scholarly journals Nup98 Localizes to Both Nuclear and Cytoplasmic Sides of the Nuclear Pore and Binds to Two Distinct Nucleoporin Subcomplexes

2003 ◽  
Vol 14 (2) ◽  
pp. 600-610 ◽  
Author(s):  
Eric R. Griffis ◽  
Songli Xu ◽  
Maureen A. Powers
Keyword(s):  
Amino Acids ◽  
Nuclear Envelope ◽  
Point Mutation ◽  
Cleavage Site ◽  
Nuclear Pore ◽  
Nucleocytoplasmic Shuttling ◽  
Double Membrane ◽  
C Terminus ◽  
Direct Binding ◽  
Autoproteolytic Cleavage

The vertebrate nuclear pore is an enormous structure that spans the double membrane of the nuclear envelope. In yeast, most nucleoporins are found symmetrically on both the nuclear and cytoplasmic sides of the structure. However, in vertebrates most nucleoporins have been localized exclusively to one side of the nuclear pore. Herein, we show, by immunofluorescence and immunoelectron microscopy, that Nup98 is found on both sides of the pore complex. Additionally, we find that the pore-targeting domain of Nup98 interacts directly with the cytoplasmic nucleoporin Nup88, a component of the Nup214, Nup88, Nup62 subcomplex. Nup98 was previously described to interact with the nuclear-oriented Nup160, 133, 107, 96 complex through direct binding to Nup96. Interestingly, the same site within Nup98 is involved in binding to both Nup88 and Nup96. Autoproteolytic cleavage of the Nup98 C terminus is required for both of these binding interactions. When cleavage is blocked by a point mutation, a minimal eight amino acids downstream of the cleavage site is sufficient to prevent most binding to either Nup96 or Nup88. Thus, Nup98 interacts with both faces of the nuclear pore, a localization in keeping with its previously described nucleocytoplasmic shuttling activity.

scholarly journals Direct binding of ESCRT protein Chm7 to phosphatidic acid–rich membranes at nuclear envelope herniations

2021 ◽  
Vol 220 (3) ◽  
Author(s):  
David J. Thaller ◽  
Danqing Tong ◽  
Christopher J. Marklew ◽  
Nicholas R. Ader ◽  
Philip J. Mannino ◽  
...  
Keyword(s):  
Amino Acids ◽  
Phosphatidic Acid ◽  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Binding Capacity ◽  
Nuclear Pore ◽  
Membrane Remodeling ◽  
Functional Importance ◽  
Direct Binding ◽  
Domain Protein

Mechanisms that control nuclear membrane remodeling are essential to maintain the integrity of the nucleus but remain to be fully defined. Here, we identify a phosphatidic acid (PA)–binding capacity in the nuclear envelope (NE)–specific ESCRT, Chm7, in budding yeast. Chm7’s interaction with PA-rich membranes is mediated through a conserved hydrophobic stretch of amino acids, which confers recruitment to the NE in a manner that is independent of but required for Chm7’s interaction with the LAP2-emerin-MAN1 (LEM) domain protein Heh1 (LEM2). Consistent with the functional importance of PA binding, mutation of this region abrogates recruitment of Chm7 to membranes and abolishes Chm7 function in the context of NE herniations that form during defective nuclear pore complex (NPC) biogenesis. In fact, we show that a PA sensor specifically accumulates within these NE herniations. We suggest that local control of PA metabolism is important for ensuring productive NE remodeling and that its dysregulation may contribute to pathologies associated with defective NPC assembly.

Abstract 52: The Role of Kindlin-2 in Integrin Activation Depends on a Short C-Terminal Region

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Jamila Hirbawi ◽  
Kamila Bledzka ◽  
Yan Qing Ma ◽  
Jun Qin ◽  
Edward F Plow
Keyword(s):  
Amino Acids ◽  
Point Mutation ◽  
Single Point ◽  
Terminal Region ◽  
Membrane Receptors ◽  
Single Point Mutation ◽  
Loss Of Function ◽  
Integrin Activation ◽  
C Terminus

Integrins are heterodimeric cell membrane receptors that regulate cell adhesion, migration, and survival. The kindlins are known to be key regulators of integrin activation, the transition from a low affinity, default state to a high affinity state for ligand. This function depends on their binding, together with talin, to the cytoplasmic tails (CT) of the β subunit of integrins. Kindlins are FERM domain containing proteins, and it is its F3 (PTB) subdomain of the FERM that is the primary binding site for integrin β CT. At its very C-terminus, beyond the F3, is a short extension of 21 amino acids, K2 660-680, and we have focused on the role of this region in the co-activator function of kindlin-2 (K2). For this analysis, we performed PAC-1 (antibody to detect activated αIIbβ3 integrin) binding assays in CHO cells stably expressing integrin α IIb β 3 that were transiently transfected with talin head domain and K2 mutants. Expression levels of all proteins were verified to be similar by western blotting and FACS. Truncation of K2 at residue 660 essentially eliminated the co-activator function of K2. Deletion of smaller segments also reduced co-activator activity by 50% to 100%. Deletion of just the last two amino acids in the sequence, W 679 V 680 , resulted in a 50% reduction in co-activator activity and a single point mutation of Y 673 A also led to a 50% loss of function. A combination mutant consisting of the W 679 V 680 deletion and the Y 673 point mutation resulted in 100% loss of kindlin-2 co-activator activity. Pull-down experiments performed using GST tagged β 3 CT and CHO lysates transfected with GFP-kindlin-2 forms suggested that the C-terminal deletion did not disrupt binding to β 3 CT. This observation was corroborated by surface plasmon resonance studies in which the binding of full-length K2 and K2Δ666C (Δ666) was compared, and their K D values for immobilized β3 CT were found to be essentially the same. Overall, these data establish an important and unanticipated role of the carboxy-terminal region of kindlin-2 in its integrin co-activator function that is not dependent of its binding to integrin.

scholarly journals Nuclear Envelope Proteins Modulating the Heterochromatin Formation and Functions in Fission Yeast

Cells ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1908 ◽  
Author(s):  
Yasuhiro Hirano ◽  
Haruhiko Asakawa ◽  
Takeshi Sakuno ◽  
Tokuko Haraguchi ◽  
Yasushi Hiraoka
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Fission Yeast ◽  
Histone Deacetylase ◽  
Nuclear Pore Complex ◽  
Molecular Mechanisms ◽  
Nuclear Pore ◽  
Double Membrane ◽  
Heterochromatin Formation ◽  
Nuclear Membranes

The nuclear envelope (NE) consists of the inner and outer nuclear membranes (INM and ONM), and the nuclear pore complex (NPC), which penetrates the double membrane. ONM continues with the endoplasmic reticulum (ER). INM and NPC can interact with chromatin to regulate the genetic activities of the chromosome. Studies in the fission yeast Schizosaccharomyces pombe have contributed to understanding the molecular mechanisms underlying heterochromatin formation by the RNAi-mediated and histone deacetylase machineries. Recent studies have demonstrated that NE proteins modulate heterochromatin formation and functions through interactions with heterochromatic regions, including the pericentromeric and the sub-telomeric regions. In this review, we first introduce the molecular mechanisms underlying the heterochromatin formation and functions in fission yeast, and then summarize the NE proteins that play a role in anchoring heterochromatic regions and in modulating heterochromatin formation and functions, highlighting roles for a conserved INM protein, Lem2.

Nucleocytoplasmic Shuttling of Porcine Parvovirus NS1 Protein Mediated by the CRM1 Nuclear Export Pathway and the Importin α/β Nuclear Import Pathway

2021 ◽  
Author(s):  
Liyan Cao ◽  
Fang Fu ◽  
Jianfei Chen ◽  
Hongyan Shi ◽  
Xin Zhang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Nonstructural Protein ◽  
Nuclear Pore ◽  
Porcine Parvovirus ◽  
Ns1 Protein ◽  
Nucleocytoplasmic Shuttling ◽  
Export Pathway ◽  
Importin Α

Porcine parvovirus (PPV) NS1, the major nonstructural protein of this virus, plays an important role in PPV replication. We show, for the first time, that NS1 dynamically shuttles between the nucleus and cytoplasm, although its subcellular localization is predominantly nuclear. NS1 contains two nuclear export signals (NESs) at amino acids 283–291 (designated NES2) and 602–608 (designated NES1). NES1 and NES2 are both functional and transferable NESs, and their nuclear export activity is blocked by leptomycin B (LMB), suggesting that the export of NS1 from the nucleus is dependent upon the chromosome region maintenance 1 (CRM1) pathway. Deletion and site-directed mutational analyses showed that NS1 contains a bipartite nuclear localization signal (NLS) at amino acids 256–274. Coimmunoprecipitation assays showed that NS1 interacts with importins α5 and α7 through its NLS. The overexpression of CRM1, importins α5 and α7 significantly promoted PPV replication, whereas the inhibition of CRM1 and importin α/β-mediated transport by specific inhibitors (LMB, importazole and ivermectin) clearly blocked PPV replication. The mutant viruses of delete NESs or NLS motif of the NS1 by using reverse genetics could not be rescued, suggesting that NESs and NLS are essential for PPV replication. Collectively, these findings suggest that NS1 shuttles between the nucleus and cytoplasm, mediated by its functional NESs and NLS, via the CRM1-dependent nuclear export pathway and the importin α/β-mediated nuclear import pathway, and PPV proliferation was inhibited if blocking NS1 nuclear import or export. Importance PPV replicates in the nucleus, and the nuclear envelope is a barrier to its entry into and egress from the nucleus. PPV NS1 is a nucleus-targeting protein that is important for viral DNA replication. Because the NS1 molecule is large (> 50 kDa), it cannot pass through the nuclear pore complex by diffusion alone, and requires specific transport receptors to permit its nucleocytoplasmic shuttling. In this study, the two functional NESs in the NS1 protein were identified, and its dependence on the CRM1 pathway for nuclear export demonstrated. The nuclear import of NS1 utilizes importins α5 and α7 in the importin α/β nuclear import pathway.

scholarly journals Direct PA-binding by Chm7 is required for nuclear envelope surveillance at herniations

2020 ◽  
Author(s):  
David J. Thaller ◽  
Danqing Tong ◽  
Christopher J. Marklew ◽  
Sapan Borah ◽  
Barbara Ciani ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Nuclear Membrane ◽  
Specific Binding ◽  
Binding Activity ◽  
Nuclear Pore ◽  
Membrane Remodeling ◽  
Functional Importance ◽  
Domain Protein

AbstractMechanisms that control nuclear membrane remodeling are essential to maintain the integrity of the nucleus but remain to be fully defined. Here, we identify a phosphatidic acid (PA)-binding activity in the nuclear envelope-specific ESCRT, Chm7, in budding yeast. PA-binding is mediated through a conserved hydrophobic stretch of amino acids, which confers specific binding to the inner nuclear membrane (INM). This INM-binding is independent but nonetheless required for interaction with the LAP2-emerin-MAN1 (LEM) domain protein, Heh1 (LEM2). Consistent with the functional importance of PA-binding, mutation of this region inhibits recruitment of Chm7 to the INM and abolishes Chm7 function in the context of nuclear envelope herniations or “blebs” that form during defective nuclear pore complex (NPC) biogenesis. In fact, we show that PA accumulates at nuclear envelope herniations. We suggest that local control of PA metabolism is important for ensuring productive nuclear envelope remodeling and that its dysregulation may contribute to pathologies associated with defective NPC assembly.

scholarly journals Specificity of human rhinovirus 2Apro is determined by combined spatial properties of four cleavage site residues

2013 ◽  
Vol 94 (7) ◽  
pp. 1535-1546 ◽  
Author(s):  
David Neubauer ◽  
Martina Aumayr ◽  
Irene Gösler ◽  
Tim Skern
Keyword(s):  
Amino Acids ◽  
Substrate Specificity ◽  
Cleavage Site ◽  
Antiviral Agents ◽  
Genetic Group ◽  
Human Rhinovirus ◽  
C Terminus ◽  
Group B

The 2A proteinase (2Apro) of human rhinoviruses cleaves the virally encoded polyprotein between the C terminus of VP1 and its own N terminus. Poor understanding of the 2Apro substrate specificity of this enzyme has hampered progress in developing inhibitors that may serve as antiviral agents. We show here that the 2Apro of human rhinovirus (HRV) 1A and 2 (rhinoviruses from genetic group A) cannot self-process at the HRV14 (a genetic group B rhinovirus) cleavage site. When the amino acids in the cleavage site of HRV2 2Apro (Ile-Ile-Thr-Thr-Ala*Gly-Pro-Ser-Asp) were singly or doubly replaced with the corresponding HRV14 residues (Asp-Ile-Lys-Ser-Tyr*Gly-Leu-Gly-Pro) at positions from P3 to P2′, HRV1A and HRV2 2Apro cleavage took place at WT levels. However, when three or more positions of the HRV1A or 2 2Apro were substituted (e.g. at P2, P1 and P2′), cleavage in vitro was essentially eliminated. Introduction of the full HRV14 cleavage site into a full-length clone of the HRV1A and transfection of HeLa cells with a transcribed RNA did not give rise to viable virus. In contrast, revertant viruses bearing cysteine at the P1 position or proline at P2′ were obtained when an RNA bearing the three inhibitory amino acids was transfected. Reversions in the enzyme affecting substrate specificity were not found in any of the in vivo experiments. Modelling of oligopeptide substrates onto the structure of HRV2 2Apro revealed no appreciable differences in residues of HRV2 and HRV14 in the respective substrate binding sites, suggesting that the overall shape of the substrate is important in determining binding efficiency.

Human D-Tyr-tRNATyr deacylase contributes to the resistance of the cell to D-amino acids

2008 ◽  
Vol 417 (1) ◽  
pp. 85-97 ◽  
Author(s):  
Gen Zheng ◽  
Wei Liu ◽  
Yanhua Gong ◽  
Hongbo Yang ◽  
Bin Yin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nuclear Envelope ◽  
Mammalian Cells ◽  
Nuclear Accumulation ◽  
Nuclear Pore ◽  
Dependent Manner ◽  
High Concentration ◽  
Cellular Resistance ◽  
Trna Editing ◽  
Dose Dependent

DTD (D-Tyr-tRNATyr deacylase) is known to be able to deacylate D-aminoacyl-tRNAs into free D-amino acids and tRNAs and therefore contributes to cellular resistance against D-amino acids in Escherichia coli and yeast. We have found that h-DTD (human DTD) is enriched in the nuclear envelope region of mammalian cells. Treatment of HeLa cells with D-Tyr resulted in nuclear accumulation of tRNATyr. D-Tyr treatment and h-DTD silencing caused tRNATyr downregulation. Furthermore, inhibition of protein synthesis by D-Tyr treatment and h-DTD silencing were also observed. D-Tyr, D-Asp and D-Ser treatment inhibited mammalian cell viability in a dose-dependent manner; overexpression of h-DTD decreased the inhibition rate, while h-DTD-silenced cells became more sensitive to the D-amino acid treatment. Our results suggest that h-DTD may play an important role in cellular resistance against D-amino acids by deacylating D-aminoacyl tRNAs at the nuclear pore. We have also found that m-DTD (mouse DTD) is specifically enriched in central nervous system neurons, its nuclear envelope localization indicates that D-aminoacyl-tRNA editing may be vital for the survival of neurons under high concentration of D-amino acids.

scholarly journals A Link between the Synthesis of Nucleoporins and the Biogenesis of the Nuclear Envelope

2001 ◽  
Vol 153 (4) ◽  
pp. 709-724 ◽  
Author(s):  
Marcello Marelli ◽  
C. Patrick Lusk ◽  
Honey Chan ◽  
John D. Aitchison ◽  
Richard W. Wozniak
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Nuclear Transport ◽  
Terminal Segment ◽  
Nuclear Pore ◽  
Membrane Structures ◽  
Double Membrane ◽  
Microscopy Analysis ◽  
Specific Association ◽  
Α Helix

The nuclear pore complex (NPC) is a multicomponent structure containing a subset of proteins that bind nuclear transport factors or karyopherins and mediate their movement across the nuclear envelope. By altering the expression of a single nucleoporin gene, NUP53, we showed that the overproduction of Nup53p altered nuclear transport and had a profound effect on the structure of the nuclear membrane. Strikingly, conventional and immunoelectron microscopy analysis revealed that excess Nup53p entered the nucleus and associated with the nuclear membrane. Here, Nup53p induced the formation of intranuclear, tubular membranes that later formed flattened, double membrane lamellae structurally similar to the nuclear envelope. Like the nuclear envelope, the intranuclear double membrane lamellae enclosed a defined cisterna that was interrupted by pores but, unlike the nuclear envelope pores, they lacked NPCs. Consistent with this observation, we detected only two NPC proteins, the pore membrane proteins Pom152p and Ndc1p, in association with these membrane structures. Thus, these pores likely represent an intermediate in NPC assembly. We also demonstrated that the targeting of excess Nup53p to the NPC and its specific association with intranuclear membranes were dependent on the karyopherin Kap121p and the nucleoporin Nup170p. At the nuclear envelope, the abilities of Nup53p to associate with the membrane and drive membrane proliferation were dependent on a COOH-terminal segment containing a potential amphipathic α-helix. The implications of these results with regards to the biogenesis of the nuclear envelope are discussed.

scholarly journals Structural features that make oligopeptides susceptible substrates for hydrolysis by recombinant thimet oligopeptidase

1997 ◽  
Vol 324 (2) ◽  
pp. 517-522 ◽  
Author(s):  
Antonio. C. M CAMARGO ◽  
Marcelo. D GOMES ◽  
Antonia. P REICHL ◽  
Emer. S FERRO ◽  
Saul JACCHIERI ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cleavage Site ◽  
Catalytic Efficiency ◽  
Structural Features ◽  
Amino Acid Residues ◽  
Systematic Analysis ◽  
Peptide Bonds ◽  
C Terminus ◽  
Thimet Oligopeptidase

A systematic analysis of the peptide sequences and lengths of several homologues of bioactive peptides and of a number of quenched-fluorescence (qf) opioid- and bradykinin-related peptides was performed to determine the main features leading the oligopeptides to hydrolysis by the recombinant rat testis thimet oligopeptidase (EC 3.4.24.15). The results indicate that a minimum substrate length of six amino acids is required and that among the oligopeptides six to thirteen amino acid residues long, their susceptibility as substrates is highly variable. Thimet oligopeptidase was able to hydrolyse, with similar catalytic efficiency, peptide bonds having hydrophobic or hydrophilic amino acids as well as proline in the P1 position of peptides, ranging from a minimum of six to a maximum of approximately thirteen amino acid residues. An intriguing observation was the shift of the cleavage site, at a Leu-Arg bond in qf dynorphin-(2–8) [qf-Dyn2–8; Abz-GGFLRRV-EDDnp, where Abz stands for o-aminobenzoyl and EDDnp for N-(2,4-dinitrophenyl) ethylenediamine], to Arg-Arg in qf-Dyn2–8Q, in which Gln was substituted for Val at its C-terminus. Similarly, a cleavage site displacement was also observed with the hydrolysis of the internally quenched-fluorescence bradykinin analogues containing Gln at the C-terminal position, namely Abz-RPPGFSPFR-EDDnp and Abz-GFSPFR-EDDnp are cleaved at the Phe-Ser bond, but Abz-RPPGFSPFRQ-EDDnp and Abz-GFSPFRQ-EDDnp are cleaved at the Pro-Phe bond.

IMPROVEMENT OF T-PA PROPERTIES BY MEANS OF SITE DIRECTED MUTAGENESIS

1987 ◽  
Author(s):  
N Haigwood ◽  
E-P Pâques ◽  
G Mullenbach ◽  
G Moore ◽  
L DesJardin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cleavage Site ◽  
Specific Activity ◽  
Biological Properties ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Mutant Proteins ◽  
C Terminus

The clinical relevance of tissue-plasminogen-activator (t-PA) as a potent thrombolytic agent has recently been established. It has however been recognized that t-PA does not fulfill all conditions required for an ideal thrombolytic pharmaceutical agent; for example, its physiological stability and its short half life in vivo necessitate the use of very large clinical doses. We have therefore attempted to develop novel mutant t-PA proteins with improved properties by creating mutants by site-directed mutagenesis in M13 bacteriophage. Seventeen mutants were designed, cloned, and expressed in CHO cells. Modifications were of three types: alterations to glycosylation sites, truncations of the N- or C-termini, and amino acids changes at the cleavage site utilized to generate the two chain form of t-PA. The mutant proteins were analyzed in vitro for specific activity, fibrin dependence of the plasminogen activation, fibrin affinity, and susceptibility to inhibition by PAI.In brief, the results are: 1) some unglycosylated and partially glycosylated molecules obtained by mutagenesis are characterized by several-fold higher specific activity than wild type t-PA; 2) truncation at the C-terminus by three amino acids yields a molecule with increased fibrin specificity; 3) mutations at the cleavage site lead zo a decreased inhibition by PAI; and 4) recombinants of these genes have been constructed and the proteins were shown to possess multiple improved properties. The use of site directed mutagenesis has proved to be a powerful instrument to modulate the biological properties of t-PA.

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