PTIP Regulates 53BP1 and SMC1 at the DNA Damage Sites

Although PTIP is implicated in the DNA damage response, through interactions with 53BP1, the function of PTIP in the DNA damage response remain elusive. Here, we show that RNF8 controls DNA damage-induced nuclear foci formation of PTIP, which in turn regulates 53BP1 localization to the DNA damage sites. In addition, SMC1, a substrate of ATM, could not be phosphorylated at the DNA damage sites in the absence of PTIP. The PTIP-dependent pathway is important for DNA double strand breaks repair and DNA damage-induced intra-S phase checkpoint activation. Taken together, these results suggest that the role of PTIP in the DNA damage response is downstream of RNF8 and upstream of 53BP1. Thus, PTIP regulates 53BP1-dependent signaling pathway following DNA damage.The DNA damage response pathways are signal transduction pathways with DNA damage sensors, mediators, and effectors, which are essential for maintaining genomic stability (1–3). Following DNA double strand breaks, histone H2AX at the DNA damage sites is rapidly phosphorylated by ATM/ATR/DNAPK (4–10), a family homologous to phosphoinositide 3-kinases (11, 12). Subsequently, phospho-H2AX (γH2AX) provides the platform for accumulation of a larger group of DNA damage response factors, such as MDC1, BRCA1, 53BP1, and the MRE11·RAD50·NBS1 complex (13, 14), at the DNA damage sites. Translocalization of these proteins to the DNA double strand breaks (DSBs)³ facilitates DNA damage checkpoint activation and enhances the efficiency of DNA damage repair (14, 15).Recently, PTIP (Pax2 transactivation domain-interacting protein, or Paxip) has been identified as a DNA damage response protein and is required for cell survival when exposed to ionizing radiation (IR) (1, 16–18). PTIP is a 1069-amino acid nuclear protein and has been originally identified in a yeast two-hybrid screening as a partner of Pax2 (19). Genetic deletion of the PTIP gene in mice leads to early embryonic lethality at embryonic day 8.5, suggesting that PTIP is essential for early embryonic development (20). Structurally, PTIP contains six tandem BRCT (BRCA1 carboxyl-terminal) domains (16–18, 21). The BRCT domain is a phospho-group binding domain that mediates protein-protein interactions (17, 22, 23). Interestingly, the BRCT domain has been found in a large number of proteins involved in the cellular response to DNA damages, such as BRCA1, MDC1, and 53BP1 (7, 24–29). Like other BRCT domain-containing proteins, upon exposure to IR, PTIP forms nuclear foci at the DSBs, which is dependent on its BRCT domains (16–18). By protein affinity purification, PTIP has been found in two large complexes. One includes the histone H3K4 methyltransferase ALR and its associated cofactors, the other contains DNA damage response proteins, including 53BP1 and SMC1 (30, 31). Further experiments have revealed that DNA damage enhances the interaction between PTIP and 53BP1 (18, 31).To elucidate the DNA damage response pathways, we have examined the upstream and downstream partners of PTIP. Here, we report that PTIP is downstream of RNF8 and upstream of 53BP1 in response to DNA damage. Moreover, PTIP and 53BP1 are required for the phospho-ATM association with the chromatin, which phosphorylates SMC1 at the DSBs. This PTIP-dependent pathway is involved in DSBs repair.     

Roles of Human AND-1 in Chromosome Transactions in S Phase

Coordinated execution of DNA replication, checkpoint activation, and postreplicative chromatid cohesion is intimately related to the replication fork machinery. Human AND-1/chromosome transmission fidelity 4 is localized adjacent to replication foci and is required for efficient DNA synthesis. In S phase, AND-1 is phosphorylated in response to replication arrest in a manner dependent on checkpoint kinase, ataxia telangiectasia-mutated, ataxia telangiectasia-mutated and Rad3-related protein, and Cdc7 kinase but not on Chk1. Depletion of AND-1 increases DNA damage, delays progression of S phase, leads to accumulation of late S and/or G₂ phase cells, and induces cell death in cancer cells. It also elevated UV-radioresistant DNA synthesis and caused premature recovery of replication after hydroxyurea arrest, indicating that lack of AND-1 compromises checkpoint activation. This may be partly due to the decreased levels of Chk1 protein in AND-1-depleted cells. Furthermore, AND-1 interacts with cohesin proteins Smc1, Smc3, and Rad21/Scc1, consistent with proposed roles of yeast counterparts of AND-1 in sister chromatid cohesion. Depletion of AND-1 leads to significant inhibition of homologous recombination repair of an I-SceI-driven double strand break. Based on these data, we propose that AND-1 coordinates multiple cellular events in S phase and G₂ phase, such as DNA replication, checkpoint activation, sister chromatid cohesion, and DNA damage repair, thus playing a pivotal role in maintenance of genome integrity.Replication fork is not only the site of DNA synthesis but also the center for coordinated execution of various chromosome transactions. The preparation for replication forks starts in the G₁ phase, when the prereplicative complex composed of origin recognition and minichromosome maintenance assembles on the chromosome. At the G₁-S boundary, Cdc45, GINS complex, and other factors join the prereplicative complex to generate a complex capable of initiating DNA replication. A series of phosphorylation events mediated by cyclin-dependent kinase and Cdc7 kinase play crucial roles in this process and facilitate the generation of active replication forks (1–6). Purification of the putative replisome complex in yeast indicated the presence of the checkpoint mediator Mrc1 and fork protection complex proteins Tof1 and Csm3 in the replication fork machinery (7), consistent with a previous report on the genome-wide analyses with chromatin immunoprecipitation analyses on chip (microarray) (8). Mcm10 is another factor present in the isolated complex, required for loading of replication protein A (RPA)² and primase-DNA polymerase α onto the replisome complex (7, 9, 10).Replication fork machinery can cope with various stresses, including shortage of the cellular nucleotide pool and replication fork blockages that interfere with its progression. Stalled replication forks activate checkpoint pathways, leading to cell cycle arrest, DNA repair, restart of DNA replication, or cell death in some cases (11–14). Single-stranded DNAs coated with RPA at the stalled replication forks are recognized by the ATR-ATR-interacting protein kinase complex and Rad17 for loading of the Rad9-Rad1-Hus1 checkpoint clamp (14–16). Factors present in the replisome complex are also known to be required for checkpoint activation. Claspin, Tim, and Tipin functionally and physically associate with sensor and effector kinases and serve as mediator/adaptors (17–23). Mcm7, a component of the replicative DNA helicase in eukaryotes, was reported to associate with the checkpoint clamp loader Rad17 (24) and to have a distinct function in checkpoint (24, 25). We recently reported that Cdc7 kinase, known to be required for DNA replication initiation, plays a role in activation of DNA replication checkpoint possibly through regulating Claspin phosphorylation (26). Thus, it appears that DNA replication and checkpoint activation functionally and physically interact with each other.Another crucial cellular event for maintenance of genome stability is sister chromatid cohesion. The cohesin complex, a conserved apparatus required for sister chromatid cohesion, contains Smc1, Smc3, and Rad21/Scc1/Mcd1 proteins. The assembled cohesin complexes are loaded onto chromatin prior to DNA replication in G₁ phase and link the sister chromosomes during S and G₂ phase until mitosis when they separate (27, 28). The mitotic cohesion defects are not rescued by supplementing cohesin in G₂ phase, and it has been suggested that establishment of sister chromatid cohesion is coupled with DNA replication (29, 30). Indeed, yeast mutants in some replisome components show defect in sister chromosome cohesion or undergo chromosome loss (31–33). Cdc7 kinase is also required for efficient mitotic chromosome cohesion (34, 35).Human AND-1 is the putative homolog of budding yeast CTF4/Pob1/CHL15 and fission yeast Mcl1/Slr3. The budding yeast counterpart was identified as a replisome component described above (7), which travels along with the replication fork (29). CTF4 is nonessential for viability, but its interactions with primase, Rad2 (FEN1 family of nuclease), and Dna2 have implicated CTF4 in lagging strand synthesis and/or Okazaki fragment processing (36–39). Yeast CTF4 and Mcl1 are involved in chromosome cohesion (33, 40, 41) and genetically interact with a cohesin, Mcd1/Rad21 (40, 42). Recently, it was reported that human AND-1 protein interacts with human primase-DNA polymerase α and Mcm10 and is required for DNA synthesis (43).Here we confirm that human AND-1 protein is required for DNA replication and efficient progression of S phase, and we further show that it facilitates replication checkpoint. Depletion of AND-1 causes accumulation of DNA damage and cell cycle arrest at late S to G₂ phase, ultimately leading to cell death. Furthermore, we also show that human AND-1 physically interacts with cohesin proteins Smc1, Smc3, Rad21/Scc1, suggesting a possibility that AND-1 may physically and functionally link replisome and cohesin complexes in vivo. Recent studies indicate that sister chromatid cohesion is required for recombinational DNA repair (44–47). Thus, we examined the requirement of AND-1 for repair of artificially induced double-stranded DNA breaks and showed that AND-1 depletion leads to significant reduction of the double strand break repair. Possible roles of AND-1 in coordination of various chromosome transactions at a replication fork and in maintenance of genome integrity during S phase will be discussed.     

APH1 Polar Transmembrane Residues Regulate the Assembly and Activity of Presenilin Complexes

Complexes involved in the γ/ϵ-secretase-regulated intramembranous proteolysis of substrates such as the amyloid-β precursor protein are composed primarily of presenilin (PS1 or PS2), nicastrin, anterior pharynx defective-1 (APH1), and PEN2. The presenilin aspartyl residues form the catalytic site, and similar potentially functional polar transmembrane residues in APH1 have been identified. Substitution of charged (E84A, R87A) or polar (Q83A) residues in TM3 had no effect on complex assembly or activity. In contrast, changes to either of two highly conserved histidines (H171A, H197A) located in TM5 and TM6 negatively affected PS1 cleavage and altered binding to other secretase components, resulting in decreased amyloid generating activity. Charge replacement with His-to-Lys substitutions rescued nicastrin maturation and PS1 endoproteolysis leading to assembly of the formation of structurally normal but proteolytically inactive γ-secretase complexes. Substitution with a negatively charged side chain (His-to-Asp) or altering the structural location of the histidines also disrupted γ-secretase binding and abolished functionality of APH1. These results suggest that the conserved transmembrane histidine residues contribute to APH1 function and can affect presenilin catalytic activity.The anterior pharynx defective-1 (APH1)⁵ protein is an essential component of presenilin-dependent complexes required for the γ/ϵ-secretase activity (1). The multicomponent γ-secretase is responsible for the intramembrane proteolysis of a variety of substrates including the amyloid-β precursor protein (APP) and Notch receptor. Notch signaling is involved in a variety of important cell fate decisions during embryogenesis and adulthood (2). The γ/ϵ-secretase cleavage of APP protein is related to the pathogenesis of Alzheimer disease by releasing the 4-kDa amyloid β-peptide (Aβ) which accumulates as senile plaques in patients with Alzheimer disease (3, 4).The γ-complexes are composed of multispanning transmembrane proteins that include APH1 (5, 6), presenilin (PS1 or PS2) (7–10), PEN2 (5), and the type 1 transmembrane nicastrin (NCT) (11). All four components are essential for proteolytic activity, and loss of any single component destabilizes the complex, resulting in the loss of substrate cleavage. Conversely, co-expression of all four components increases γ-secretase activity (12–14). During the maturation of the complexes, presenilins undergo an endoproteolytic cleavage to generate amino- and carboxyl-terminal fragments which remain associated as heterodimers in the active high molecular weight complexes (15–18). Although the exact function of presenilins has been debated (19, 20), it has been proposed that the presenilins are aspartyl proteases with two transmembrane residues constituting the catalytic subunit (21). Analogous aspartyl catalytic dyads are found in the signal peptide peptidases (21, 22). Contributions from the other components are under investigation, and it has been shown, for example, that the large ectodomain of NCT plays a key role in substrate recognition (23, 24). It has also been shown that other proteins can regulate activity such as TMP21, a member of p24 cargo protein, which binds to the presenilin complexes and selectively modulates γ but not ϵ cleavage (25, 26).APH1 is a seven-transmembrane protein with a topology such that the amino terminus is oriented with the endoplasmic reticulum and the carboxyl terminus resides in the cytoplasm (6, 27). It is also expressed as different isoforms encoded by two genes in humans (APH1a on chromosome 1; APH1b on chromosome 15) or three genes in rodents (APH1a on chromosome 3; APH1b and APH1c on chromosome 9). APH1a has 55% sequence similarity with APH1b/APH1c, whereas APH1b and APH1c share 95% similarity. In addition to these different genes, APH1a is alternatively spliced to generate a short (APH1aS) and a long isoform (APH1aL). These two isoforms differ by the addition of 18 residues on the carboxyl-terminal part of APH1aL (28, 29). Deletion of APH1a in mice is embryonically lethal and is associated with developmental and patterning defects similar to those found in Notch, NCT, or PS1 null embryos (30, 31). In contrast to the essential nature of APH1a, the combined APH1b/c-deficient mice survive into adulthood (31). This suggests that APH1a is the major homologue involved in presenilin-dependent function during embryonic development. In addition, these different APH1 variants are constituents of distinct, proteolytically active presenilin-containing complexes and may, therefore, make unique contributions to γ-secretase activity (30–32).Despite their importance to complex formation and function, the exact role of the APH1 isoforms in presenilin-dependent γ/ϵ-secretase activity remains under investigation. In the current study, several highly conserved polar and charged residues located within the transmembrane domains of APH1 were identified. Mutagenesis of two conserved histidine residues embedded in TM5 and TM6 (His-171 and His-197) lead to alterations in γ-secretase complex maturation and activity. The histidine residues contribute to APH1 function and are involved in stabilizing interactions with other γ-secretase components. These key histidines may also be physically localized near the presenilin active site and involved in the γ-secretase activity as shown by the decreased activity of γ-secretase complexes that are assembled with the His-mutants.     

Broad Coverage Identification of Multiple Proteolytic Cleavage Site Sequences in Complex High Molecular Weight Proteins Using Quantitative Proteomics as a Complement to Edman Sequencing

Proteolytic processing modifies the pleiotropic functions of many large, complex, and modular proteins and can generate cleavage products with new biological activity. The identification of exact proteolytic cleavage sites in the extracellular matrix laminins, fibronectin, and other extracellular matrix proteins is not only important for understanding protein turnover but is needed for the identification of new bioactive cleavage products. Several such products have recently been recognized that are suggested to play important cellular regulatory roles in processes, including angiogenesis. However, identifying multiple cleavage sites in extracellular matrix proteins and other large proteins is challenging as N-terminal Edman sequencing of multiple and often closely spaced cleavage fragments on SDS-PAGE gels is difficult, thus limiting throughput and coverage. We developed a new liquid chromatography-mass spectrometry approach we call amino-terminal oriented mass spectrometry of substrates (ATOMS) for the N-terminal identification of protein cleavage fragments in solution. ATOMS utilizes efficient and low cost dimethylation isotopic labeling of original N-terminal and proteolytically generated N termini of protein cleavage fragments followed by quantitative tandem mass spectrometry analysis. Being a peptide-centric approach, ATOMS is not dependent on the SDS-PAGE resolution limits for protein fragments of similar mass. We demonstrate that ATOMS reliably identifies multiple proteolytic sites per reaction in complex proteins. Fifty-five neutrophil elastase cleavage sites were identified in laminin-1 and fibronectin-1 with 34 more identified by matrix metalloproteinase cleavage. Hence, our degradomics approach offers a complimentary alternative to Edman sequencing with broad applicability in identifying N termini such as cleavage sites in complex high molecular weight extracellular matrix proteins after in vitro cleavage assays. ATOMS can therefore be useful in identifying new cleavage products of extracellular matrix proteins cleaved by proteases in pathology for bioactivity screening.Recently, considerable efforts have been deployed to develop high throughput proteomic screens to identify protease substrates in complex biological samples (1–8). Validation of substrates identified by these approaches or identification of cleavage sites by in vitro incubation of candidate substrates with the protease of interest is generally performed by SDS-PAGE analysis and Edman degradation and sequencing. However, the complexity of large modular proteins renders Edman sequencing of proteolytic fragments difficult to apply because each of the numerous proteolytic fragments should be analyzed separately, and high coverage of cleavage sites is rarely attained (9). Cleavage site identification after protein degradation is also very difficult for small peptide products less than 4 kDa. Consequently, the precise cleavage sites in complex extracellular matrix proteins such as laminin and fibronectin by important tissue and inflammatory cell proteases such as the matrix metalloproteinases (MMPs)1 and neutrophil elastase are mostly unknown.These limitations of Edman sequencing are problematic in the study of tissue remodeling and proteolysis in pathology. Neutrophil elastase and several MMPs such as MMP2, MMP8, and MMP9 play key roles in inflammation (10, 11), tissue healing (12, 13), and carcinogenesis (14, 15) and are well known for degrading extracellular matrix proteins (16). More recently, signaling functions for MMPs are increasingly recognized as one of their most important roles by the precise processing of cytokines or their binding proteins (17). In addition, several important examples are now known of cryptic binding sites being exposed after precise protein cleavage or new proteins termed neoproteins (18) being released upon limited cleavage of extracellular matrix proteins and having completely different functions compared with their parent molecule, including several with importance in angiogenesis (19–25). Many such sites or neoproteins are generated by inflammatory proteases or proteases of the coagulation and fibrinolysis systems (24, 25), and this is a burgeoning field of discovery that is often hampered by difficulties in their N-terminal sequencing.In light of this limitation, we developed, validated, and used a new method for targeted and simultaneous N-terminal sequencing of one or a small number of protein N termini or cleavage products we call amino-terminal oriented mass spectrometry of substrates (ATOMS). We applied ATOMS for the analysis of cleavage sites generated in laminin-1 and fibronectin-1 by neutrophil elastase and neutrophil and tissue MMPs. Laminin-1 (LM-111), a trimeric glycoprotein composed of the α1, β1, and γ1 chains, is ubiquitously expressed in epithelium and endothelium. Proteolytic processing of laminins greatly affects cellular behavior and is also implicated in cancer cell migration (20, 26–29). Another important extracellular matrix protein is plasma fibronectin (also known as fibronectin isoform 1) and its cellular isoforms, which are homodimers linked by a disulfide bridge at the C terminus (30) that are important for cell adhesion and intracellular signaling (31–34). Fibronectin is susceptible to proteolysis (35, 36), which affects its biological functions (37–39). However, the cleavage sites within these two molecules by inflammatory MMPs and neutrophil elastase are largely unknown. Here we identified a total of 55 neutrophil elastase cleavage sites in LM-111 and fibronectin-1 and 34 MMP cleavage sites, demonstrating the capacity of ATOMS to identify multiple N-terminal sequences in solution. ATOMS also outperformed N-terminal Edman sequencing with 50% more cleavage sites identified by ATOMS, representing a significant advance in N-terminal sequencing technology. The utility of the method is broadly applicable for the analysis of multiple cleavages in other very large molecules and so offers great potential to accurately identify and rapidly sequence multiple cryptic bioactive protein fragments liberated following proteolytic processing.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

The Herpes Simplex Virus Type 1 Cleavage/Packaging Protein,UL32, Is Involved in Efficient Localization of Capsids to Replication Compartments

Six genes, including UL32, have been implicated in the cleavage and packaging of herpesvirus DNA into preassembled capsids. We have isolated a UL32 insertion mutant which is capable of near-wild-type levels of viral DNA synthesis; however, the mutant virus is unable to cleave and package viral DNA, consistent with the phenotype of a previously isolated temperature-sensitive herpes simplex virus type 1 mutant, tsN20 (P. A. Schaffer, G. M. Aron, N. Biswal, and M. Benyesh-Melnick, Virology 52:57–71, 1973). A polyclonal antibody which recognizes UL32 was previously used by Chang et al. (Y. E. Chang, A. P. Poon, and B. Roizman, J. Virol. 70:3938–3946, 1996) to demonstrate that UL32 accumulates predominantly in the cytoplasm of infected cells. In this report, a functional epitope-tagged version of UL32 showed that while UL32 is predominantly cytoplasmic, some nuclear staining which colocalizes with the major DNA binding protein (ICP8, UL29) in replication compartments can be detected. We have also used a monoclonal antibody (5C) specific for the hexon form of major capsid protein VP5 to study the distribution of capsids during infection. In cells infected with wild-type KOS (6 and 8 h postinfection), 5C staining patterns indicate that capsids are present in nuclei within replication compartments. These results suggest that cleavage and packaging occur in replication compartments at least at 6 and 8 h postinfection. Cells infected with the UL32 mutant exhibit a hexon staining pattern which is more diffusely distributed throughout the nucleus and which is not restricted to replication compartments. We propose that UL32 may play a role in “bringing” preassembled capsids to the sites of DNA packaging and that the failure to localize to replication compartments may explain the cleavage/packaging defect exhibited by this mutant. These results suggest that the UL32 protein is required at a step distinct from those at which other cleavage and packaging proteins are required and may be involved in the correct localization of capsids within infected cells.During infection of cells with herpes simplex virus type 1 (HSV-1), the large concatemeric products of DNA replication are cleaved to unit length and packaged into preassembled capsids. Capsids are icosahedral structures composed of 150 hexons and 12 pentons. Three types of capsids (A, B, and C) can be isolated from infected cells by velocity centrifugation (20). C capsids contain the viral DNA genome; B capsids contain the scaffolding protein; and A capsids contain neither DNA nor the scaffolding protein. Pulse chase experiments with another alphaherpesvirus, equine herpesvirus 1, indicate that at least some B capsids can package DNA and mature into infectious virions, while A capsids cannot (46). By analogy with the bacteriophages, these results suggest that B capsids represent procapsids which are intermediates in the packaging process. However, a new intermediate in the assembly process has recently been identified (41, 62). These newly identified capsid forms observed in in vitro assembly extracts have the same protein content as B capsids but are more spherical; these capsids are unstable and adopt the more angular form characteristic of B capsids after prolonged incubation in vitro. These results suggest that the unstable spherical forms may represent the true procapsid intermediate (41, 62).In many bacteriophages, the procapsid contains at least three essential components: an icosahedrally arranged protein shell, an internal scaffold, and a dodecameric ring called the portal vertex through or around which the phage DNA is taken up (8, 11, 18). For HSV-1, the outer shell is composed of four proteins: the major capsid protein, VP5; a small protein bound to hexons, VP26; and a triplex structure made up of heterotrimers of VP19C and VP23 (reviewed in reference 56). VP24, VP21, and VP22a are found in the interior of the capsid and are encoded by overlapping genes UL26 and UL26.5; VP21 and VP22a are present in B but not A or C capsids and are considered to make up the internal scaffold (reviewed in reference 56). Although bacteriophages contain a portal vertex, no such structure has been observed in HSV-1 capsids. Whether the herpesviruses have a unique portal vertex through which viral DNA is taken up is unclear; it is possible that this type of unique vertex is only needed in viruses which have a tail. Capsids indistinguishable from those isolated from HSV-1-infected cells have been observed in extracts from insect cells infected with recombinant baculoviruses bearing HSV-1 capsid genes (42, 60). Therefore, it is clear that these proteins are sufficient for capsid assembly in vitro; however, it is not known whether capsids formed in vitro are competent for DNA uptake. It is possible that minor components of capsids play important roles in genome encapsidation.In addition to the capsid proteins, at least six genes are essential for the encapsidation of viral DNA: the UL6, UL15, UL25, UL28, UL32, and UL33 genes. Temperature-sensitive (ts) strains with mutations in these genes have similar phenotypes, in that viral DNA can be replicated but not cleaved and packaged (1, 2, 4, 6, 48, 51, 54, 55, 66). Strains with null mutations in the UL6, UL15, UL25, UL28, and UL33 genes have been isolated and characterized, thereby confirming the roles of these genes in cleavage and packaging (5, 27, 37, 45, 59, 68). Despite the identification of these required genes, the mechanism by which viral DNA is cleaved and packaged is not understood, nor has the role of any of the gene products been determined. The UL6 and UL25 proteins have been detected in A, B, and C capsids as well as in virions (3, 28, 37, 44); however, the precise role of these two proteins in capsids remains to be determined.A ts UL32 mutant, tsN20, defective in cleavage and packaging, has been reported previously (51). Because mutants with lesions resulting in temperature sensitivity are often prone to problems associated with incomplete penetrance at the nonpermissive temperature, we isolated a UL32 insertion mutant, hr64. Characterization of hr64 confirms that UL32 is essential for cleavage and packaging. Previous studies demonstrated that UL32 localizes to the cytoplasm of infected cells (13). We have used a functional epitope-tagged version of UL32 to confirm that in infected cells, this protein is mainly cytoplasmic, although some nuclear staining was observed.HSV-1 DNA replication occurs in globular nuclear domains termed “replication compartments” initially identified by ICP8 (UL29) staining patterns in an immunofluorescence assay (49). All seven replication proteins have now been localized within replication compartments (10, 24, 29–31, 43) as has regulatory protein ICP4 (26, 50). Ward et al. have recently reported that at late times after infection (18 h), capsids accumulate in the nucleus in regions distinct from replication compartments (64). These authors suggest that these regions represent assembly stations in which DNA is packaged. We report herein, however, that at 6 and 8 h postinfection, capsids colocalize with ICP8 in replication compartments. This suggests that at these early times, cleavage and packaging occur within replication compartments. Furthermore, we report that in cells infected with the UL32 mutant virus, capsids are distributed throughout the nucleus, accumulating in regions outside the replication compartments. This suggests that UL32 may play a role in the efficient localization of capsids in infected cells.     

Proteome-wide Substrate Analysis Indicates Substrate Exclusion as a Mechanism to Generate Caspase-7 Versus Caspase-3 Specificity

Caspase-3 and -7 are considered functionally redundant proteases with similar proteolytic specificities. We performed a proteome-wide screen on a mouse macrophage lysate using the N-terminal combined fractional diagonal chromatography technology and identified 46 shared, three caspase-3-specific, and six caspase-7-specific cleavage sites. Further analysis of these cleavage sites and substitution mutation experiments revealed that for certain cleavage sites a lysine at the P5 position contributes to the discrimination between caspase-7 and -3 specificity. One of the caspase-7-specific substrates, the 40 S ribosomal protein S18, was studied in detail. The RPS18-derived P6–P5′ undecapeptide retained complete specificity for caspase-7. The corresponding P6–P1 hexapeptide still displayed caspase-7 preference but lost strict specificity, suggesting that P′ residues are additionally required for caspase-7-specific cleavage. Analysis of truncated peptide mutants revealed that in the case of RPS18 the P4–P1 residues constitute the core cleavage site but that P6, P5, P2′, and P3′ residues critically contribute to caspase-7 specificity. Interestingly, specific cleavage by caspase-7 relies on excluding recognition by caspase-3 and not on increasing binding for caspase-7.Caspases, a family of evolutionarily conserved proteases, mediate apoptosis, inflammation, proliferation, and differentiation by cleaving many cellular substrates (1–3). The apoptotic initiator caspases (caspase-8, -9, and -10) are activated in large signaling platforms and propagate the death signal by cleavage-induced activation of executioner caspase-3 and -7 (4, 5). Most of the cleavage events occurring during apoptosis have been attributed to the proteolytic activity of these two executioner caspases, which can act on several hundreds of proteins (2, 3, 6, 7). The substrate degradomes of the two main executioner caspases have not been determined but their identification is important to gaining greater insight in their cleavage specificity and biological functions.The specificity of caspases was rigorously profiled by using combinatorial tetrapeptide libraries (8), proteome-derived peptide libraries (9), and sets of individual peptide substrates (10, 11). The results of these studies indicate that specificity motifs for caspase-3 and -7 are nearly indistinguishable with the canonical peptide substrate, DEVD, used to monitor the enzymatic activity of both caspase-3 and -7 in biological samples. This overlap in cleavage specificity is manifested in their generation of similar cleavage fragments from a variety of apoptosis-related substrates such as inhibitor of caspase-activated DNase, keratin 18, PARP,1 protein-disulfide isomerase, and Rho kinase I (for reviews, see Refs. 2, 3, and 7). This propagated the view that these two caspases have completely redundant functions during apoptosis. Surprisingly, mice deficient in one of these caspases (as well as mice deficient in both) have distinct phenotypes. Depending on the genetic background of the mice, caspase-3-deficient mice either die before birth (129/SvJ) or develop almost normally (C57BL/6J) (12–14). This suggests that dynamics in the genetic background, such as increased caspase-7 expression, compensate for the functional loss of caspase-3 (15). In the C57BL/6J background, caspase-7 single deficient mice are also viable, whereas caspase-3 and -7 double deficient mice die as embryos, further suggesting redundancy (12–14). However, because caspase-3 and -7 probably arose from gene duplication between the Cephalochordata-Vertebrata diversion (16), they might have acquired different substrate specificities during evolution. Caspase-3 and -7 do exhibit different activities on a few arbitrarily identified natural substrates, including BID, X-linked inhibitor of apoptosis protein, gelsolin, caspase-6, ataxin-7, and co-chaperone p23 (17–20). In addition, caspase-3 generally cleaves more substrates during apoptosis than caspase-7 and therefore appears to be the major executioner caspase. Moreover, a recent report describing caspase-1-dependent activation of caspase-7, but not of caspase-3, in macrophages in response to microbial stimuli supports the idea of a non-redundant function for caspase-7 downstream of caspase-1 (21).Commercially available “caspase-specific” tetrapeptide substrates are widely used for specific caspase detection, but they display substantial promiscuity and cannot be used to monitor individual caspases in cells (22, 23). Detecting proteolysis by measuring the release of C-terminal fluorophores, such as 7-amino-4-methylcoumarin (amc), restricts the specificity of these peptide substrates to non-prime cleavage site residues, which may have hampered the identification of specific cleavage events. To address this limitation, a recently developed proteomics technique, called proteomic identification of protease cleavage sites, was used to map both non-prime and prime preferences for caspase-3 and -7 on a tryptic peptide library (9). However, no clear distinction in peptide recognition motifs between caspase-3 and -7 could be observed (9). Because not all classical caspase cleavage sites are processed (7), structural or post-translational higher order constraints are likely involved in steering the cleavage site selectivity. Peptide-based approaches generally overlook such aspects.We made use of the COFRADIC N-terminal peptide sorting methodology (24–26) to profile proteolytic events of caspase-3 and -7 in a macrophage proteome labeled by triple stable isotope labeling by amino acids in cell culture (SILAC), which allowed direct comparison of peak intensities in peptide MS spectra and consequent quantification of N termini that are equally, preferably, or exclusively generated by the action of caspase-3 or -7 (26, 27). We identified 55 cleavage sites in 48 protein substrates, encompassing mutual, preferred, and unique caspase-3 and -7 cleavage sites.