Nuclear Localization Signal Residues Research Articles

Structures of importin-α bound to the wild-type and an internal deletion mutant of the bipartite nuclear localization signal of HIF-1α

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor that plays an important role as a master regulator of oxygen homeostasis. The activity of HIF-1 is regulated in part by dynamic intracellular trafficking of its α subunit (HIF-1α) that can shuttle between the nucleus and cytoplasm. It has been shown that nuclear localization of HIF-1α requires a variant of classic nuclear localization signal (NLS) and that an internal deletion of the amino acid residues (residues 724–751) in the NLS almost abolish the nuclear localization. Here we report the X-ray crystal structure of the nuclear import adaptor importin-α1 bound to the wild-type HIF-1α NLS at 1.8 Å resolution and of importin-α1 bound to the Δ724-751 mutant of the HIF-1α NLS at 1.9 Å resolution. In the wild-type structure, two basic clusters in the HIF-1α NLS made extensive interactions with importin-α1 on two sites (the major site and the minor site). In the mutant structure, the NLS residues still interacted extensively with the major site on importin-α1, but the interactions with the minor site were not observed. The structural data, together with computational analyses of binding free energies, indicate that the loss of the minor-site interactions inhibit nuclear accumulation of HIF-1α.

Rare Angiogenin and Ribonuclease 4 variants associated with amyotrophic lateral sclerosis exhibit loss-of-function: a comprehensive in silico study

Amyotrophic Lateral Sclerosis (ALS), a debilitating neurodegenerative disorder is related to mutations in a number of genes, and certain genes of the Ribonuclease (RNASE) superfamily trigger ALS more frequently. Even though missense mutations in Angiogenin (ANG) and Ribonuclease 4 (RNASE4) have been previously shown to cause ALS through loss-of-function mechanisms, understanding the role of rare variants with a plausible explanation of their functional loss mechanisms is an important mission. The study aims to understand if any of the rare ANG and RNASE4 variants catalogued in Project MinE consortium caused ALS due to loss of ribonucleolytic or nuclear translocation or both these activities. Several in silico analyses in combination with extensive molecular dynamics (MD) simulations were performed on wild-type ANG and RNASE4, along with six rare variants (T11S-ANG, R122H-ANG, D2E-RNASE4, N26K-RNASE4, T79A-RNASE4 and G119S-RNASE4) to study the structural and dynamic changes in the catalytic triad and nuclear localization signal residues responsible for ribonucleolytic and nuclear translocation activities respectively. Our comprehensive analyses comprising 1.2μs simulations with a focus on physicochemical, structural and dynamic properties reveal that T11S-ANG, N26K-RNASE4 and T79A-RNASE4 variants would result in loss of ribonucleolytic activity due to conformational switching of catalytic His114 and His116 respectively but none of the variants would lose their nuclear translocation activity. Our study not only highlights the importance of rare variants but also demonstrates that elucidating the structure-function relationship of mutant effectors is crucial to gain insights into ALS pathophysiology and in developing effective therapeutics.

A molecular dynamics based investigation reveals the role of rare Ribonuclease 4 variants in amyotrophic lateral sclerosis susceptibility

Missense mutations in certain genes of the Ribonuclease (RNASE) superfamily cause amyotrophic lateral sclerosis (ALS) through loss of either ribonucleolytic or nuclear translocation or both of these activities. While rare ANG/RNASE5 variants have been previously shown to be ALS causative, it is not yet known if any of the reported rare RNASE4 variants can also trigger ALS. The study aims to understand whether rare variants of RNASE4 can manifest ALS through similar loss-of-function mechanisms. Molecular dynamics (MD) simulations were performed on wild-type and all reported rare RNASE4 variants to study the structural and dynamic changes in the catalytic triad and nuclear localization signal residues responsible for ribonucleolytic and nuclear translocation activities respectively. Our systematic study comprising a total of 2.1 μs MD simulations reveal that three rare variants M29I, H72P and R95W would lose their ribonucleolytic activity as a result of conformational alteration of catalytic residue His116, and the R31T and R32W variants would lose their nuclear translocation ability due to local folding and reduced solvent accessibility of 30QRR32 residues. Our results show that five among the 20 known rare variants in RNASE4 (M29I, H72P, R95W, R31T and R32W) are possibly deleterious and may manifest ALS due to loss-of-function mechanisms. Overall, this is the first study to demonstrate that although rare and not yet clinically correlated, certain rare RNASE4 variants could cause ALS due to their loss-of-function characteristics and highlights the need to discover novel RNASE variants for a comprehensive understanding of structure-function-disease relationships and design effective therapeutics.

Prediction of Functional Loss of Human Angiogenin Mutants Associated with ALS by Molecular Dynamics Simulations

Several missense mutations in the coding region of angiogenin (ANG) gene have been identified in Amyotrophic Lateral Sclerosis (ALS) patients. These mutations lead to loss of either ribonucleolytic activity or nuclear translocation activity or both of ANG (protein encoded by ANG gene) causing ALS. We present here a cohesive and comprehensive picture of the molecular origins of functional loss of all ALS associated ANG mutants, emerging via extensive molecular dynamics simulations. Our method effectively predicts that conformational change of His114 results in loss of ribonucleolytic activity and that reduction of solvent accessible surface area of nuclear localization signal residues 31RRR33 results in loss of nuclear translocation activity. These predictions hold true, without exception, for all ANG mutants studied and can be employed to infer whether a new ANG mutation is causative of ALS or benign ahead of experimental findings.

158 A molecular dynamics simulation-based prediction of deleterious angiogenin mutations causing amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective death of motor neurons leading to paralysis and death between 3–5 years of diagnosis. Through whole genome association studies, several single nucleotide polymorphisms (SNPs) encoding missense mutations in angiogenin (ANG) protein have been identified as one of the primary factors causing ALS. Structural studies of ANG show that catalytic triad comprising His13, Lys40, and His114 residues imparts ribonucleolytic activity while nuclear localization signal residues 31RRR33 are responsible for nuclear translocation activity. Loss of either ribonucleolytic activity or nuclear translocation activity or both of these functions due to mutations cause ALS. However, the mechanisms of loss-of-functions of ANG mutants are not completely understood. Here, we present a cohesive and comprehensive picture of functional loss mechanisms of all known ALS-associated ANG mutants by extensive molecular dynamics (MD) simulations (Padhi, Kumar, Vasaikar, Jayaram, & Gomes, 2012; AK, 2013). Our studies show that conformational switching of catalytic residue His114 is responsible for the loss of ribonucleolytic activity while reduction in solvent-accessible surface area (SASA) of 31RRR33 as a result of local folding is responsible for the loss of nuclear translocation activity (Padhi et al., 2012; AK, 2013). Our prediction of loss-of-functions of 17 ANG mutants correlated positively with the reported experimental results. We have subsequently developed a fast molecular dynamics method based on certain global attributes / dynamic markers that can be used to determine whether a mutation is deleterious or benign. To make our method accessible to researchers and clinicians, we created a web server-based tool, ANGDelMut, freely available at http://bioschool.iitd.ernet.in/research.htm, where a user can submit new mutations to ascertain whether they cause ALS. We hope that our method will benefit the community at large and will pave the way for the development of a successful therapy for patients suffering from ALS.

Mechanisms of Loss of Functions of Human Angiogenin Variants Implicated in Amyotrophic Lateral Sclerosis

BackgroundMutations in the coding region of angiogenin (ANG) gene have been found in patients suffering from Amyotrophic Lateral Sclerosis (ALS). Neurodegeneration results from the loss of angiogenic ability of ANG (protein coded by ANG). In this work, we performed extensive molecular dynamics (MD) simulations of wild-type ANG and disease associated ANG variants to elucidate the mechanism behind the loss of ribonucleolytic activity and nuclear translocation activity, functions needed for angiogenesis.Methodology/Principal FindingsMD simulations were carried out to study the structural and dynamic differences in the catalytic site and nuclear localization signal residues between WT-ANG (Wild-type ANG) and six mutants. Variants K17I, S28N, P112L and V113I have confirmed association with ALS, while T195C and A238G single nucleotide polymorphisms (SNPs) encoding L35P and K60E mutants respectively, have not been associated with ALS. Our results show that loss of ribonucleolytic activity in K17I is caused by conformational switching of the catalytic residue His114 by 99°. The loss of nuclear translocation activity of S28N and P112L is caused by changes in the folding of the residues 31RRR33 that result in the reduction in solvent accessible surface area (SASA). Consequently, we predict that V113I will exhibit loss of angiogenic properties by loss of nuclear translocation activity and L35P by loss of both ribonucleolytic activity and nuclear translocation activity. No functional loss was inferred for K60E. The MD simulation results were supported by hydrogen bond interaction analyses and molecular docking studies.Conclusions/SignificanceConformational switching of catalytic residue His114 seems to be the mechanism causing loss of ribonucleolytic activity and reduction in SASA of nuclear localization signal residues 31RRR33 results in loss of nuclear translocation activity in ANG mutants. Therefore, we predict that L35P mutant, would exhibit loss of angiogenic functions, and hence would correlate with ALS while K60E would not show any loss.

Sensing actin dynamics: Structural basis for G-actin-sensitive nuclear import of MAL

The coordination of cytoskeletal actin dynamics with gene expression reprogramming is emerging as a crucial mechanism to control diverse cellular processes, including cell migration, differentiation and neuronal circuit assembly. The actin-binding transcriptional coactivator MAL (also known as MRTF-A/MKL1/BSAC) senses G-actin concentration and transduces Rho GTPase signals to serum response factor (SRF). MAL rapidly shuttles between the cytoplasm and the nucleus in unstimulated cells but Rho-induced depletion of G-actin leads to MAL nuclear accumulation and activation of transcription of SRF:MAL-target genes. Although the molecular and structural basis of actin-regulated nucleocytoplasmic shuttling of MAL is not understood fully, it is proposed that nuclear import of MAL is mediated by importin α/β heterodimer, and that G-actin competes with importin α/β for the binding to MAL. Here we present structural, biochemical and cell biological evidence that MAL has a classical bipartite nuclear localization signal (NLS) in the N-terminal ‘RPEL’ domain containing Arg-Pro-X-X-X-Glu-Leu (RPEL) motifs. The NLS residues of MAL adopt an extended conformation and bind along the surface groove of importin-α, interacting with the major- and minor-NLS binding sites. We also present a crystal structure of wild-type MAL RPEL domain in complex with five G-actins. Comparison of the importin-α- and actin-complexes revealed that the binding of G-actins to MAL is associated with folding of NLS residues into a helical conformation that is inappropriate for importin-α recognition.

Characterization of the cassava geminivirus transcription activation protein putative nuclear localization signal

The geminivirus transcription activation protein (TrAP) localizes to the nucleus and contains a putative nuclear localization signal (NLS) ( 28PRRRR 32) on the N-terminus. The role of individual residues of this putative NLS on nuclear localization and symptom induction was investigated using TrAP of East African cassava mosaic Cameroon virus (EACMCV). Subcellular localization was conducted using the green fluorescent protein (GFP). Results showed that the proline residue at position 28 (Pro-28) is essential for nuclear localization whereas individually, none of the four contiguous arginines is necessary for nuclear targeting. The role of each of the five NLS amino acid residues on TrAP-mediated disease phenotype and gene silencing suppression was investigated by expressing these mutants in Nicotiana benthamiana from the PVX vector and under the control of the Cauliflower mosaic virus 35S promoter. Results showed that all five residues of the NLS play a role on disease phenotype production in N. benthamiana plants. Furthermore, each of the NLS residues appeared to be required for suppression of VIGS but appeared not to be required for the ability of TrAP to transactivate transcription and interact with adenosine kinase (ADK).

Mutations in the nuclear localization signal of nsP2 influencing RNA synthesis, protein expression and cytotoxicity of Semliki Forest virus

The cytotoxicity of Semliki Forest virus (SFV) infection is caused partly by the non-structural protein nsP2, an essential component of the SFV replicase complex. Due to the presence of a nuclear localization signal (NLS), nsP2 also localizes in the nucleus of infected cells. The present study analysed recombinant SFV replicons and genomes with various deletions or substitutions in the NLS, or with a proline-to-glycine mutation at position 718 of nsP2 (P718G). Deletion of one or two arginine residues from the NLS or substitution of two of the arginines with aspartic acid resulted in a virus with a temperature-sensitive phenotype, and substitution of all three arginines was lethal. Thus, most of the introduced mutations severely affected nsP2 functioning in viral replication; in addition, they inhibited the ability of SFV to induce translational shut-off and kill infected cells. SFV replicons with a P718G mutation or replacement of the NLS residues 648RRR650 with RDD were found to be the least cytotoxic. Corresponding replicons expressed non-structural proteins at normal levels, but had severely reduced genomic RNA synthesis and were virtually unable to replicate and transcribe co-electroporated helper RNA. The non-cytotoxic phenotype was maintained in SFV full-length genomes harbouring the corresponding mutations; however, during a single cycle of cell culture, these were converted to a cytotoxic phenotype, probably due to the accumulation of compensatory mutations.

Adenovirus Core Protein pVII Is Translocated into the Nucleus by Multiple Import Receptor Pathways

Adenoviruses are nonenveloped viruses with an approximately 36-kb double-stranded DNA genome that replicate in the nucleus. Protein VII, an abundant structural component of the adenovirus core that is strongly associated with adenovirus DNA, is imported into the nucleus contemporaneously with the adenovirus genome shortly after virus infection and may promote DNA import. In this study, we evaluated whether protein VII uses specific receptor-mediated mechanisms for import into the nucleus. We found that it contains potent nuclear localization signal (NLS) activity by transfection of cultured cells with protein VII fusion constructs and by microinjection of cells with recombinant protein VII fusions. We identified three NLS-containing regions in protein VII by deletion mapping and determined important NLS residues by site-specific mutagenesis. We found that recombinant protein VII and its NLS-containing domains strongly and specifically bind to importin alpha, importin beta, importin 7, and transportin, which are among the most abundant cellular nuclear import receptors. Moreover, these receptors can mediate the nuclear import of protein VII fusions in vitro in permeabilized cells. Considered together, these data support the hypothesis that protein VII is a major NLS-containing adaptor for receptor-mediated import of adenovirus DNA and that multiple import pathways are utilized to promote efficient nuclear entry of the viral genome.

Signaling through the Smad Pathway by Insulin-like Growth Factor-binding Protein-3 in Breast Cancer Cells: RELATIONSHIP TO TRANSFORMING GROWTH FACTOR-β1 SIGNALING

We previously demonstrated in T47D cells transfected to express the transforming growth factor-beta receptor type II (TGF-betaRII) that insulin-like growth factor binding protein-3 (IGFBP-3) could stimulate Smad2 and Smad3 phosphorylation, potentiate TGF-beta1-stimulated Smad phosphorylation, and cooperate with exogenous TGF-beta1 in cell growth inhibition (Fanayan, S., Firth, S. M., Butt, A. J., and Baxter, R. C. (2000) J. Biol. Chem. 275, 39146-39151). This study further explores IGFBP-3 signaling through the Smad pathway. Like TGF-beta1, natural and recombinant IGFBP-3 stimulated the time- and dose-dependent phosphorylation of TGF-betaR1 as well as Smad2 and Smad3. This effect required the presence of TGF-betaRII. IGFBP-3 mutated in carboxyl-terminal nuclear localization signal residues retained activity in TGF-betaR1 and Smad phosphorylation, whereas IGFBP-5 was inactive. Immunoneutralization of endogenous TGF-beta1 suggested that TGF-beta1 was not essential for IGFBP-3 stimulation of this pathway, but it increased the effect of IGFBP-3. IGFBP-3, like TGF-beta1, elicited a rapid decline in immunodetectable Smad4 and Smad4.Smad2 complexes. IGFBP-3 and nuclear localization signal mutant IGFBP-3 stimulated the activation of the plasminogen activator inhibitor-1 promoter but was not additive with TGF-beta, suggesting that this end point is not a direct marker of the IGFBP-3 effect on cell proliferation. This study defines a signaling pathway for IGFBP-3 from a cell surface receptor to nuclear transcriptional activity, requiring TGF-betaRII but not dependent on the nuclear translocation of IGFBP-3. The precise mechanism by which IGFBP-3 interacts with the TGF-beta receptor system remains to be established.