Poly(ADP-ribose) polymerase-1: association with nuclear lamins in rodent liver cells

The distribution of poly(ADP-ribose) polymerase-1 (PARP-1) over different nuclear compartments was studied by nuclear fractionation procedures and Western analysis revealing a prominent role of the nuclear matrix. This structure is operationally defined by the solubility properties of the A- and B-type lamins under defined experimental conditions. We consistently observed that most of the nuclear matrix-associated PARP-1 partitioned, in an active form, with the insoluble, lamin-enriched protein fractions that were prepared by a variety of established biochemical procedures. These PARP-1-protein interactions resisted salt extraction, disulfide reduction, RNase and DNase digestion. An inherent ability of PARP-1 to reassemble with the lamins became evident after a cycle of solubilization/dialysis using either urea or Triton X-100 and disulfide reduction, indicating that these interactions were dominated by hydrophobic forces. Together with in vivo crosslinking and co-immunoprecipitation experiments our results show that the lamins are prominent PARP-1-binding partners which could contribute to the functional sequestration of the enzyme on the nuclear matrix.     

PARP-1与中枢神经系统疾病的关系

聚腺苷酸二磷酸核糖基聚合酶1[poly（ADP-ribose）pdymerase-1,PARP-1]是-种广泛存在于真核生物细胞核中的蛋白酶,在中枢神经系统的疾病发生中扮演着重要的角色。在外界损伤刺激下,PARP-1易被受损的DNA激活,进而通过不同途径影响神经细胞的生理功能,引起细胞炎性反应,甚至导致细胞死亡,触发中枢神经系统疾病的发生。抑制PARP-1在治疗慢性和急性的中枢神经系统疾病的作用也越来越受到重视。     

A role for nuclear lamins in nuclear envelope assembly.

The molecular interactions responsible for nuclear envelope assembly after mitosis are not well understood. In this study, we demonstrate that a peptide consisting of the COOH-terminal domain of Xenopus lamin B3 (LB3T) prevents nuclear envelope assembly in Xenopus interphase extracts. Specifically, LB3T inhibits chromatin decondensation and blocks the formation of both the nuclear lamina-pore complex and nuclear membranes. Under these conditions, some vesicles bind to the peripheral regions of the chromatin. These "nonfusogenic" vesicles lack lamin B3 (LB3) and do not bind LB3T; however, "fusogenic" vesicles containing LB3 can bind LB3T, which blocks their association with chromatin and, subsequently, nuclear membrane assembly. LB3T also binds to chromatin in the absence of interphase extract, but only in the presence of purified LB3. Additionally, we show that LB3T inhibits normal lamin polymerization in vitro. These findings suggest that lamin polymerization is required for both chromatin decondensation and the binding of nuclear membrane precursors during the early stages of normal nuclear envelope assembly.     

The DNA topoisomerase IIbeta binding protein 1 (TopBP1) interacts with poly (ADP-ribose) polymerase (PARP-1)

We investigated the physical association of the DNA topoisomerase IIbeta binding protein 1 (TopBP1), involved in DNA replication and repair but also in regulation of apoptosis, with poly(ADP-ribose) polymerase-1 (PARP-1). This enzyme plays a crucial role in DNA repair and interacts with many DNA replication/repair factors. It was shown that the sixth BRCA1 C-terminal (BRCT) domain of TopBP1 interacts with a protein fragment of PARP-1 in vitro containing the DNA-binding and the automodification domains. More significantly, the in vivo interaction of endogenous TopBP1 and PARP-1 proteins could be shown in HeLa-S3 cells by co-immunoprecipitation. TopBP1 and PARP-1 are localized within overlapping regions in the nucleus of HeLa-S3 cells as shown by immunofluorescence. Exposure to UVB light slightly enhanced the interaction between both proteins. Furthermore, TopBP1 was detected in nuclear regions where poly(ADP-ribose) (PAR) synthesis takes place and is ADP-ribosylated by PARP-1. Finally, cellular (ADP-ribosyl)ating activity impairs binding of TopBP1 to Myc-interacting zinc finger protein-1 (Miz-1). The results indicate an influence of post-translational modifications of TopBP1 on its function during DNA repair.     

Partial cleavage of A-type lamins concurs with their total disintegration from the nuclear lamina during apoptosis

Although activated caspase 6 is capable of cleaving both A- and B-type lamins during apoptosis, the higher-order structure of the nuclear lamina may cause a differential breakdown of these two types of lamins. In order to obtain a better understanding of the dynamics and the consequences of the rapid, coordinated breakdown of the lamina complex, we applied the green fluorescent protein (GFP) technology in living cells, in which the fate of individual caspase cleavage fragments of A- and B-type lamins was examined. CHO-K1 cells were stably transfected with cDNA constructs encoding N-terminally GFP-labelled hybrids of lamin A, lamin Adelta10, lamin C or lamin B1. The course of the apoptotic process, induced by the kinase inhibitor staurosporine or by the proteasome inhibitor MG132, was monitored by digital imaging microscopy or confocal microscopy. Time-lapse recordings showed that parallel to DNA condensation N-terminally GFP-tagged A-type lamins became diffusely dispersed throughout the nucleoplasm and rapidly translocated to the cytoplasm. In contrast, the majority of GFP-lamin B1 signal remained localised at the nuclear periphery, even after extensive DNA condensation. Comparison of lamin B1-GFP signal with A-type lamin antibody staining in the same apoptotic cells confirmed the temporal differences between A- and B-type lamina dispersal. Immunoblotting revealed only a partial cleavage of A-type lamins and an almost complete cleavage of lamin B1 during apoptosis. In contrast to lamin B1 in normal cells, this cleaved lamin B1, which is apparently still associated with the nuclear membrane, can be completely extracted by methanol or ethanol. Fluorescence loss of intensity after photobleaching experiments showed that in apoptotic cells A-type lamin-GFP molecules diffuse almost freely in both nucleoplasm and cytoplasm, while the lamin B1-GFP fragments remain more stably associated with the nuclear membrane, which is confirmed by co-localisation immunofluorescence studies with a nucleoporin p62 antibody. Our results therefore clearly show a differential behaviour of A- and B-type lamins during apoptosis, suggesting not only distinct differences in the organisation of the lamina filaments, but also that caspase cleavage of only a small fraction of A-type lamins is needed for its complete disintegration.     

The fates of chicken nuclear lamin proteins during mitosis: evidence for a reversible redistribution of lamin B2 between inner nuclear membrane and elements of the endoplasmic reticulum

In chicken, three structurally distinct nuclear lamin proteins have been described. According to their migration on two-dimensional gels, these proteins have been designated as lamins A, B1, and B2. To investigate the functional relationship between chicken lamins and their mammalian counterparts, we have examined here the state of individual chicken lamin proteins during mitosis. Current models proposing functional specializations of mammalian lamin subtypes are in fact largely based on the observation that during mitosis mammalian lamin B remains associated with membrane vesicles, whereas lamins A and C become freely soluble. Cell fractionation experiments combined with immunoblotting show that during mitosis both chicken lamins B1 and B2 remain associated with membranes, whereas lamin A exists in a soluble form. In situ immunoelectron microscopy carried out on mitotic cells also reveals membrane association of lamin B2, whereas the distribution of lamin A is random. From these results we conclude that both chicken lamins B1 and B2 may functionally resemble mammalian lamin B. Interestingly, immunolabeling of mitotic cells revealed an association of lamin B2 with extended membrane cisternae that resembled elements of the endoplasmic reticulum. Quantitatively, we found that all large endoplasmic reticulum-like membranes present in metaphase cells were decorated with lamin B2-specific antibodies. Given that labeling of these mitotic membranes was lower than labeling of interphase nuclear envelopes, it appears likely that during mitotic disassembly and reassembly of the nuclear envelope lamin B2 may reversibly distribute between the inner nuclear membrane and the endoplasmic reticulum.     

Dynamic properties of germ line-specific lamin B3: the role of the shortened rod domain

The mammalian lamin B2 gene codes for two proteins, the somatic lamin B2 and the germ line-specific lamin B3. Lamin B3 lacks the N-terminus and a part of the alpha-helical rod domain present in lamin B2. These domains are substituted by 84 amino acids unique for lamin B3. When ectopically expressed in somatic cells, lamin B3 causes severe deformation of nuclei which adopt a hook-like configuration. Accordingly, it was proposed that lamin B3 provides the germ line cells with a more flexible nuclear periphery that facilitates spermatogenesis-specific nuclear reorganization events. Here we investigated which protein domains of lamin B3 are responsible for nuclear deformation in transfected cells, and how stable is the nuclear periphery of these cells. Expression of wild-type and mutant lamins evidenced that nuclear deformations are due to the shortened rod domain of lamin B3. Cell fractionation experiments revealed that lamin B3 can be solubilized more easily than lamin B2. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) analyses of transfected cells showed that lamin B3 has an increased mobility compared to B2. Our results lead to the conclusion that lamin B3 reduces the stability of the nuclear periphery. They are also consistent with the notion that lamin B3 is relevant to specific properties of the nuclear envelope during spermiogenesis.     

Defective control of mitotic and post-mitotic checkpoints in poly(ADP-ribose) polymerase-1(-/-) fibroblasts after mitotic spindle disruption

Poly(ADP-ribose) polymerase-1 (PARP), a DNA damage-responsive nuclear enzyme present in higher eukaryotes, is well-known for its roles in protecting the genome after DNA damage. However, even without exogenous DNA damage, PARP may play a role in stabilizing the genome because cells or mice deficient in PARP exhibit various signs of genomic instability, such as tetraploidy, aneuploidy, chromosomal abnormalities and susceptibility to spontaneous carcinogenesis. Normally, cell cycle checkpoints ensure elimination of cells with genomic abnormalities. Therefore, we examined efficiency of mitotic and post-mitotic checkpoints in PARP-/- and PARP+/+ mouse embryonic fibroblasts treated with mitotic spindle disrupting agent colcemid. PARP+/+ cells, like most mammalian cells, eventually escaped from spindle disruption-induced mitotic checkpoint arrest by 60 h. In contrast, PARP-/- cells rapidly escaped from mitotic arrest within 24 h by downregulation of cyclin B1/CDK-1 kinase activity. After escaping from mitotic arrest; both the PARP genotypes arrive in G1 tetraploid state, where they face post-mitotic checkpoints which either induce apoptosis or prevent DNA endoreduplication. While all the G1 tetraploid PARP+/+ cells were eliminated by apoptosis, the majority of the G1 tetraploid PARP-/- cells became polyploid by resisting apoptosis and carrying out DNA endoreduplication. Introduction of PARP in PARP-/- fibroblasts partially increased the stringency of mitotic checkpoint arrest and fully restored susceptibility to G1 tetraploidy checkpoint-induced apoptosis; and thus prevented formation of polyploid cells. Our results suggest that PARP may serve as a guardian angel of the genome even without exogenous DNA damage through its role in mitotic and post-mitotic G1 tetraploidy checkpoints.     

PARP-1 Inhibition Is Neuroprotective in the R6/2 Mouse Model of Huntington’s Disease

Poly (ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that is involved in physiological processes as DNA repair, genomic stability, and apoptosis. Moreover, published studies demonstrated that PARP-1 mediates necrotic cell death in response to excessive DNA damage under certain pathological conditions. In Huntington’s disease brains, PARP immunoreactivity was described in neurons and in glial cells, thereby suggesting the involvement of apoptosis in HD. In this study, we sought to determine if the PARP-1 inhibitor exerts a neuroprotective effect in R6/2 mutant mice, which recapitulates, in many aspects, human HD. Transgenic mice were treated with the PARP-1 inhibitor INO-1001 mg/Kg daily starting from 4 weeks of age. After transcardial perfusion, histological and immunohistochemical studies were performed. We found that INO 1001-treated R6/2 mice survived longer and displayed less severe signs of neurological dysfunction than the vehicle treated ones. Primary outcome measures such as striatal atrophy, morphology of striatal neurons, neuronal intranuclear inclusions and microglial reaction confirmed a neuroprotective effect of the compound. INO-1001 was effective in significantly increasing activated CREB and BDNF in the striatal spiny neurons, which might account for the beneficial effects observed in this model. Our findings show that PARP-1 inhibition could be considered as a valid therapeutic approach for HD.     

Nuclear envelope breakdown requires overcoming the mechanical integrity of the nuclear lamina

In prophase cells, lamin B1 is the major component of the nuclear lamina, a filamentous network underlying the nucleoplasmic side of the nuclear membrane, whereas lamin A/C is dissociated from the scaffold. In vivo fluorescence microscopy studies have shown that, during the G2/M transition, the first gap in the nuclear envelope (NE) appears before lamin B1 disassembly and is caused by early spindle microtubules impinging on the NE. This result suggests that the mechanical tearing of the NE by microtubules plays a central role to the progression of mitosis. To investigate whether this microtubule-induced NE deformation is sufficient for NE breakdown, we assess the mechanical resilience of a reconstituted lamin B1 network. Quantitative rheological methods demonstrate that human lamin B1 filaments form stiff networks that can resist much greater deformations than those caused by microtubules impinging on the NE. Moreover, lamin B1 networks possess an elastic stiffness, which increases under tension, and an exceptional resilience against shear deformations. These results demonstrate that both mechanical tearing of the lamina and biochemical modification of lamin B1 filaments are required for NE breakdown.     

Ran及其结合与调控蛋白在核膜装配过程中的调控作用

核膜在细胞周期中呈现高度的动态性：在细胞分裂的前中期,核膜崩解并分散到细胞质中;在细胞分裂的后期,核膜开始在染色体的表面重新装配,最终形成完整的核膜结构。近期的研究发现,Ran GTP酶、物质转运蛋白importinβ、内层核膜蛋白LBR（lamin B receptor）以及核孔复合体蛋白nucleoporins在核膜重建的过程中起关键性调控作用,并受到细胞周期调控因子p34cdc2激酶的调节。LBR是一个八次跨膜的膜蛋白,主要定位于内层核膜。在细胞分裂的早期,随着核膜崩解,LBR与核膜崩解而生成的小膜泡一起分散到细胞质中;在细胞分裂的后期,通过LBR与importinβ相互结合,含有LBR的膜泡被importinβ携带至染色质的表面参与核膜重建。目前已知p34cdc2激酶对LBR与importinβ介导的核膜重建起重要调控作用。Nucleoporins是核孔复合体主要组分。随核膜崩解,核孔复合体解聚成nucleoporins,分散到细胞质中,或结合到其他亚细胞成分上。细胞分裂后期,核孔复合体伴随核膜装配而组装。     

Protein kinase C-beta II Is an apoptotic lamin kinase in polyomavirus-transformed, etoposide-treated pyF111 rat fibroblasts

The role of protein kinase C-beta(II) (PKC-beta(II)) in etoposide (VP-16)-induced apoptosis was studied using polyomavirus-transformed pyF111 rat fibroblasts in which PKC-beta(II) specific activity in the nuclear membrane (NM) doubled and the enzyme was cleaved into catalytic fragments. No PKC-beta(II) complexes with lamin B1 and/or active caspases were immunoprecipitable from the NM of proliferating untreated cells, but large complexes of PKC-beta(II) holoprotein and its catalytic fragments with lamin B1, active caspase-3 and -6, and inactive phospho-CDK-1, but not PKC-beta(I) or PKC-delta, could be immunoprecipitated from the NM of VP-16-treated cells, suggesting that PKC-beta(II) is an apoptotic lamin kinase. By 30 min after normal nuclei were mixed with cytoplasms from VP-16-treated, but not untreated, cells, PKC-beta(II) holoprotein had moved from the apoptotic cytoplasm to the normal NM, and lamin B1 was phosphorylated before cleavage by caspase-6. Lamin B1 phosphorylation was partly reduced, but its cleavage was completely prevented, despite the presence of active caspase-6, by adding a selective PKC-betas inhibitor, hispidin, to the apoptotic cytoplasms. Thus, a PKC-beta(II) response to VP-16 seems necessary for lamin B1 cleavage by caspase-6 and nuclear lamina dissolution in apoptosing pyF111 fibroblasts. The possibility of PKC-beta(II) being an apoptotic lamin kinase in these cells was further suggested by lamin B1-bound PKC-delta being inactive or only slightly active and by PKC-alpha not combining with the lamin.     

The apparent absence of lamin B1 and emerin in many tissue nuclei is due to epitope masking

Summary Immunolocalization studies have concluded that the nuclear membrane protein, emerin, is absent from many cell types and that lamin B1 is absent from adult heart and skeletal muscle. We now show that epitope masking in the nucleus is often responsible for failure to detect emerin and lamins in human, rat and pig tissues. Human heart cardiomyocyte nuclei were negative for lamin B1 using a commercial mAb, but were positive using two other lamin B1 antibodies, mAb8D1 and pAbB1-cbs. Rat hippocampal neuronal nuclei were immunostained by mAb8D1, but not pAbB1-cbs, while the commercial antibody stained only a subset. These data suggest that different regions of the lamin B1 molecule are masked in different tissues. Similarly, pig spleen had fewer emerin-positive nuclei than lung (5% vs. 32%), although their emerin content was similar by Western blotting. As mAbs against six epitopes gave the same result, the whole emerin molecule is either masked or redistributed in a subset of cells. Our findings argue that immunostaining evidence can be misleading for expression of nuclear envelope proteins. Problems with lamin B1 immunostaining can be avoided by using mAb8D1, but use of antibodies recognizing different epitopes may reveal cell-specific protein interactions in the nucleus.     

Mechanics in human fibroblasts and progeria: Lamin A mutation E145K results in stiffening of nuclei

The lamina is a filamentous meshwork beneath the inner nuclear membrane that confers mechanical stability to nuclei. The E145K mutation in lamin A causes Hutchinson‐Gilford progeria syndrome (HGPS). It affects lamin filament assembly and induces profound changes in the nuclear architecture. Expression of wild‐type and E145K lamin A in Xenopus oocytes followed by atomic force microscopy (AFM) probing of isolated oocyte nuclei has shown significant changes in the mechanical properties of the lamina. Nuclei of oocytes expressing E145K lamin A are stiffer than those expressing wild‐type lamin A. Here we present mechanical measurements by AFM on dermal fibroblasts obtained from a 4‐year‐old progeria patient bearing the E145K lamin A mutation and compared it to fibroblasts obtained from 2 healthy donors of 10 and 61 years of age, respectively. The abnormal shape of nuclei expressing E145K lamin A was analyzed by fluorescence microscopy. Lamina thickness was measured using electron micrographs. Fluorescence microscopy showed alterations in the actin network of progeria cells. AFM probing of whole dermal fibroblasts did not demonstrate significant differences in the elastic moduli of nuclear and cytoplasmic cell regions. In contrast, AFM measurements of isolated nuclei showed that nuclei of progeria and old person's cells are significantly stiffer than those of the young person, indicating that the process of aging, be it natural or abnormal, increases nuclear stiffness. Our results corroborate AFM data obtained using Xenopus oocyte nuclei and prove that the presence of E145K lamin A abnormally increases nuclear stiffness.     

The inner nuclear membrane protein p58 associates in vivo with a p58 kinase and the nuclear lamins.

p58, also referred to as the lamin B receptor, is an intrinsic protein of the inner nuclear membrane that binds in vitro to lamin B. Previous studies have demonstrated that p58 is phosphorylated in vivo and removal of its phosphate moieties affects lamin B binding. Using affinity-purified antipeptide antibodies, we have now immunoisolated p58 from bird erythrocyte lysates under isotonic, non-denaturing conditions. Analysis of the immunopurified material shows that five distinct proteins are tightly and specifically associated with p58. Two of these polypeptides can be identified as nuclear lamins A and B. The immunoisolate also contains a kinase activity that phosphorylates p58 in vivo and in vitro, exclusively at serine residues, as indicated by phosphoamino acid analysis and two-dimensional phosphopeptide mapping. Cell fractionation experiments and in vitro phosphorylation assays demonstrate that the p58 kinase resides in the nuclear envelope and is distinct from protein kinase A and cdc2 kinase, for both of which p58 is an in vitro substrate. These data suggest that p58 is interacting in vivo with a p58 kinase and the nuclear lamins.     

PARP-1, PARP-2 and ATM in the DNA damage response: functional synergy in mouse development

Poly(ADP-ribosyl)ation is an immediate DNA damage-dependent posttranslational modification of histones and nuclear proteins that contributes to the survival of injured proliferating cells. Poly(ADP-ribose) polymerases (PARPs) now constitute a superfamily of 18 proteins, encoded by different genes and displaying a common conserved catalytic domain. PARP-1 (113kDa), the founding member, and PARP-2 (62kDa) are both involved in DNA-break sensing and signaling when single strand break repair (SSBR) or base excision repair (BER) pathways are engaged. The generation by homologous recombination of deficient mouse models have confirmed the caretaker function of PARP-1 and PARP-2 in mammalian cells under genotoxic stress. This review summarizes our present knowledge on their physiological role in the cellular response to DNA damage and on the genetic interactions between PARP-1, PARP-2, Atm that play an essential role during early embryogenesis.