Unbiased proteomic screen for binding proteins to modified lysines on histone H3

We report a sensitive peptide pull‐down approach in combination with protein identification by LC‐MS/MS and qualitative abundance measurements by spectrum counting to identify proteins binding to histone H3 tail containing dimethyl lysine 4 (H3K4me2), dimethyl lysine 9 (H3K9me2), or acetyl lysine 9 (H3K9ac). Our study identified 86 nuclear proteins that associate with the histone H3 tail peptides examined, including seven known direct binders and 16 putative direct binders with conserved PHD finger, bromodomain, and WD40 domains. The reliability of our proteomic screen is supported by the fact that more than one‐third of the proteins identified were previously described to associate with histone H3 tail directly or indirectly. To our knowledge, the results presented here are the most comprehensive analysis of H3K4me2, H3K9me2, and H3K9ac associated proteins and will provide a useful resource for researchers studying the mechanisms of histone code effector proteins.     

Two complexes that contain histones are required for nucleosome assembly in vitro: role of nucleoplasmin and N1 in Xenopus egg extracts

The composition and function of histone storage complexes of Xenopus eggs have been investigated using monoclonal antibodies. We show that core histones are contained in two distinct complexes: H2A and H2B are associated with nucleoplasmin, and H3 and H4 are associated with nuclear protein N1. Immunodepletion analyses demonstrate that both complexes are required for nucleosome core assembly by extracts in vitro, the product being a simple sum of the histones from each complex. In addition, the majority of the stored H2A is shown to be an unusual form that migrates close to the position of H3 by SDS-polyacrylamide gel electrophoresis and resembles a variant synthesized in a cell-cycle-independent manner in mammalian cells.     

Identification of nuclear type II [(3)H]estradiol binding sites as histone H4

[3H]Luteolin binds covalently to uterine nuclear type II sites [B. Markaverich, K. Shoulars, M.A. Alejandro, T. Brown, Steroids 66 (2001) 707] and was used to identify this protein(s). SDS-PAGE analyses of [3H]luteolin-labeled type II site preparations revealed specific binding to 11- and 35-kDa proteins. The 11-kDa protein was identified as histone H4 by amino acid sequencing. Western blotting confirmed that the 11- and 35-kDa proteins were acetylated forms of histone H4. Anti-histone H4 antibodies (but not H2A, H2B, or H3 antibodies) quantitatively immunoadsorbed type II binding sites from nuclear extracts. Binding analyses by [3H]estradiol exchange, using luteolin as a competitor, detected specific type II binding activity to histone H4 (but not histones H2A, H2B, or H3) generated in a rabbit reticulocyte lysate translation system and confirmed that histone H4 is the type II site.     

Demonstration of exogenous nuclear histone H3 binding to mitochondria and subsequent cytochrome c release in cauliflower

The life cycle of a cell is partly regulated by the programmed cell death (PCD) process. From development to demise, a cell's PCD process must respond to external signals and internal factors mediated by mitochondria. Previous studies show that the release of histones into the cytosol caused by DNA damage or loss of nuclear integrity is correlated with apoptosis in mammalian cells. These released histones bind to mitochondria and permeabilize its inner and outer membranes, which causes the release of cytochrome c into the cytosol that leads to caspase activation and the demise of the cell. Owing to the high conservation of histones, we hypothesize that histone‐mediated cytochrome c release from mitochondria may be conserved across a wide range of eukaryotes. We investigated this histone–mitochondrial interaction in cauliflower using density‐gradient purified mitochondria and exogenous histones from a crude histone fraction, then added the exogenous histone fractions to the purified cauliflower mitochondria and analyzed the mitochondrial pellets and supernatants by immunoblotting against cytochrome c and H3. Our data clearly shows that histone‐enriched fractions elicited cytochrome c release from mitochondria, and that mitochondria bind exogenous histone H3.     

Antibodies to the N-terminus of calpactin II (p35) affect Ca2+ binding and phosphorylation by the epidermal growth factor receptor in vitro

Calpactins I and II are related 39-kilodalton (kDa) proteins that interact with phospholipids and actin in a calcium-dependent manner and are substrates of tyrosine protein kinases. They contain a short amino-terminal tail attached to a 36-kDa core domain. Monoclonal antibodies (Mabs) were raised to bovine calpactin II and used as site-specific probes of its structure and function. All of the antibodies reacted with native calpactin II and gave rise to a single band of 39 kDa among total cell protein displayed on Western blots. Most of the antibodies (9/14) reacted with determinants on the tail as shown by Western blots and competition with a synthetic tail peptide. Four antibodies reacted with determinants on the core and a 10-kDa tryptic fragment. Antibody-calpactin II complexes were tested for their ability to interact with lipid, actin, and Ca2+ and to serve as substrates of the epidermal growth factor (EGF) receptor tyrosine protein kinase. Whereas none of the antibodies had a detectable effect on actin binding, two anticore antibodies reduced calpactin's affinity for phospholipid. Ca2+-binding sites are known to reside within the core region, yet most antitail antibodies markedly increased the affinity of calpactin II for Ca2+, with four Ca2+-binding sites observed. Antitail antibodies either (i) abolished or (ii) greatly stimulated (10-fold) the phosphorylation of calpactin II by the EGF receptor. These results suggest that the interactions between calpactin II and Ca2+, phospholipid, or the EGF receptor are more complex than previously thought and can be modulated by interactions occurring in the tail.     

JMJD5 cleaves monomethylated histone H3 N‐tail under DNA damaging stress

The histone H3 N‐terminal protein domain (N‐tail) is regulated by multiple posttranslational modifications, including methylation, acetylation, phosphorylation, and by proteolytic cleavage. However, the mechanism underlying H3 N‐tail proteolytic cleavage is largely elusive. Here, we report that JMJD5, a Jumonji C (JmjC) domain‐containing protein, is a Cathepsin L‐type protease that mediates histone H3 N‐tail proteolytic cleavage under stress conditions that cause a DNA damage response. JMJD5 clips the H3 N‐tail at the carboxyl side of monomethyl‐lysine (Kme1) residues. In vitro H3 peptide digestion reveals that JMJD5 exclusively cleaves Kme1 H3 peptides, while little or no cleavage effect of JMJD5 on dimethyl‐lysine (Kme2), trimethyl‐lysine (Kme3), or unmethyl‐lysine (Kme0) H3 peptides is observed. Although H3 Kme1 peptides of K4, K9, K27, and K36 can all be cleaved by JMJD5 in vitro, K9 of H3 is the major cleavage site in vivo, and H3.3 is the major H3 target of JMJD5 cleavage. Cleavage is enhanced at gene promoters bound and repressed by JMJD5 suggesting a role for H3 N‐tail cleavage in gene expression regulation.     

Mitochondrial base excision repair of uracil and AP sites takes place by single-nucleotide insertion and long-patch DNA synthesis

Base excision repair (BER) corrects a variety of small base lesions in DNA. The UNG gene encodes both the nuclear (UNG2) and the mitochondrial (UNG1) forms of the human uracil-DNA glycosylase (UDG). We prepared mitochondrial extracts free of nuclear BER proteins from human cell lines. Using these extracts we show that UNG is the only detectable UDG in mitochondria, and mitochondrial BER (mtBER) of uracil and AP sites occur by both single-nucleotide insertion and long-patch repair DNA synthesis. Importantly, extracts of mitochondria carry out repair of modified AP sites which in nuclei occurs through long-patch BER. Such lesions may be rather prevalent in mitochondrial DNA because of its proximity to the electron transport chain, the primary site of production of reactive oxygen species. Furthermore, mitochondrial extracts remove 5' protruding flaps from DNA which can be formed during long-patch BER, by a "flap endonuclease like" activity, although flap endonuclease (FEN1) is not present in mitochondria. In conclusion, combined short- and long-patch BER activities enable mitochondria to repair a broader range of lesions in mtDNA than previously known.     

Destabilization of the outer and inner mitochondrial membranes by core and linker histones

Background

Extensive DNA damage leads to apoptosis. Histones play a central role in DNA damage sensing and may mediate signals of genotoxic damage to cytosolic effectors including mitochondria.

Methodology/Principal Findings

We have investigated the effects of histones on mitochondrial function and membrane integrity. We demonstrate that both linker histone H1 and core histones H2A, H2B, H3, and H4 bind strongly to isolated mitochondria. All histones caused a rapid and massive release of the pro-apoptotic intermembrane space proteins cytochrome c and Smac/Diablo, indicating that they permeabilize the outer mitochondrial membrane. In addition, linker histone H1, but not core histones, permeabilized the inner membrane with a collapse of the membrane potential, release of pyridine nucleotides, and mitochondrial fragmentation.

Conclusions

We conclude that histones destabilize the mitochondrial membranes, a mechanism that may convey genotoxic signals to mitochondria and promote apoptosis following DNA damage.     

An alternate form of Ku80 is required for DNA end-binding activity in mammalian mitochondria

Mammalian mitochondrial DNA end-binding activity is nearly indistinguishable from that of nuclear Ku. This observation led to the hypothesis that mitochondrial DNA end-binding activity is in part dependent upon Ku80 gene expression. To test this hypothesis, we assayed for Ku activity in mitochondrial extracts prepared from the xrs-5 hamster cell line that lacks Ku80 mRNA expression. Mitochondrial protein extracts prepared from this cell line lacked the DNA end-binding activity found in similar extracts prepared from wild-type cells. Azacytidine-reverted xrs-5 cells that acquired nuclear DNA end-binding activity also acquired mitochondrial DNA end-binding activity. Western blot analysis of human mitochondrial protein extracts using a monoclonal antibody specific for an N-terminal epitope of Ku80 identified a protein with an apparent molecular weight of 68 kDa. This mitochondrial protein was not detected by a monoclonal antibody specific for an epitope at the C-terminal end of Ku80. Consistently, while both the N- and C-terminal Ku80 monoclonal antibodies supershifted the nuclear DNA end-binding complex on an electrophoretic mobility shift assay, only the N-terminal monoclonal antibody supershifted the mitochondrial DNA end-binding complex. To confirm that the 68 kDa Ku protein was not a consequence of nuclear protein contamination of mitochondrial preparations, highly purified intact nuclei and mitochondria were treated with proteinase K which traverses the pores of intact nuclei but gains limited access into intact mitochondria. Ku80 in purified intact nuclei was sensitive to treatment with this protease, while the 68 kDa Ku protein characteristic of purified intact mitochondria was resistant. Further, immunocytochemical analysis revealed the co-localization of the N-terminal specific Ku80 monoclonal antibody with a mitochondrial-targeted green fluorescence protein. Mitochondrial localization of the C-terminal Ku80 monoclonal antibody was not observed. These data are consistent with the hypothesis that a C-terminally truncated form of Ku80 is localized in mammalian mitochondria where it functions in a DNA end-binding activity.     

Protein oxidation in the leaves and roots of cucumber plants (Cucumis sativus L.), mutant MSC16 and wild type

Reactive oxygen species (ROS) may cause irreversible carbonylation of proteins, resulting in structural and/or functional modifications. Carbonylated proteins were analyzed and compared in tissue extracts or purified mitochondria isolated from the leaves and roots of wild-type (WT) or MSC16 mutant cucumber plants. For analysis of the oxidized protein formation and degradation, several techniques were applied: Western blotting, quantitative, spectrophotometric assay of carbonyl concentration and protease activity measurements. Oxidized proteins were tagged with 2,4-dinitrophenylhydrazine (DNPH) and detected with anti-DNP antibodies. Western blots of 1D gels indicated that, in the leaves of both WT and MSC16 plants, certain oxidized proteins have chloroplastic origin. In MSC16 plants, protein oxidation is probably higher in chloroplasts than in mitochondria. Carbonyl concentration is similar in MSC16 and WT leaf extracts, but this may be the result of twice as high protease activity observed in MSC16 leaf extracts and indicates that chloroplastic proteases may effectively remove the oxidized proteins from chloroplasts. In mitochondria of both WT and MSC16 leaves, the levels of oxidized proteins and protease activity are similar. In MSC16 root extracts, the carbonyl concentration is lower and protease activity is similar as compared to WT plants. Nevertheless, in MSC16 root mitochondria, the 30% lower carbonyl concentration, lower band abundance for oxidized proteins and over 50% higher protease activity indicate that mitochondrial proteases are involved in degradation of the oxidatively damaged proteins. In matrix and membrane subfractions, the levels of oxidized proteins are similar in leaf mitochondria or lower in root mitochondria from MSC16 as compared to WT plants. The results show that the oxidized protein degradation network in MSC16 cucumber mutants is well developed, thus becoming a survival factor for plants with mitochondrial dysfunctions.     

Histone H1.2 is translocated to mitochondria and associates with Bak in bleomycin-induced apoptotic cells

Bleomycin induces single- and double-stranded breaks in DNA, with consequent mitochondrial membrane aberrations that lead to the apoptotic cell death. It is poorly understood how DNA damage-inducing apoptotic signals are transmitted to mitochondria, from which apoptotic factors are released into the cytoplasm. Here, we investigated the localization of histone H1.2 in the bleomycin-treated human squamous carcinoma SCCTF cells. The presence of DNA double-strand breaks in the bleomycin-treated cells was examined by Western analysis using antibody against phosphorylated histone H2AX (gamma-H2AX). Incubation of SCCTF cells for 48 h with 10 microM bleomycin induced apoptosis, as determined by cleavage of lamin B1 to 28 kDa fragment and DNA ladder formation. The mitochondrial permeabilization causing apoptotic feature was also detected with MitoCapture in the bleomycin-treated cells. Histone H1.2 was translocated from the nucleus to the mitochondria after treatment with bleomycin and co-localized with Bak in mitochondria. Our present results suggest that histone H1.2 plays an important role in transmitting apoptotic signals from the nucleus to the mitochondria following double-stranded breaks of DNA by bleomycin.     

TAT-mediated protein transduction and targeted delivery of fusion proteins into mitochondria of breast cancer cells

The protein transduction domain (PTD) from the HIV-1 TAT protein has been widely utilized to deliver biologically active macromolecules, including full-length proteins, into a variety of cell types in vitro and in vivo. Without additional targeting signals, the intracellular localization of the proteins delivered in this fashion appears to be cytoplasmic, nuclear or, as recently reported, endosomal. In this study, we show that the presence of the mitochondrial targeting signal (MTS) from hMnSOD on the N-terminus of TAT-fusion proteins directs them into mitochondria of breast cancer cells. We generated and purified fusion proteins containing GFP (MTS-GFP-TAT) or Exonuclease III (MTS-ExoIII-TAT) from Escherichia coli. The results of Western blots of subcellular fractions and fluorescent microscopic analyses revealed efficient protein transduction and mitochondrial localization of the fusion proteins. Specific exonuclease activity was found in the mitochondrial extracts isolated from MTS-ExoIII-TAT transduced cells. This increased exonuclease activity reduced the repair of mtDNA damage following oxidative stress. This diminished mtDNA repair led to a decrease in survival of breast cancer cells. Thus, the present study demonstrates the applicability of this new approach for intramitochondrial targeting of TAT-fusion proteins capable of modulating mitochondrial function and cell survival.     

A Distinct Switch in Interactions of the Histone H4 Tail Domain upon Salt-dependent Folding of Nucleosome Arrays

The core histone tail domains mediate inter-nucleosomal interactions that direct folding and condensation of nucleosome arrays into higher-order chromatin structures. The histone H4 tail domain facilitates inter-array interactions by contacting both the H2A/H2B acidic patch and DNA of neighboring nucleosomes (1, 2). Likewise, H4 tail-H2A contacts stabilize array folding (3). However, whether the H4 tail domains stabilize array folding via inter-nucleosomal interactions with the DNA of neighboring nucleosomes remains unclear. We utilized defined oligonucleosome arrays containing a single specialized nucleosome with a photo-inducible cross-linker in the N terminus of the H4 tail to characterize these interactions. We observed that the H4 tail participates exclusively in intra-array interactions with DNA in unfolded arrays. These interactions are diminished during array folding, yet no inter-nucleosome, intra-array H4 tail-DNA contacts are observed in condensed chromatin. However, we document contacts between the N terminus of the H4 tail and H2A. Installation of acetylation mimics known to disrupt H4-H2A surface interactions did not increase observance of H4-DNA inter-nucleosomal interactions. These results suggest the multiple functions of the H4 tail require targeted distinct interactions within condensed chromatin.