Proline-tyrosine Nuclear Localization Signal Research Articles

Defective recognition of a nonclassical nuclear localization signal in neurodevelopmental disorders

In this issue of Structure, Gonzalez etal. present the cryo-EM structure of Karyopherin-β2 bound to the proline-tyrosine nuclear localization signal (PY-NLS) of heterogeneous nuclear ribonucleoprotein H2 (HNRNPH2). The structure advances our understanding of not only the diversity of PY-NLSs but also the pathogenic mechanisms arising from HNRNPH2 variants.

A new Karyopherin-β2 binding PY-NLS epitope of HNRNPH2 linked to neurodevelopmental disorders

The HNRNPH2 proline-tyrosine nuclear localization signal (PY-NLS) is mutated in HNRNPH2-related X-linked neurodevelopmental disorder, causing the normally nuclear HNRNPH2 to accumulate in the cytoplasm. We solved the cryoelectron microscopy (cryo-EM) structure of Karyopherin-β2/Transportin-1 bound to the HNRNPH2 PY-NLS to understand importin-NLS recognition and disruption in disease. HNRNPH2 206RPGPY210 is a typical R-X2-4-P-Y motif comprising PY-NLS epitopes 2 and 3, followed by an additional Karyopherin-β2-binding epitope, we term epitope 4, at residues 211DRP213; no density is present for PY-NLS epitope 1. Disease variant mutations at epitopes 2-4 impair Karyopherin-β2 binding and cause aberrant cytoplasmic accumulation in cells, emphasizing the role of nuclear import defect in disease. Sequence/structure analysis suggests that strong PY-NLS epitopes 4 are rare and thus far limited to close paralogs of HNRNPH2, HNRNPH1, and HNRNPF. Epitope 4-binidng hotspot Karyopherin-β2 W373 corresponds to close paralog Karyopherin-β2b/Transportin-2 W370, a pathological variant site in neurodevelopmental abnormalities, suggesting that Karyopherin-β2b/Transportin-2-HNRNPH2/H1/F interactions may be compromised in the abnormalities.

A minimal construct of nuclear-import receptor Karyopherin-β2 defines the regions critical for chaperone and disaggregation activity

Karyopherin-β2 (Kapβ2) is a nuclear-import receptor that recognizes proline-tyrosine nuclear localization signals of diverse cytoplasmic cargo for transport to the nucleus. Kapβ2 cargo includes several disease-linked RNA-binding proteins with prion-like domains, such as FUS, TAF15, EWSR1, hnRNPA1, and hnRNPA2. These RNA-binding proteins with prion-like domains are linked via pathology and genetics to debilitating degenerative disorders, including amyotrophic lateral sclerosis, frontotemporal dementia, and multisystem proteinopathy. Remarkably, Kapβ2 prevents and reverses aberrant phase transitions of these cargoes, which is cytoprotective. However, the molecular determinants of Kapβ2 that enable these activities remain poorly understood, particularly from the standpoint of nuclear-import receptor architecture. Kapβ2 is a super-helical protein comprised of 20 HEAT repeats. Here, we design truncated variants of Kapβ2 and assess their ability to antagonize FUS aggregation and toxicity in yeast and FUS condensation at the pure protein level and in human cells. We find that HEAT repeats 8 to 20 of Kapβ2 recapitulate all salient features of Kapβ2 activity. By contrast, Kapβ2 truncations lacking even a single cargo-binding HEAT repeat display reduced activity. Thus, we define a minimal Kapβ2 construct for delivery in adeno-associated viruses as a potential therapeutic for amyotrophic lateral sclerosis/frontotemporal dementia, multisystem proteinopathy, and related disorders.

Engineered Nuclear Import Receptor Karyopherin‐β2 Chaperones Aberrant Phase Transitions of Disease‐Associated Cargo

Karyopherin‐β2 (Kapβ2) is a nuclear import receptor (NIR) that localizes proteins bearing a proline‐tyrosine nuclear localization signal (PY‐NLS) to the nucleus, including the RNA‐binding protein (RBP) FUS. Recent work has demonstrated that NIRs can chaperone RBPs in vitro and in cells, preventing and reversing their self‐assembly and aggregation. However, when the PY‐NLS is mutated, Kapβ2 no longer efficiently chaperones its cargo. Here, we focus on the case of FUSP525L, an RBP NLS mutant associated with a highly aggressive form of juvenile ALS. Using a structural approach, we have engineered Kapβ2 to recognize mutant FUS and demonstrate improved chaperone activity in vitro, in human cells, and in mice.

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.

Engineered nuclear import receptor karyopherin-B2 chaperones aberrant phase transitions of disease-associated cargo

Karyopherin-β2 (Kapβ2) is a nuclear import receptor (NIR) that localizes proteins bearing a proline-tyrosine nuclear localization signal (PY-NLS) to the nucleus, including the RNA-binding protein (RBP) FUS. Recent work has demonstrated that NIRs can chaperone RBPs in vitro and in cells, preventing and reversing their self-assembly and aggregation. However, when the PY-NLS is mutated, Kapβ2 no longer efficiently chaperones its cargo. Here, we focus on the case of FUSP525L, an RBP NLS mutant associated with a highly aggressive form of juvenile ALS.

Mechanism of karyopherin-\u03b22 binding and nuclear import of ALS variants FUS(P525L) and FUS(R495X)

Mutations in the RNA-binding protein FUS cause familial amyotropic lateral sclerosis (ALS). Several mutations that affect the proline-tyrosine nuclear localization signal (PY-NLS) of FUS cause severe juvenile ALS. FUS also undergoes liquid–liquid phase separation (LLPS) to accumulate in stress granules when cells are stressed. In unstressed cells, wild type FUS resides predominantly in the nucleus as it is imported by the importin Karyopherin-β2 (Kapβ2), which binds with high affinity to the C-terminal PY-NLS of FUS. Here, we analyze the interactions between two ALS-related variants FUS(P525L) and FUS(R495X) with importins, especially Kapβ2, since they are still partially localized to the nucleus despite their defective/missing PY-NLSs. The crystal structure of the Kapβ2·FUS(P525L)PY-NLS complex shows the mutant peptide making fewer contacts at the mutation site, explaining decreased affinity for Kapβ2. Biochemical analysis revealed that the truncated FUS(R495X) protein, although missing the PY-NLS, can still bind Kapβ2 and suppresses LLPS. FUS(R495X) uses its C-terminal tandem arginine-glycine-glycine regions, RGG2 and RGG3, to bind the PY-NLS binding site of Kapβ2 for nuclear localization in cells when arginine methylation is inhibited. These findings suggest the importance of the C-terminal RGG regions in nuclear import and LLPS regulation of ALS variants of FUS that carry defective PY-NLSs.

Effect of nuclear import receptors on liquid-liquid phase separation.

Low-complexity (LC) sequences, regions that are predominantly made up of limited amino acids, are often observed in eukaryotic nuclear proteins. The role of these LC sequences has remained unclear for decades. Recent studies have shown that LC sequences are important in the formation of membrane-less organelles via liquid–liquid phase separation (LLPS). The RNA binding protein, fused in sarcoma (FUS), is the most widely studied of the proteins that undergo LLPS. It forms droplets, fibers, or hydrogels using its LC sequences. The N-terminal LC sequence of FUS is made up of Ser, Tyr, Gly, and Gln, which form a labile cross-β polymer core while the C-terminal Arg-Gly-Gly repeats accelerate LLPS. Normally, FUS localizes to the nucleus via the nuclear import receptor karyopherin β2 (Kapβ2) with the help of its C-terminal proline-tyrosine nuclear localization signal (PY-NLS). Recent findings revealed that Kapβ2 blocks FUS mediated LLPS, suggesting that Kapβ2 is not only a transport protein but also a chaperone which regulates LLPS during the formation of membrane-less organelles. In this review, we discuss the effects of the nuclear import receptors on LLPS.

High-mobility group box 1 links sensing of reactive oxygen species by huntingtin to its nuclear entry

Huntington's disease (HD) is a neurodegenerative, age-onset disorder caused by a CAG DNA expansion in exon 1 of the HTT gene, resulting in a polyglutamine expansion in the huntingtin protein. Nuclear accumulation of mutant huntingtin is a hallmark of HD, resulting in elevated mutant huntingtin levels in cell nuclei. Huntingtin is normally retained at the endoplasmic reticulum via its N17 amphipathic α-helix domain but is released by oxidation of Met-8 during reactive oxygen species (ROS) stress. Huntingtin enters the nucleus via an importin β1- and 2-dependent proline-tyrosine nuclear localization signal (PY-NLS), which has a unique intervening sequence in huntingtin. Here, we have identified the high-mobility group box 1 (HMGB1) protein as an interactor of the intervening sequence within the PY-NLS. Nuclear levels of HMGB1 positively correlated with varying levels of nuclear huntingtin in both HD and normal human fibroblasts. We also found that HMGB1 interacts with the huntingtin N17 region and that this interaction is enhanced by the presence of ROS and phosphorylation of critical serine residues in the N17 region. We conclude that HMGB1 is a huntingtin N17/PY-NLS ROS-dependent interactor, and this protein bridging is essential for relaying ROS sensing by huntingtin to its nuclear entry during ROS stress. ROS may therefore be a critical age-onset stress that triggers nuclear accumulation of mutant huntington in Huntington's disease.

Nuclear Import Receptor Inhibits Phase Separation of FUS through Binding to Multiple Sites

Liquid-liquid phase separation (LLPS) is believed to underlie formation of biomolecular condensates, cellular compartments that concentrate macromolecules without surrounding membranes. Physical mechanisms that control condensate formation/dissolution are poorly understood. The RNA-binding protein fused in sarcoma (FUS) undergoes LLPS invitro and associates with condensates in cells. We show that the importin karyopherin-β2/transportin-1 inhibits LLPS of FUS. This activity depends on tight binding of karyopherin-β2 to the C-terminal proline-tyrosine nuclear localization signal (PY-NLS) of FUS. Nuclear magnetic resonance (NMR) analyses reveal weak interactions of karyopherin-β2 with sequence elements and structural domains distributed throughout the entirety of FUS. Biochemical analyses demonstrate that most of these same regions also contribute to LLPS of FUS. The data lead to a model where high-affinity binding of karyopherin-β2 to the FUS PY-NLS tethers the proteins together, allowing multiple, distributed weak intermolecular contacts to disrupt FUS self-association, blocking LLPS. Karyopherin-β2 may act analogously to control condensates in diverse cellular contexts.

Distinctive Nuclear Localization Signals in the Oomycete Phytophthora sojae.

To date, nuclear localization signals (NLSs) that target proteins to nuclei in oomycetes have not been defined, but have been assumed to be the same as in higher eukaryotes. Here, we use the soybean pathogen Phytophthora sojae as a model to investigate these sequences in oomycetes. By establishing a reliable in vivo NLS assay based on confocal microscopy, we found that many canonical monopartite and bipartite classical NLSs (cNLSs) mediated nuclear import poorly in P. sojae. We found that efficient localization of P. sojae nuclear proteins by cNLSs requires additional basic amino acids at distal sites or collaboration with other NLSs. We found that several representatives of another well-characterized NLS, proline-tyrosine NLS (PY-NLS) also functioned poorly in P. sojae. To characterize PY-NLSs in P. sojae, we experimentally defined the residues required by functional PY-NLSs in three P. sojae nuclear-localized proteins. These results showed that functional P. sojae PY-NLSs include an additional cluster of basic residues for efficient nuclear import. Finally, analysis of several highly conserved P. sojae nuclear proteins including ribosomal proteins and core histones revealed that these proteins exhibit a similar but stronger set of sequence requirements for nuclear targeting compared with their orthologs in mammals or yeast.

Karyopherin-β2 Recognition of a PY-NLS Variant that Lacks the Proline-Tyrosine Motif

Karyopherin-β2 or Transportin-1 binds proline-tyrosine nuclear localization signals (PY-NLSs) in its cargos. PY-NLSs are described by structural disorder, overall positive charge, and binding epitopes composed of an N-terminal hydrophobic or basic motif and a C-terminal R-X2-5P-Y motif. The N-terminal tail of histone H3 binds Kapβ2 with high affinity but does not contain a recognizable PY-NLS. The crystal structure of the Kapβ2-H3 tail shows residues 11-27 of H3 binding to the PY-NLS site of Kapβ2. H3residues 11TGGKAPRK18 bind the site for PY-NLS Epitope 1 (N-terminal hydrophobic/basic motif), which is most important for Kapβ2-binding. H3 residue Arg26 occupies the PY-NLS Epitope 2 position (usually arginine of R-X2-5P-Y) but PY-NLS Epitope 3 (proline-tyrosine motif) is missing in the H3 tail. Histone H3 thus provides an example of a PY-NLS variant with no proline-tyrosine or homologous proline-hydrophobic motif. The H3 tail uses a very strong Epitope 1 to compensate for loss of the often-conserved proline-tyrosine epitope.

R521C and R521H mutations in FUS result in weak binding with Karyopherinβ2 leading to Amyotrophic lateral sclerosis: a molecular docking and dynamics study

Fused in sarcoma (FUS) gene encodes the RNA binding protein FUS. This gene is mapped to chromosome 16p11.2. The FUS protein binds with karyopherineβ2 (Kapβ2) through its proline/tyrosine nuclear localization signal (PY-NLS) that helps in the localization of FUS protein within the nucleus. Arginine residue in 521 position (R521) of PY-NLS plays a vital role in the binding of FUS protein with Kapβ2. Mutations in this position (R521C and R521H) are the most predominant mutations associated with amyotrophic lateral sclerosis (ALS). However, the mechanism by which these mutations lead to ALS is poorly understood. We examined the binding behaviour of the mutants FUS (R521C) and FUS (R521H) with Kapβ2 through protein–protein docking and molecular dynamics simulation. The binding patterns of mutants were compared with the binding behaviour of wild FUS–Kapβ2. Our results suggest that these mutants have relatively weak binding affinity with Kapβ2 when compared with wild FUS–Kapβ2 as indicated by the lesser number of interactions found between the mutant FUS and Kapβ2. Hence, these mutations weakens the binding and this results in the cytoplasmic mislocalization of mutant FUS; and thereby it increases the severity of ALS.

Nuclear localization signals for four distinct karyopherin-β nuclear import systems.

The Karyopherin-β family of proteins mediates nuclear transport of macromolecules. Nuclear versus cytoplasmic localization of proteins is often suggested by the presence of NLSs (nuclear localization signals) or NESs (nuclear export signals). Import-Karyopherin-βs or Importins bind to NLSs in their protein cargos to transport them through nuclear pore complexes into the nucleus. Until recently, only two classes of NLS had been biochemically and structurally characterized: the classical NLS, which is recognized by the Importin-α/β heterodimer and the PY-NLS (proline-tyrosine NLS), which is recognized by Karyopherin-β2 or Transportin-1. Structures of two other Karyopherin-βs, Kap121 and Transportin-SR2, in complex with their respective cargos were reported for the first time recently, revealing two new distinct classes of NLSs. The present paper briefly describes the classical NLS, reviews recent literature on the PY-NLS and provides in-depth reviews of the two newly discovered classes of NLSs that bind Kap121p and Transportin-SR respectively.

Reactive oxygen species-mediated DJ-1 monomerization modulates intracellular trafficking involving karyopherin β2.

Mutations in DJ-1 are a cause of recessive, early-onset Parkinson's disease (PD). Although oxidative stress and mitochondrial integrity have been implicated in PD, it is largely unknown why neurons degenerate. DJ-1 is involved in oxidative stress-mediated responses and in mitochondrial maintenance; however, its specific function remains vague. Here we show that DJ-1 exhibits neuronal dynamic intracellular trafficking, with dimeric/monomeric cycling modulated by the oxidative environment. We demonstrate that oxidative stress enhances monomerization of wild-type cytosolic DJ-1, leading to nuclear recruitment. The pathogenic DJ-1/E163K variant is unable to homodimerize but is retained in the cytosol upon wild-type DJ-1 heterodimerization. We found that this wild-type/pathogenic heterodimer is disrupted by oxidative stress, leading to DJ-1/E163K mitochondrial translocation. We further demonstrated that endogenously expressed wild-type DJ-1 is imported into neuronal nuclei as a monomer and that nucleo-cytoplasmic transport is oxidative stress mediated. We identified a novel proline-tyrosine nuclear localization signal (PY-NLS) in DJ-1, and we found that nuclear monomeric DJ-1 import is mediated by an oxidative stress-dependent interaction with karyopherin β2. Our study provides evidence that oxidative stress-mediated intracellular trafficking of DJ-1, mediated by dynamic DJ-1 dimeric/monomeric cycling, is implicated in PD pathogenesis.

Intranuclear Aggregation of Mutant FUS/TLS as a Molecular Pathomechanism of Amyotrophic Lateral Sclerosis

Dominant mutations in FUS/TLS cause a familial form of amyotrophic lateral sclerosis (fALS), where abnormal accumulation of mutant FUS proteins in cytoplasm has been observed as a major pathological change. Many of pathogenic mutations have been shown to deteriorate the nuclear localization signal in FUS and thereby facilitate cytoplasmic mislocalization of mutant proteins. Several other mutations, however, exhibit no effects on the nuclear localization of FUS in cultured cells, and their roles in the pathomechanism of fALS remain obscure. Here, we show that a pathogenic mutation, G156E, significantly increases the propensities for aggregation of FUS in vitro and in vivo. Spontaneous in vitro formation of amyloid-like fibrillar aggregates was observed in mutant but not wild-type FUS, and notably, those fibrils functioned as efficient seeds to trigger the aggregation of wild-type protein. In addition, the G156E mutation did not disturb the nuclear localization of FUS but facilitated the formation of intranuclear inclusions in rat hippocampal neurons with significant cytotoxicity. We thus propose that intranuclear aggregation of FUS triggered by a subset of pathogenic mutations is an alternative pathomechanism of FUS-related fALS diseases.

A Proline‐Tyrosine Nuclear Localization Signal (PY‐NLS) Is Required for the Nuclear Import of Fission Yeast PAB2, but Not of Human PABPN1

Nuclear poly(A)-binding proteins (PABPs) are evolutionarily conserved proteins that play key roles in eukaryotic gene expression. In the fission yeast Schizosaccharomyces pombe, the major nuclear PABP, Pab2, functions in the maturation of small nucleolar RNAs as well as in nuclear RNA decay. Despite knowledge about its nuclear functions, nothing is known about how Pab2 is imported into the nucleus. Here, we show that Pab2 contains a proline-tyrosine nuclear localization signal (PY-NLS) that is necessary and sufficient for its nuclear localization and function. Consistent with the role of karyopherin β2 (Kapβ2)-type receptors in the import of PY-NLS cargoes, we show that the fission yeast ortholog of human Kapβ2, Kap104, binds to recombinant Pab2 and is required for Pab2 nuclear localization. The absence of arginine methylation in a basic region N-terminal to the PY-core motif of Pab2 did not affect its nuclear localization. However, in the context of a sub-optimal PY-NLS, we found that Pab2 was more efficiently targeted to the nucleus in the absence of arginine methylation, suggesting that this modification can affect the import kinetics of a PY-NLS cargo. Although a sequence resembling a PY-NLS motif can be found in the human Pab2 ortholog, PABPN1, our results indicate that neither a functional PY-NLS nor Kapβ2 activity are required to promote entry of PABPN1 into the nucleus of human cells. Our findings describe the mechanism by which Pab2 is imported into the nucleus, providing the first example of a PY-NLS import system in fission yeast. In addition, this study suggests the existence of alternative or redundant nuclear import pathways for human PABPN1.

DLocalMotif: a discriminative approach for discovering local motifs in protein sequences

Local motifs are patterns of DNA or protein sequences that occur within a sequence interval relative to a biologically defined anchor or landmark. Current protein motif discovery methods do not adequately consider such constraints to identify biologically significant motifs that are only weakly over-represented but spatially confined. Using negatives, i.e. sequences known to not contain a local motif, can further increase the specificity of their discovery. This article introduces the method DLocalMotif that makes use of positional information and negative data for local motif discovery in protein sequences. DLocalMotif combines three scoring functions, measuring degrees of motif over-representation, entropy and spatial confinement, specifically designed to discriminatively exploit the availability of negative data. The method is shown to outperform current methods that use only a subset of these motif characteristics. We apply the method to several biological datasets. The analysis of peroxisomal targeting signals uncovers several novel motifs that occur immediately upstream of the dominant peroxisomal targeting signal-1 signal. The analysis of proline-tyrosine nuclear localization signals uncovers multiple novel motifs that overlap with C2H2 zinc finger domains. We also evaluate the method on classical nuclear localization signals and endoplasmic reticulum retention signals and find that DLocalMotif successfully recovers biologically relevant sequence properties. http://bioinf.scmb.uq.edu.au/dlocalmotif/

The FUS about arginine methylation in ALS and FTLD

In a time where links between amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) neurodegeneration are becoming increasingly clear, it is important to establish the convergent and divergent mechanisms responsible for this. Accordingly, Dormann et al (2012) have identified that methylation of the Fused in sarcoma (FUS) RGG3 domain is involved in the cytoplasmic mislocalisation of ALS‐FUS mutants, through a transportin‐dependent mechanism. By contrast, hypomethylation in this domain may play a role in the aberrant accumulation of FUS in FTLD‐FUS. This work showcases arginine methylation as a phenomenon to watch out for in neurodegenerative pathology.

Arginine methylation next to the PY-NLS modulates Transportin binding and nuclear import of FUS.

Fused in sarcoma (FUS) is a nuclear protein that carries a proline-tyrosine nuclear localization signal (PY-NLS) and is imported into the nucleus via Transportin (TRN). Defects in nuclear import of FUS have been implicated in neurodegeneration, since mutations in the PY-NLS of FUS cause amyotrophic lateral sclerosis (ALS). Moreover, FUS is deposited in the cytosol in a subset of frontotemporal lobar degeneration (FTLD) patients. Here, we show that arginine methylation modulates nuclear import of FUS via a novel TRN-binding epitope. Chemical or genetic inhibition of arginine methylation restores TRN-mediated nuclear import of ALS-associated FUS mutants. The unmethylated arginine-glycine-glycine domain preceding the PY-NLS interacts with TRN and arginine methylation in this domain reduces TRN binding. Inclusions in ALS-FUS patients contain methylated FUS, while inclusions in FTLD-FUS patients are not methylated. Together with recent findings that FUS co-aggregates with two related proteins of the FET family and TRN in FTLD-FUS but not in ALS-FUS, our study provides evidence that these two diseases may be initiated by distinct pathomechanisms and implicates alterations in arginine methylation in pathogenesis.