplasmic transport in ALS/FTD

The cytoplasmic mislocalization and aggregation of TAR DNA-binding protein-43 (TDP-43) is a common histopathological hallmark of the amyotrophic lateral sclerosis and frontotemporal dementia disease spectrum (ALS/FTD). However, the composition of aggregates and their contribution to the disease process remain unknown. Here we used proximity-dependent biotin identification (BioID) to interrogate the interactome of detergent-insoluble TDP-43 aggregates and found them enriched for components of the nuclear pore complex and nucleocytoplasmic transport machinery. Aggregated and disease-linked mutant TDP-43 triggered the sequestration and/or mislocalization of nucleoporins and transport factors, and interfered with nuclear protein import and RNA export in mouse primary cortical neurons, human fibroblasts and induced pluripotent stem cell–derived neurons. Nuclear pore pathology is present in brain tissue in cases of sporadic ALS and those involving genetic mutations in TARDBP and C9orf72. Our data strongly implicate TDP-43-mediated nucleocytoplasmic transport defects as a common disease mechanism in ALS/FTD.

Importin α1 is required for nuclear import of herpes simplex virus proteins and capsid assembly in fibroblasts and neurons.

Herpesviruses are large DNA viruses which depend on many nuclear functions, and therefore on host transport factors to ensure specific nuclear import of viral and host components. While some import cargoes bind directly to certain transport factors, most recruit importin β1 via importin α. We identified importin α1 in a small targeted siRNA screen to be important for herpes simplex virus (HSV-1) gene expression. Production of infectious virions was delayed in the absence of importin α1, but not in cells lacking importin α3 or importin α4. While nuclear targeting of the incoming capsids, of the HSV-1 transcription activator VP16, and of the viral genomes were not affected, the nuclear import of the HSV-1 proteins ICP4 and ICP0, required for efficient viral transcription, and of ICP8 and pUL42, necessary for DNA replication, were reduced. Furthermore, quantitative electron microscopy showed that fibroblasts lacking importin α1 contained overall fewer nuclear capsids, but an increased proportion of mature nuclear capsids indicating that capsid formation and capsid egress into the cytoplasm were impaired. In neurons, importin α1 was also not required for nuclear targeting of incoming capsids, but for nuclear import of ICP4 and for the formation of nuclear capsid assembly compartments. Our data suggest that importin α1 is specifically required for the nuclear localization of several important HSV1 proteins, capsid assembly, and capsid egress into the cytoplasm, and may become rate limiting in situ upon infection at low multiplicity or in terminally differentiated cells such as neurons.

Abstract B12: Investigating the specificity of the small molecule inhibitor INI-43 for Kpnβ1 in cancer cells

Abstract Karyopherin β1 (Kpnβ1) is the major nuclear import protein involved in the shuttling of cargo proteins and mRNAs into the cell nucleus. Recent studies have found that Kpnβ1 levels are significantly upregulated in cancer cells, and that targeted inhibition of Kpnβ1 with siRNA results in reduced proliferation and the induction of apoptosis, suggesting that Kpnβ1 is important for cancer cell survival. Inhibitor of Nuclear Import-43 (INI-43) is a small molecule inhibitor identified in our laboratory as having the potential to interfere with the nuclear import function of Kpnβ1. It has been shown to inhibit the nuclear localization of both Kpnβ1 and known Kpnβ1 cargoes such as NFκB and NFAT, as well as inhibit the proliferation of cancer cells of different tissue origins. This study aimed to investigate the specificity of INI-43 for Kpnβ1 by performing experiments to determine whether expression of exogenous Kpnβ1-GFP was able to rescue cells from the effects of INI-43 treatment. Results show that Kpnβ1 overexpression rescues cell viability, as well as the inhibitory effects that INI-43 has on the nuclear import of NFκB/p65 and NFAT. Overexpression of Kpnβ1 also rescues cells from an INI-43 induced G2/M cell cycle block. In addition, treatment of cells with INI-43 enhances degradation of Kpnβ1, but not of other members of the Karyopherin family. These results suggest that the small molecule inhibitor INI-43 is acting, at least in part, by targeting Kpnβ1. Citation Format: Sarah Carden, Pauline van der Watt, Patrizia Lavia, Virna Leaner. Investigating the specificity of the small molecule inhibitor INI-43 for Kpnβ1 in cancer cells [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr B12.

Mechanotransduction: Enforcing protein import.

Applying force to the nucleus reduces the diffusion barrier at nuclear pores and promotes nuclear import of certain proteins, including the transcription regulator YAP, depending on their molecular properties.

PRMT5: A novel regulator of Hepatitis B virus replication and an arginine methylase of HBV core.

In mammals, protein arginine methyltransferase 5, PRMT5, is the main type II enzyme responsible for the majority of symmetric dimethylarginine formation in polypeptides. Recent study reported that PRMT5 restricts Hepatitis B virus (HBV) replication through epigenetic repression of HBV DNA transcription and interference with encapsidation of pregenomic RNA. Here we demonstrate that PRMT5 interacts with the HBV core (HBc) protein and dimethylates arginine residues within the arginine-rich domain (ARD) of the carboxyl-terminus. ARD consists of four arginine rich subdomains, ARDI, ARDII, ARDIII and ARDIV. Mutation analysis of ARDs revealed that arginine methylation of HBc required the wild-type status of both ARDI and ARDII. Mass spectrometry analysis of HBc identified multiple potential ubiquitination, methylation and phosphorylation sites, out of which lysine K7 and arginines R150 (within ARDI) and R156 (outside ARDs) were shown to be modified by ubiquitination and methylation, respectively. The HBc symmetric dimethylation appeared to be linked to serine phosphorylation and nuclear import of HBc protein. Conversely, the monomethylated HBc retained in the cytoplasm. Thus, overexpression of PRMT5 led to increased nuclear accumulation of HBc, and vice versa, down-regulation of PRMT5 resulted in reduced levels of HBc in nuclei of transfected cells. In summary, we identified PRMT5 as a potent controller of HBc cell trafficking and function and described two novel types of HBc post-translational modifications (PTMs), arginine methylation and ubiquitination.

The truncated NLR protein TIR-NBS13 is a MOS6/IMPORTIN-α3 interaction partner required for plant immunity.

Importin-α proteins mediate the translocation of nuclear localization signal (NLS)-containing proteins from the cytoplasm into the nucleus through nuclear pore complexes (NPCs). Genetically, Arabidopsis IMPORTIN-α3/MOS6 (MODIFIER OF SNC1, 6) is required for basal plant immunity and constitutive disease resistance activated in autoimmune mutant snc1 (suppressor of npr1-1, constitutive 1), suggesting that MOS6 plays a role in the nuclear import of proteins involved in plant defense signaling. Here, we sought to identify and characterize defense-regulatory cargo proteins and interaction partners of MOS6. We conducted both in silico database analyses and affinity purification of functional epitope-tagged MOS6 from pathogen-challenged stable transgenic plants coupled with mass spectrometry. We show that among the 13 candidate MOS6 interactors we selected for further functional characterization, the TIR-NBS-type protein TN13 is required for resistance against Pseudomonas syringae pv. tomato (Pst) DC3000 lacking the type-III effector proteins AvrPto and AvrPtoB. When expressed transiently in N. benthamiana leaves, TN13 co-immunoprecipitates with MOS6, but not with its closest homolog IMPORTIN-α6, and localizes to the endoplasmic reticulum (ER), consistent with a predicted N-terminal transmembrane domain in TN13. Our work uncovered the truncated NLR protein TN13 as a component of plant innate immunity that selectively binds to MOS6/IMPORTIN-α3 in planta. We speculate that the release of TN13 from the ER membrane in response to pathogen stimulus, and its subsequent nuclear translocation, is important for plant defense signal transduction.

Hikeshi modulates the proteotoxic stress response in human cells: Implication for the importance of the nuclear function of HSP70s.

Hikeshi mediates the heat stress-induced nuclear import of heat-shock protein 70 (HSP70s: HSP70/HSC70). Dysfunction of Hikeshi causes some serious effects in humans; however, the cellular function of Hikeshi is largely unknown. Here, we investigated the effects of Hikeshi depletion on the survival of human cells after proteotoxic stress and found opposite effects in HeLa and hTERT-RPE1 (RPE) cells; depletion of Hikeshi reduced the survival of HeLa cells, but increased the survival of RPE cells in response to proteotoxic stress. Hikeshi depletion sustained heat-shock transcription factor 1 (HSF1) activation in HeLa cells after recovery from stress, but introduction of a nuclear localization signal-tagged HSC70 in Hikeshi-depleted HeLa cells down-regulated HSF1 activity. In RPE cells, the HSF1 was efficiently activated, but the activated HSF1 was not sustained after recovery from stress, as in HeLa cells. Additionally, we found that p53 and subsequent up-regulation of p21 were higher in the Hikeshi-depleted RPE cells than in the wild-type cells. Our results indicate that depletion of Hikeshi renders HeLa cells proteotoxic stress-sensitive through the abrogation of the nuclear function of HSP70s required for HSF1 regulation. Moreover, Hikeshi depletion up-regulates p21 in RPE cells, which could be a cause of its proteotoxic stress resistant.