Nuclear Localization Signal Motif Research Articles

Mutant KRAS-activated circATXN7 fosters tumor immunoescape by sensitizing tumor-specific T cells to activation-induced cell death

Mutant KRAS (KRASMUT) is often exploited by cancers to shape tumor immunity, but the underlying mechanisms are not fully understood. Here we report that tumor-specific cytotoxic T lymphocytes (CTLs) from KRASMUT cancers are sensitive to activation-induced cell death (AICD). circATXN7, an NF-κB-interacting circular RNA, governs T cell sensitivity to AICD by inactivating NF-κB. Mechanistically, histone lactylation derived from KRASMUT tumor cell-produced lactic acid directly activates transcription of circATXN7, which binds to NF-κB p65 subunit and masks the p65 nuclear localization signal motif, thereby sequestering it in the cytoplasm. Clinically, circATXN7 upregulation in tumor-specific CTLs correlates with adverse clinical outcomes and immunotherapeutic resistance. Genetic ablation of circAtxn7 in CD8+ T cells leads to mutant-selective tumor inhibition, while also increases anti-PD1 efficacy in multiple tumor models in female mice. Furthermore, targeting circATXN7 in adoptively transferred tumor-reactive CTLs improves their antitumor activities. These findings provide insight into how lymphocyte-expressed circRNAs contribute to T-cell fate decisions and anticancer immunotherapies.

Allelic variation of BBX24 is a dominant determinant controlling red coloration and dwarfism in pear.

Variation in anthocyanin biosynthesis in pear fruit provides genetic germplasm resources for breeding, while dwarfing is an important agronomic trait, which is beneficial to reduce the management costs and allow for the implementation of high-density cultivation. Here, we combined bulked segregant analysis (BSA), quantitative trait loci (QTL), and structural variation (SV) analysis to identify a 14-bp deletion which caused a frame shift mutation and resulted in thepremature translation termination of a B-box (BBX) family of zinc transcription factor, PyBBX24, and its allelic variation termed PyBBX24ΔN14. PyBBX24ΔN14 overexpression promotes anthocyanin biosynthesis in pear, strawberry, Arabidopsis, tobacco, and tomato, while that of PyBBX24 did not. PyBBX24ΔN14 directly activates the transcription of PyUFGT and PyMYB10 through interaction with PyHY5. Moreover, stable overexpression of PyBBX24ΔN14 exhibits a dwarfing phenotype in Arabidopsis, tobacco, and tomato plants. PyBBX24ΔN14 can activate the expression of PyGA2ox8 via directly binding to its promoter, thereby deactivating bioactive GAsand reducing the plant height. However, the nuclear localization signal (NLS) and Valine-Proline (VP) motifs in the C-terminus of PyBBX24 reverse these effects. Interestingly, mutations leading to premature termination of PyBBX24 were also identified in red sports of un-related European pear varieties. We conclude that mutations in PyBBX24 gene link both an increase in pigmentation and a decrease in plantheight.

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.

Identification, functional characterization and expression pattern of interferon-gamma (IFN-γ) and interferon-gamma receptor 1 (IFNGR1) in Nibea albiflora

Interferon-gamma (IFN-γ) is an inflammatory cytokine that plays a crucial role in regulating both innate and cell-mediated immune responses by binding to a receptor complex made up of IFNGR1 and IFNGR2. In this study, the complete cDNA of IFN-γ and IFNGR1 from Nibea albiflora were cloned and functionally characterized (named NaIFN-γ and NaIFNGR1), whose complete cDNA sequences were 1593 bp and 2792 bp, encoding 201 and 399 amino acids, respectively. Multiple sequence alignment and phylogenetic analysis showed that the concluded amino acids sequences of NaIFN-γ and NaIFNGR1 shared high identity with their teleost orthologues including the IFN-γ signature and nuclear localization signal (NLS) motif in NaIFN-γ and FN Ⅲ domain in NaIFNGR1. Real-time PCR showed that NaIFN-γ and NaIFNGR1 constitutively expressed in all tested tissues, such as the head-kidney, spleen, liver, kidney, gill, muscle, blood, and intestine with the highest expression of NaIFN-γ and NaIFNGR1 appearing in the liver and gill, respectively. After experiencing stimulation with Polyinosinic-polycytidylic acid (Poly (I:C)), Vibrio alginolyticus (V. alginolyticus) or Vibrio parahaemolyticus (V. parahaemolyticus), NaIFN-γ and NaIFNGR1 mRNA were up-regulated with the time-dependent model. Due to the presence of a nuclear localization signal (NLS), the subcellular localization revealed that NaIFN-γ dispersed throughout the cytoplasm and nucleus. NaIFNGR1, as a member of Cytokine receptor family B, was primarily expressed on the cell membrane. When NaIFN-γ and NaIFNGR1 were co-transfected, their fluorescence signals overlapped on the membrane of HEK 293T cells indicating the potential interaction between IFN-γ and IFNGR1. The GST-pull-down results further showed that NaIFN-γ could directly interact with the extracellular region of NaIFNGR1, further confirming the affinity between IFN-γ and IFNGR1. Taken together, the results firstly demonstrated that the NaIFN-γ ligand-receptor system existed in N.albiflora and played a pivotal part in N.albiflora's immune response against pathogenic bacterial infections, which contributed to the better understanding of the role of IFN-γ in the immunomodulatory mechanisms of teleost.

Multi-functional regulation of cGAS by the nuclear localization signal2 (NLS2) motif: Nuclear localization, enzyme activity and protein degradation

Cyclic GMP-AMP synthase (cGAS), which recognizes double-stranded DNA (dsDNA) and activates the innate immune system, is mainly localized in the cytosol, but also shows nuclear localization. Here, we sought to determine the role of nuclear cGAS by mutating known nuclear localization signal (NLS) motifs in cGAS and assessing its functionality by monitoring phosphorylation of the downstream target, interferon regulatory factor-3 (IRF3). Interestingly, NLS2-mutated cGAS failed to promote phosphorylation of IRF3, reflecting the loss of its ability to produce cyclic GMP-AMP (cGAMP). We further found that insertion of an NLS from SV40 large T antigen could not restore this loss of activity, indicating that this loss was attributable to the mutation of NLS2 itself, but not dependent on the inability of cGAS to enter the nucleus. NLS2-mutant cGAS protein also showed decreased stability dependent on polyubiquitination, an effect that was independent of both its loss of catalytic function and its inability to enter into the nucleus. Collectively, these findings indicate that the NLS2 motif of cGAS is not only involved in regulating the subcellular localization of cGAS protein but also influences its stability and enzymatic activity through independent mechanisms, highlighting the novel roles of NLS2 in regulating the intracellular functions of cGAS.

Structural Characterization of Porcine Adeno-Associated Virus Capsid Protein with Nuclear Trafficking Protein Importin Alpha Reveals a Bipartite Nuclear Localization Signal.

Adeno-associated viruses (AAV) are important vectors for gene therapy, and accordingly, many aspects of their cell transduction pathway have been well characterized. However, the specific mechanisms that AAV virions use to enter the host nucleus remain largely unresolved. We therefore aimed to reveal the interactions between the AAV Cap protein and the nuclear transport protein importin alpha (IMPα) at an atomic resolution. Herein we expanded upon our earlier research into the Cap nuclear localization signal (NLS) of a porcine AAV isolate, by examining the influence of upstream basic regions (BRs) towards IMPα binding. Using a high-resolution crystal structure, we identified that the IMPα binding determinants of the porcine AAV Cap comprise a bipartite NLS with an N-terminal BR binding at the minor site of IMPα, and the previously identified NLS motif binding at the major site. Quantitative assays showed a vast difference in binding affinity between the previously determined monopartite NLS, and bipartite NLS described in this study. Our results provide a detailed molecular view of the interaction between AAV capsids and the nuclear import receptor, and support the findings that AAV capsids enter the nucleus by binding the nuclear import adapter IMPα using the classical nuclear localization pathway.

Functional dissection of N-terminal nuclear trafficking signals of SETDB1.

SETDB1 is a histone H3-lysine 9-specific methyltransferase that fulfills epigenetic functions inside the nucleus; however, when overexpressed, SETDB1 majorily localizes in the cytoplasm. SETDB1 has a single nuclear-localization-signal (NLS) motif and two successive nuclear-export-signal (NES1 and NES2) motifs in the N-terminus, suggesting that SETDB1 localization is the consequence of a balance between the two antithetic motifs. Here, we performed a series of motif deletions to characterize their effects on the cellular movement of SETDB1. Given the cytoplasmic localization of GFP-SETDB1 in the whole form, without the NES motifs, GFP-SETDB1 was not nuclear, and 3xNLS addition plus NES removal held the majority of GFP-SETDB1 within the nucleus. The results indicated that the cytoplasmic localization of GFP-SETDB1 is the combined result of weak NLS and robust NESs. In ATF7IP-overexpressing cells, GFP-SETDB1 entered the nucleus only in the presence of the NES1 motif; neither the NES2 nor NLS motif was necessary. Since subcellular fractionation results showed that ATF7IP was nuclear-only, an intermediary protein may interact specifically with the NES1 motif after stimulation by ATF7IP. When GFP-SETDB1 had either NES1 or NES2, it was precipitated (in immunoprecipitation) and colocalized (in immunofluorescence) with ATF7IP, indicating that GFP-SETDB1 interacts with ATF7IP through the NES motifs in the nucleus. The regulated nuclear entry of SETDB1 is assumed to set a tight restriction on its abundance within the nucleus, thereby ensuring balanced nuclear SETDB1 levels.

Cloning, Exogenous Expression and Function Analysis of Interferon-γ from Gadus macrocephalus.

Interferon γ (IFN–γ) is now considered to be one of the key molecules in the regulation of innate and adaptive immunity. The function of IFN–γ is best described in humans, but less of IFN–γ in fish species has been described at protein level. In the present study, IFN–γ from Gadus macrocephalus (GmIFN–γ) has been examined in terms of bioinformatics, prokaryotic expression, yeast expression, antiviral activity and immune regulatory function. The cDNA of GmIFN–γ contains an open reading frame of 570 nucleotides, coding 189 amino acids. The mature protein contains a nuclear localization signal motif and an obvious IFN–γ signature sequence at the C-terminal. GmIFN–γ is very similar to that of Atlantic cod, with homology up to 89.89%, but less than 32% to other species. GmIFN–γ can be detected in the gills, spleen, intestine, brain and kidney. Interestingly, during early development, a strong signal of GmIFN–γ was not detected until 40 days post hatching. Prokaryotic expression plasmid pET–32a–GmIFN–γ was constructed, and the expression products in BL21 were confirmed by Mass Spectrometry. Meanwhile, the plasmid pGAPZA–GmIFN–γ with Myc tag was constructed and transmitted into Pichia pastoris yeast GS115, and the products were tested using Western blot. The purified GmIFN–γ from either BL21 or yeast has a strong antivirus (Spring viremia of carp virus) effect. The vector of pcDNA3.1–GmIFN–γ was expressed in EPC cell lines; high transcript levels of MHC class I chain-related protein A (MICA) gene were detected; and the exogenous GmIFN–γ protein could also induce MICA expression, indicating that GmIFN–γ could stimulate immune response. The yeast GS115 with GmIFN–γ protein, which is an inclusion body, was given to zebrafish orally, and the transcript of zebrafish IFN–γ was upregulated significantly; however, genes of the interferon type–I signal pathway were not well stimulated.

Dual targeting of Saccharomyces cerevisiae Pso2 to mitochondria and the nucleus, and its functional relevance in the repair of DNA interstrand crosslinks.

Repair of DNA interstrand crosslinks involves a functional interplay among different DNA surveillance and repair pathways. Previous work has shown that interstrand crosslink-inducing agents cause damage to Saccharomyces cerevisiae nuclear and mitochondrial DNA, and its pso2/snm1 mutants exhibit a petite phenotype followed by loss of mitochondrial DNA integrity and copy number. Complex as it is, the cause and underlying molecular mechanisms remains elusive. Here, by combining a wide range of approaches with in vitro and in vivo analyses, we interrogated the subcellular localization and function of Pso2. We found evidence that the nuclear-encoded Pso2 contains 1 mitochondrial targeting sequence and 2 nuclear localization signals (NLS1 and NLS2), although NLS1 resides within the mitochondrial targeting sequence. Further analysis revealed that Pso2 is a dual-localized interstrand crosslink repair protein; it can be imported into both nucleus and mitochondria and that genotoxic agents enhance its abundance in the latter. While mitochondrial targeting sequence is essential for mitochondrial Pso2 import, either NLS1 or NLS2 is sufficient for its nuclear import; this implies that the 2 nuclear localization signal motifs are functionally redundant. Ablation of mitochondrial targeting sequence abrogated mitochondrial Pso2 import, and concomitantly, raised its levels in the nucleus. Strikingly, mutational disruption of both nuclear localization signal motifs blocked the nuclear Pso2 import; at the same time, they enhanced its translocation into the mitochondria, consistent with the notion that the relationship between mitochondrial targeting sequence and nuclear localization signal motifs is competitive. However, the nuclease activity of import-deficient species of Pso2 was not impaired. The potential relevance of dual targeting of Pso2 into 2 DNA-bearing organelles is discussed.

Tumor suppressor BAP1 nuclear import is governed by transportin-1.

Subcellular localization of the deubiquitinating enzyme BAP1 is deterministic for its tumor suppressor activity. While the monoubiquitination of BAP1 by an atypical E2/E3-conjugated enzyme UBE2O and BAP1 auto-deubiquitination are known to regulate its nuclear localization, the molecular mechanism by which BAP1 is imported into the nucleus has remained elusive. Here, we demonstrated that transportin-1 (TNPO1, also known as Karyopherin β2 or Kapβ2) targets an atypical C-terminal proline-tyrosine nuclear localization signal (PY-NLS) motif of BAP1 and serves as the primary nuclear transporter of BAP1 to achieve its nuclear import. TNPO1 binding dissociates dimeric BAP1 and sequesters the monoubiquitination sites flanking the PY-NLS of BAP1 to counteract the function of UBE2O that retains BAP1 in the cytosol. Our findings shed light on how TNPO1 regulates the nuclear import, self-association, and monoubiquitination of BAP1 pertinent to oncogenesis.

A Putative Effector CcSp84 of Cytospora chrysosperma Localizes to the Plant Nucleus to Trigger Plant Immunity.

Cytospora chrysosperma is the main causal agent of poplar canker disease in China, especially in some areas with poor site conditions. Pathogens secrete a large number of effectors to interfere the plant immunity and promote their infection and colonization. Nevertheless, the roles of effectors in C. chrysosperma remain poorly understood. In this study, we identified and functionally characterized a candidate effector CcSp84 from C. chrysosperma, which contained a nuclear localization signal motif at the C-terminal and was highly induced during infection stages. Transient expression of CcSp84 in Nicotiana benthamiana leaves could trigger cell death. Additionally, deletion of CcSp84 significantly reduced fungal virulence to the polar twigs, while no obvious defects were observed in fungal growth and sensitivity to H2O2. Confocal microscopy revealed that CcSp84 labeled with a green fluorescent protein (GFP) was mainly accumulated in the plant nucleus. Further analysis revealed that the plant nucleus localization of CcSp84 was necessary to trigger plant immune responses, including ROS accumulation, callose deposition, and induced expression of jasmonic acid and ethylene defense-related genes. Collectively, our results suggest that CcSp84 is a virulence-related effector, and plant nucleus localization is required for its functions.

Isolation and Genomic Characterization of Avian Reovirus From Wild Birds in South Korea.

Avian reoviruses (ARVs) cause severe arthritis, tenosynovitis, pericarditis, and depressed growth in chickens, and these conditions have become increasingly frequent in recent years. Studies on the role of wild birds in the epidemiology of ARVs are insufficient. This study provides information about currently circulating ARVs in wild birds by gene detection using diagnostic RT-PCR, virus isolation, and genomic characterization. In this study, we isolated and identified 10 ARV isolates from 7,390 wild birds' fecal samples, including migratory bird species (bean goose, Eurasian teal, Indian spot-billed duck, and mallard duck) from 2015 to 2019 in South Korea. On comparing the amino acid sequences of the σC-encoding gene, most isolates, except A18-13, shared higher sequence similarity with the commercial vaccine isolate S1133 and Chinese isolates. However, the A18-13 isolate is similar to live attenuated vaccine av-S1133 and vaccine break isolates (SD09-1, LN09-1, and GX110116). For the p10- and p17-encoding genes, all isolates have identical fusion associated small transmembrane (FAST) protein and nuclear localization signal (SNL) motif to chicken-origin ARVs. Phylogenetic analysis of the amino acid sequences of the σC-encoding gene revealed that all isolates were belonged to genotypic cluster I. For the p10- and p17-encoding genes, the nucleotide sequences of all isolates indicated close relationship with commercial vaccine isolate S1133 and Chinese isolates. For the σNS-encoding gene, the nucleotide sequences of all isolates indicated close relationship with the Californian chicken-origin isolate K1600657 and belonged to chicken-origin ARV cluster. Our data indicates that wild birds ARVs were derived from the chicken farms. This finding suggests that wild birds serve as natural carriers of such viruses for domestic poultry.

A Gypsy element contributes to the nuclear retention and transcriptional regulation of the resident lncRNA in locusts

ABSTRACT The majority of long noncoding RNAs (lncRNAs) contain transposable elements (TEs). PAHAL, a nuclear-retained lncRNA that is inserted by a Gypsy retrotransposon, has been shown to be a vital regulator of phenylalanine hydroxylase (PAH) gene expression that controls dopamine biosynthesis and behavioural aggregation in the migratory locust. However, the role of the Gypsy retrotransposon in the transcriptional regulation of PAHAL remains unknown. Here, we identified a Gypsy retrotransposon (named Gypsy element) as an inverted long terminal repeat located in the 3′ end of PAHAL, representing a feature shared by many other lncRNAs in the locust genome. The embedded Gypsy element contains a RNA nuclear localization signal motif, which promotes the stable accumulation of PAHAL in the nucleus. The Gypsy element also provides high-affinity SRSF2 binding sites for PAHAL that induce the recruitment of SRSF2, resulting in the PAHAL-mediated transcriptional activation of PAH. Thus, our data demonstrate that TEs provide discrete functional domains for lncRNA organization and highlight the contribution of TEs to the regulatory significance of lncRNAs.

Nuclear Exportin 1 (XPO1) Binds to the Nuclear Localization/Export Signal of the Turnip Mosaic Virus NIb to Promote Viral Infection.

The nuclear localization signal (NLS) and nuclear export signal (NES) are key signatures of proteins for controlling nuclear import and export. The NIb protein of turnip mosaic virus (TuMV) is an RNA-dependent RNA polymerase (RdRP) that is absolutely required for viral genome replication. Previous studies have shown that NIb is a nucleocytoplasmic shuttling protein and contains four putative NES and four putative NLS motifs. Here, we analyzed the function of these NESs and NLSs, and identified two functional NESs and one functional NLS. Mutation of the identified functional NESs or NLS inhibited viral RNA accumulation and systemic infection. Exportin 1 (XPO1) is a nuclear export receptor that binds directly to cargo proteins harboring a leucine-rich NES and translocates them to the cytoplasm. We found that XPO1 contains two NIb-binding domains, which recognize the NLS and NES of NIb, respectively, to mediate the nucleocytoplasmic transport of NIb and promote viral infection. Taken together, these data suggest that the nucleocytoplasmic transport of NIb is modulated by XPO1 through its interactions with the functional NLS and NES of NIb to promote viral infection.

Bombyx mori U-shaped regulates the melanization cascade and immune response via binding with the Lozenge protein.

Zinc finger protein, an important transcription factor, regulates gene expression associated with various physiological and pathological processes. U-shaped, belong to the Friend of GATA (FOG) transcription factor, plays a crucial role in hematopoiesis by interacting with the GATA transcription factor as a co-factor. However, little is known about its functions in insects. In the present study, a U-shaped cDNA was identified and characterized from the silkworm Bombyx mori and its potential roles in innate immunity investigated. The predicted silkworm U-shaped amino acid sequence contained a classical nuclear localization signal (NLS) motif "GESSPKRRRR" at position 450-459, and arginine residues at position 456 and 478 are the critical sites of the NLS. U-shaped mRNA was detected in all tested tissues of the B. mori; however, the highest levels were found in the hemocytes. U-shaped mRNA expression levels were upregulated in the hemocyte after the Escherichia coli and Staphylococcus aureus challenge. Furthermore, U-shaped knockdown significantly reduced the melanization process and suppressed the expression of melanization-associated genes, including PPO1, PPO2, PPAE and BAEE. In addition, U-shaped interacts with Lozenge protein to regulate the innate immune response of the insect. Our results revealed that U-shaped binds directly to Lozenge protein to modulate the melanization process and innate immune responses in silkworm.

Autographa Californica Multiple Nucleopolyhedrovirus orf13 Is Required for Efficient Nuclear Egress of Nucleocapsids.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf13 (ac13) is a conserved gene in all sequenced alphabaculoviruses. However, its function in the viral life cycle remains unknown. In this study, we found that ac13 was a late gene and that the encoded protein, bearing a putative nuclear localization signal motif, colocalized with the nuclear lamina. Deletion of ac13 did not affect viral genome replication, nucleocapsid assembly or occlusion body (OB) formation, but reduced virion budding from infected cells by approximately 400-fold compared with the wild-type virus. Deletion of ac13 substantially impaired the egress of nucleocapsids from the nucleus to the cytoplasm, while the OB morphogenesis was unaffected. Taken together, our results indicated that ac13 was required for efficient nuclear egress of nucleocapsids during virion budding, but was dispensable for OB formation.

NF45/NF90-mediated rDNA transcription provides a novel target for immunosuppressant development.

Herein, we demonstrate that NFAT, a key regulator of the immune response, translocates from cytoplasm to nucleolus and interacts with NF45/NF90 complex to collaboratively promote rDNA transcription via triggering the directly binding of NF45/NF90 to the ARRE2‐like sequences in rDNA promoter upon T‐cell activation in vitro. The elevated pre‐rRNA level of T cells is also observed in both mouse heart or skin transplantation models and in kidney transplanted patients. Importantly, T‐cell activation can be significantly suppressed by inhibiting NF45/NF90‐dependent rDNA transcription. Amazingly, CX5461, a rDNA transcription‐specific inhibitor, outperformed FK506, the most commonly used immunosuppressant, both in terms of potency and off‐target activity (i.e., toxicity), as demonstrated by a series of skin and heart allograft models. Collectively, this reveals NF45/NF90‐mediated rDNA transcription as a novel signaling pathway essential for T‐cell activation and as a new target for the development of safe and effective immunosuppressants.

Role of LEF5 Nuclear Localization Signaling in the Production of Helicoverpa armigera Nucleopolyhedrovirus Progeny

Late Expression Factor 5 (LEF-5) of Helicoverpa armigera nucleopolyhedrovirus (HaNPV) functions as an initiation factor and plays an important role in the production of infectious viral progeny. Previous study revealed that LEF-5 was localized to the nuclear in infection cells. Bioinformatic analysis identified two putative nuclear localization signals (NLSs), i.e., NLS145-176 and NLS241-271, in the LEF5 amino acid sequence from 145 to 176 and from 241 to 271. However, it was still elusive what were the roles of the two putative NLSs in HaNPV DNA replication and its late expression. In this work, we used GFP as gene reporter to examine the roles of the two LEF5 NLSs in the production of viral progeny. Several HaNPV mutants were constructed in which two NLSs were either truncated or deleted. We found that the deletion of NLS145-176 and NLS241-271 would not prevent the HaNPV LEF-5 from the localization into the nucleus while the HaNPV LEF-5 simultaneously presented in cytoplasm. The lef-5 knockout would not affect the early gene expression and DNA replication. However, the expression of typical late transcripts was dramatically reduced. The mutations in the LEF5 NLS motifs resulted in reduced levels of infectious virus production. (This work was supported by the National Natural Science Foundation of China, Grants No. 31500132).

HNF1A-MODY Mutations in Nuclear Localization Signal Impair HNF1A-Import Receptor KPNA6 Interactions.

Mutations in hepatocyte nuclear factor (HNF)1A gene cause the most common form of Maturity-onset diabetes of the young (MODY), a monogenic subtype of diabetes mellitus. Functional characterization of mutant proteins reveals that mutations may disrupt DNA binding capacity, transactivation ability and nuclear localization of HNF1A depending on the position of the mutation. Previously identified Arg271Trp and Ser345Tyr mutations in HNF1A were found to be defective in nuclear localization. Arg271 residue resides in a region similar to classical nuclear localization signal (NLS) motif, while Ser345 does not. Importin α family members recognize NLS motifs on cargo proteins and subsequently translocate them into nucleus. Here, we first investigated the nuclear localization mechanism of wild type HNF1A protein. For this purpose, we analyzed the interaction of HNF1A with three mouse homolog importin α proteins (KPNA2, KPNA4 and KPNA6) by co-immunoprecipitation assay and molecular docking simulation. Hereby, KPNA6 was identified as the main import receptor, which is responsible for the transport of HNF1A into the nucleus. Immunolocalization studies in mouse pancreatic cells (Min6) also confirmed the co-localization of HNF1A and KPNA6 in the cytoplasm. Secondly, the interaction between KPNA6 and mutant HNF1A proteins (Arg271Trp and Ser345Tyr) was assessed. Co-immunoprecipitation studies revealed a reduced interaction compared to wild type HNF1A. Our study demonstrated for the first time that HNF1A transcription factor is recognized and transported by importin/karyopherin import family, and mutations in NLS motifs may disrupt the interaction leading to nuclear localization abnormalities and MODY phenotype.

Trypanosoma cruzi Importin α: ability to bind to a functional classical nuclear localization signal of the bipartite type.

Importin α, a transport factor in the classical pathway of nuclear transport of proteins in eukaryotes, has not been experimentally studied in trypanosomatids. A chimeric fluorescent version of this protein (TcImportin α-EGFP) expressed in transfected epimastigotes of Trypanosoma cruzi is characterized here. Initially, the cellular localization of the tagged protein was analysed in exponentially growing and non-growing quiescent cells in a stationary phase. In growing epimastigotes, the fluorescence signal appeared to be mostly localized in the nucleolus, with additional minor fluorescent dots observed close to the nuclear periphery. In the stationary phase, both aged epimastigotes and metacyclic trypomastigotes presented with dispersed fluorescence of a granular form within the nucleoplasm of the cells that predominantly localized in poorly DAPI-stained regions. On the other hand, the ability of a tagged (6×His) version of TcImportin α to bind the nuclear protein cargo TcRPA31 (TcRPA31-EGFP) was determined by pull-down assays of co-transfected cultures. In addition, the results from the in vitro analyses with these tagged recombinant proteins showed that the functional nuclear localization signal (NLS) previously mapped to TcRPA31 was sufficient to sustain binding to TcImportin α. Moreover, the second cluster of basic amino acids within this bipartite NLS (formerly termed element B) was found to be essential for complex formation, as previously described for the nuclear translocation of these fluorescent chimeras. To our knowledge, this approach is the first in which Importin α was experimentally researched in kinetoplastids. The ability of TcImportin α to bind the NLS motif analysed here, is an essential feature expected for its potential functional role as a soluble transport factor.