Quantitative Analysis of Yeast Checkpoint Protein Kinase Activity by Combined Mass Spectrometry Enzyme Assays.

Virtually all eukaryotic cell functions and signaling pathways are regulated by protein phosphorylation. The Rad53 kinase plays crucial roles in the DNA damage response in Saccharomyces cerevisiae and is widely used as a surrogate marker for DNA damage checkpoint activation by diverse genotoxic agents. Most currently available assays for Rad53 activation are based on either electrophoretic mobility shifts or semiquantitative in situ autophosphorylation activity on protein blots. Here, we describe direct quantitative measures to assess Rad53 activity using immunoprecipitation kinase assays and quantitative mass spectrometric analysis of Rad53 activation loop autophosphorylation states. Both assays employ a highly specific Rad53 antibody, and thus enable the analysis of the untagged endogenous protein under physiological conditions. The principles of these assays are readily transferable to other protein kinases for which immunoprecipitation-grade antibodies are available, and thus potentially applicable to a wide range of eukaryotic signaling pathways beyond yeast.

Aiolos collaborates with Blimp-1 to regulate the survival of multiple myeloma cells.

The transcriptional repressor B lymphocyte-induced maturation protein-1 (Blimp-1) has crucial roles in the control of plasma cell differentiation and in maintaining survival of plasma cells. However, how Blimp-1 ensures the survival of plasma cell malignancy, multiple myeloma (MM), has remained elusive. Here we identified Aiolos, an anti-apoptotic transcription factor of MM cells, as a Blimp-1-interacting protein by mass spectrometry. ChIP coupled with DNA microarray was used to profile the global binding of Aiolos and Blimp-1 to endogenous targets in MM cells, which revealed their co-binding to a large number of genes, including apoptosis-related genes. Accordingly, Blimp-1 and Aiolos regulate similar transcriptomes in MM cells. Analysis of the binding motifs for Blimp-1 and Aiolos uncovered a partial motif that was similar across sites for both proteins. Aiolos promotes the binding of Blimp-1 to target genes and thereby enhances Blimp-1-dependent transcriptional repression. Furthermore, treatment with an anti-MM agent, lenalidomide, caused ubiquitination and proteasomal degradation of Blimp-1, leading to the de-repression of a new Blimp-1 direct target, CULLIN 4A (CUL4A), and reduced Aiolos levels. Accordingly, lenalidomide-induced cell death was partially rescued by reintroduction of Blimp-1 or knockdown of CUL4A. Thus, we demonstrated the functional impacts and underlying mechanisms of the interaction between Aiolos and Blimp-1 in maintaining MM cell survival. We also showed that interruption of Blimp-1/Aiolos regulatory pathways contributes to lenalidomide-mediated anti-MM activity.

PHRF1 promotes genome integrity by modulating non-homologous end-joining.

Methylated histone readers are critical for chromatin dynamics, transcription, and DNA repair. Human PHRF1 contains a plant homeodomain (PHD) that recognizes methylated histones and a RING domain, which ubiquitinates substrates. A recent study reveals that PHRF1 is a tumor suppressor that promotes TGF-ß cytostatic signaling through TGIF ubiquitination. Also, PHRF1 is a putative phosphorylation substrate of ataxia telangiectasia-mutated/ataxia telangiectasia and Rad3-related kinases; however, the role of PHRF1 in DNA damage response is unclear. Here we report a novel function of PHRF1 in modulating non-homologous end-joining (NHEJ). PHRF1 quickly localizes to DNA damage lesions upon genotoxic insults. Ablation of PHRF1 decreases the efficiency of plasmid-based end-joining, whereas PHRF1 overexpression leads to an elevated NHEJ in H1299 reporter cells. Immunoprecipitation and peptide pull-down assays verify that PHRF1 constitutively binds to di- and trimethylated histone H3 lysine 36 (H3K36) (H3K36me2 and H3K36me3) via its PHD domain. Substitution of S915DT917E to ADAE in PHRF1 decreases its affinity for NBS1. Both PHD domain and SDTE motif are required for its NHEJ-promoting activity. Furthermore, PHRF1 mediates PARP1 polyubiquitination for proteasomal degradation. These results suggest that PHRF1 may combine with H3K36 methylation and NBS1 to promote NHEJ and stabilize genomic integrity upon DNA damage insults.

CC-chemokine ligand 18/pulmonary activation-regulated chemokine expression in the CNS with special reference to traumatic brain injuries and neoplastic disorders.

Pulmonary activation-regulated chemokine (PARC) now designated CC-chemokine ligand 18 (CCL18) has been shown to play a significant role in the pathogenesis of various tissue injuries and diseases in a proinflammatory or immune suppressive way to limit or support the inflammation or disease. While much is known about the roles of CCL18/PARC in non-neural tissues, its expression in the CNS has remained largely unexplored and controversial. Using reverse transcription polymerase chain reaction (RT-PCR) and double immunohistochemical staining, we analyzed the expression of CCL18/PARC in the human brain with special reference to traumatic brain injuries and tumors. The RT-PCR analysis revealed the expression of CCL18/PARC mRNA both in the traumatic brain and glioma tissues examined. Immunoexpression of CCL18/PARC protein was consistently detected in all cases of traumatic brain injuries examined by immunohistochemical staining. Double immunofluorescence labeling has extended the study that CCL18/PARC positive cells were macrophages/microglia, astrocytes or neurons. The CCL18/PARC expression was localized in macrophage-like cells in two of eight glioblastoma tissues whose cancer cells were CCL18/PARC negative. Unexpectedly, CCL18/PARC mRNA weakly and constitutively expressed by glioblastoma cell line was upregulated after endotoxin stimulation. The present results indicated a significant production of CCL18/PARC in different CNS traumatic and neoplasm tissues by specific cellular elements expressing the chemokine. An anti-inflammatory mechanism jointly exerted by these cells via CCL18/PARC may be involved in the CNS immunity after traumatic injury and tumorigenesis.

Evaluation of optimal cerebral perfusion pressure in severe traumatic brain injury.

Traumatic brain injury (TBI) is a major cause of death and disability. In the 2000 guidelines, one of the suggestions for TBI treatment was to maintain cerebral perfusion pressure (CPP) < or = 70 mmHg. But in the 2003 guidelines, the suggestion was changed to < or = 60 mmHg. There have been some discrepancies of opinions about this recommendation in recent publications. In this study, we retrospectively reviewed 305 severe TBI (STBI) patients with Glasgow Coma Scales (GCS) < or = 8 between January 1, 2002 and March 31, 2003. The study group was stratified according to use or nonuse of intracranial pressure (ICP) monitoring, ICP levels, ages, and GCS levels in order to test the correlation between CCP and the prognosis. The patients < 50-year-old, with higher GCS level, with ICP monitoring, and with ICP levels < 20 mmHg had lower mortality rates and better prognosis (GOS) (p < 0.05 or 0.001). The patients in the GCS 3-5 subgroup had a significantly lower mortality and better prognosis if the CPP value was maintained higher than 70 mmHg (p < 0.05) The optimal CPP maintained < or = 60 mmHg did not fit in all STBI patients. Our study concludes that it is critical to maintain CPP substantially higher in lower GCS level patients.

Human p16gamma, a novel transcriptional variant of p16(INK4A), coexpresses with p16(INK4A) in cancer cells and inhibits cell-cycle progression.

The INK4A locus encodes two tumor suppressor genes, p16(INK4A) and p14(ARF), transcribed using alternative exons 1alpha or 1beta spliced onto the same exons 2 and 3. Both p16(INK4A) and p14(ARF) are capable of inhibiting the cell-cycle progression, albeit in different manner; p16(INK4A) is phosphorylation of retinoblastoma (pRB) dependent while p14(ARF) is p53-dependent. In this study, we report the discovery of a novel variant of p16(INK4A), termed p16gamma, in a primary T-cell acute lymphoblastic leukemia (T-ALL) patient sample and a neuroblastoma cell line, which was expressed at both the transcriptional and translational levels. Cloning and sequencing of the p16gamma cDNA revealed that p16gamma was identical to p16(INK4A), except that it contained an in-frame insertion of 197 bp between exons 2 and 3. p16gamma expression was detected in the majority of p16(INK4A)-expressing primary T-ALL and B-ALL patient samples and other p16(INK4A)-expressing tumor samples, but was only barely detectable in some normal mononuclear cells and other non-tumor samples. Structural analysis by nuclear magnetic resonance and circular dichroism confirmed that p16gamma, like p16(INK4A), is also an ankyrin-repeat protein. Functional analysis of p16gamma revealed that p16gamma protein interacted with cyclin D-dependent kinase4 and inhibited its kinase activity. Using a luciferase reporter assay, the transfection of p16gamma repressed the E2F response, the downstream target of pRB, with an efficacy equivalent to that of p16(INK4A). Moreover p16gamma, like p16(INK4A), induced cell-cycle arrest at G(0)/G(1), and inhibited cell growth in colony formation assay.

Suggestive evidence for the involvement of the second calcium and surface loop in interfacial binding: monoclinic and trigonal crystal structures of a quadruple mutant of phospholipase A2.

The crystal structures of the monoclinic and trigonal forms of the quadruple mutant K53,56,120,121M of recombinant bovine pancreatic phospholipase A2 (PLA2) have been solved and refined at 1.9 and 1.1 A resolution, respectively. Interestingly, the monoclinic form reveals the presence of the second calcium ion. Furthermore, the surface-loop residues are ordered and the conformation of residues 62-66 is similar to that observed in other structures containing the second calcium ion. On the other hand, in the trigonal form the surface loop is disordered and the second calcium is absent. Docking studies suggest that the second calcium and residues Lys62 and Asp66 from the surface loop could be involved in the interaction with the polar head group of the membrane phospholipid. It is hypothesized that the two structures of the quadruple mutant, monoclinic and trigonal, represent the conformations of PLA2 at the lipid interface and in solution, respectively. A docked structure with a phospholipid molecule and with a transition-state analogue bound, one at the active site coordinating to the catalytic calcium and the other at the second calcium site, but both at the i-face, is presented.

Atomic resolution structure of the double mutant (K53,56M) of bovine pancreatic phospholipase A2.

The structure of the double mutant K53,56M has previously been refined at 1.9 A resolution using room-temperature data. The present paper reports the crystal structure of the same mutant K53,56M refined against 1.1 A data collected using synchrotron radiation. A total of 116 main-chain atoms from 29 residues and 44 side chains are modelled in alternate conformations. Most of the interfacial binding residues are found to be disordered and alternate conformations could be recognized. The second calcium ion-binding site residue Glu92 adopts two alternate conformations. The minor and major conformations of Glu92 correspond to the second calcium ion bound and unbound states.

Third calcium ion found in an inhibitor-bound phospholipase A2.

The lipolytic enzyme phospholipase A2 plays a crucial role in lipid metabolism and catalyzes hydrolysis of the fatty-acid ester bond at the sn-2 position of phospholipids. Here, the crystal structure (1.7 A resolution) of the triple mutant (K53,56,121M) of bovine pancreatic phospholipase A2 complexed with an organic molecule, p-methoxybenzoic acid (anisic acid), is reported. Residues 60-70 (the surface-loop residues) are ordered and adopt conformations which are different from those normally found in structures in which a second calcium ion is present. It is interesting to note that for the first time a third calcium ion has been identified. In addition, four Tris (2-amino-2-hydroxymethyl-1,3-propanediol) molecules were located. It is believed that one of the Tris molecules plays a role in clamping the third calcium ion and that another is involved in controlling the dynamics of the surface loop through hydrogen bonds.

Atomic resolution (0.97 A) structure of the triple mutant (K53,56,121M) of bovine pancreatic phospholipase A2.

The enzyme phospholipase A2 catalyzes the hydrolysis of the sn-2 acyl chain of phospholipids, forming fatty acids and lysophospholipids. The crystal structure of a triple mutant (K53,56,121M) of bovine pancreatic phospholipase A2 in which the lysine residues at positions 53, 56 and 121 are replaced recombinantly by methionines has been determined at atomic resolution (0.97 A). The crystal is monoclinic (space group P2), with unit-cell parameters a = 36.934, b = 23.863, c = 65.931 A, beta = 101.47 degrees. The structure was solved by molecular replacement and has been refined to a final R factor of 10.6% (Rfree = 13.4%) using 63,926 unique reflections. The final protein model consists of 123 amino-acid residues, two calcium ions, one chloride ion, 243 water molecules and six 2-methyl-2,4-pentanediol molecules. The surface-loop residues 60-70 are ordered and have clear electron density.

Crystal structures of the free and anisic acid bound triple mutant of phospholipase A2.

Phospholipase A2 catalyses the hydrolysis of the ester bond of 3-sn-phosphoglycerides. Here, we report the crystal structures of the free and anisic acid-bound triple mutant (K53,56,120M) of bovine pancreatic phospholipase A2. In the bound triple mutant structure, the small organic molecule p-anisic acid is found in the active site, and one of the carboxylate oxygen atoms is coordinated to the functionally important primary calcium ion. The other carboxylate oxygen atom is hydrogen bonded to the phenolic hydroxyl group of Tyr69. In addition, the bound anisic acid molecule replaces one of the functionally important water molecules in the active site. The residues 60-70, which are in a loop (surface loop), are disordered in most of the bovine pancreatic phospholipase A2 structures. It is interesting to note that these residues are ordered in the bound triple mutant structure but are disordered in the free triple mutant structure. The organic crystallization ingredient 2-methyl-2,4-pentanediol is found near the active site of the free triple mutant structure. The overall tertiary folding and stereochemical parameters for the final models of the free and anisic acid-bound triple mutant are virtually identical.

Solution structure of a viral DNA polymerase X and evidence for a mutagenic function.

The African swine fever virus DNA polymerase X (ASFV Pol X or Pol X), the smallest known nucleotide polymerase, has recently been reported to be an extremely low fidelity polymerase that may be involved in strategic mutagenesis of the viral genome. Here we report the solution structure of Pol X. The structure, unique within the realm of nucleotide polymerases, consists of only palm and fingers subdomains. Despite the absence of a thumb subdomain, which is important for DNA binding in other polymerases, we show that Pol X binds DNA with very high affinity. Further structural analyses suggest a novel mode of DNA binding that may contribute to low fidelity synthesis. We also demonstrate that the ASFV DNA ligase is a low fidelity ligase capable of sealing a nick that contains a G-G mismatch. This supports the hypothesis of a virus-encoded, mutagenic base excision repair pathway consisting of a tandem Pol X/ligase mutator.

Virtual reality orthopedic surgery simulator.

This paper describes a highly interactive virtual reality orthopedic surgery simulator. The simulator allows surgeons to use various surgical instruments to operate on virtual rigid anatomic structures, such bones, prostheses and bone grafts, to simulate every procedure on the rigid structures for complex orthopedic surgeries, including arthroplasty, corrective or open osteotomy, open reduction of fractures and amputation. A comparative study of the simulator with paper simulation was performed and showed that interns and residents found the simulator to be a useful learning tool, and that visiting doctors could use it effectively for planning verification and rehearsal of operations.

DNA polymerase beta: pre-steady-state kinetic analyses of dATP alpha S stereoselectivity and alteration of the stereoselectivity by various metal ions and by site-directed mutagenesis.

The first pre-steady-state kinetic analysis of the stereoselectivity of a DNA polymerase, Pol beta from rat brain, toward Rp and Sp isomers of dATPalphaS, and alteration of the stereoselectivity by various metal ions and by site-directed mutagenesis are reported. Diastereomers of dATPalphaS were synthesized by enzymatic methods to >98% purity. The rate of polymerization (k(pol)) and the apparent dissociation constant (K(d,app)) were measured with dATP, Rp-dATPalphaS, and Sp-dATPalphaS in the presence of Mg(2+), Mn(2+), or Cd(2+). The results indicate that wild type (WT) polymerase (Pol) beta can incorporate both Sp- and Rp-dATPalphaS in the presence of Mg(2+), but Sp is the preferred isomer. The stereoselectivity, defined as (k(pol)/K(d))(Sp)/(k(pol)/K(d))(Rp) (abbreviated Sp/Rp ratio), is 57.5 in the presence of Mg(2+). When Mg(2+) was substituted with Mn(2+) and Cd(2+), the Sp/Rp ratio decreased to 7.6 and 21, respectively. These results are discussed in relation to the crystal structures of various Pol beta complexes, as well as previous steady-state kinetic studies of other DNA polymerases. In addition, the D276R mutant was designed to introduce a potential extra hydrogen bonding interaction between the arginine side chain and the pro-Sp oxygen of the alpha-phosphate of dNTP. The kinetic data of the D276R mutant showed a pronounced relaxation of stereoselectivity of dATPalphaS (Sp/Rp ratio = 1.5, 3.7, and 1.5 for Mg(2+), Mn(2+), and Cd(2+), respectively). Furthermore, the D276R mutant showed a 5-fold enhanced reactivity toward Rp-dATPalphaS relative to WT Pol beta, suggesting that this mutant Pol beta can be used to incorporate Rp-dNTPalphaS into DNA oligomers.

Insight into the catalytic mechanism of DNA polymerase beta: structures of intermediate complexes.

The catalytic reaction mediated by DNA polymerases is known to require two Mg(II) ions, one associated with dNTP binding and the other involved in metal ion catalysis of the chemical step. Here we report a functional intermediate structure of a DNA polymerase with only one metal ion bound, the DNA polymerase beta-DNA template-primer-chromium(III).2'-deoxythymidine 5'-beta,gamma-methylenetriphosphate [Cr(III).dTMPPCP] complex, at 2.6 A resolution. The complex is distinct from the structures of other polymerase-DNA-ddNTP complexes in that the 3'-terminus of the primer has a free hydroxyl group. Hence, this structure represents a fully functional intermediate state. Support for this contention is provided by the observation of turnover in biochemical assays of crystallized protein as well as from the determination that soaking Pol beta crystals with Mn(II) ions leads to formation of the product complex, Pol beta-DNA-Cr(III).PCP, whose structure is also reported. An important feature of both structures is that the fingers subdomain is closed, similar to structures of other ternary complexes in which both metal ion sites are occupied. These results suggest that closing of the fingers subdomain is induced specifically by binding of the metal-dNTP complex prior to binding of the catalytic Mg(2+) ion. This has led us to reevaluate our previous evidence regarding the existence of a rate-limiting conformational change in Pol beta's reaction pathway. The results of stopped-flow studies suggest that there is no detectable rate-limiting conformational change step.

Involvement of the Arg-Asp-His catalytic triad in enzymatic cleavage of the phosphodiester bond.

Phosphatidylinositol-specific phospholipase C (PI-PLC) catalyzes the cleavage of the P-O bond in phosphatidylinositol via intramolecular nucleophilic attack of the 2-hydroxyl group of inositol on the phosphorus atom. Our earlier stereochemical and site-directed mutagenesis studies indicated that this reaction proceeds by a mechanism similar to that of RNase A, and that the catalytic site of PI-PLC consists of three major components analogous to those observed in RNase A, the His32 general base, the His82 general acid, and Arg69 acting as a phosphate-activating residue. In addition, His32 is associated with Asp274 in forming a catalytic triad with inositol 2-hydroxyl, and His82 is associated with Asp33 in forming a catalytic diad. The focus of this work is to provide a global view of the mechanism, assess cooperation between various catalytic residues, and determine the origin of enzyme activation by the hydrophobic leaving group. To this end, we have investigated kinetic properties of Arg69, Asp33, and His82 mutants with phosphorothioate substrate analogues which feature leaving groups of varying hydrophobicity and pK(a). Our results indicate that interaction of the nonbridging pro-S oxygen atom of the phosphate group with Arg69 is strongly affected by Asp33, and to a smaller extent by His82. This result in conjunction with those obtained earlier can be rationalized in terms of a novel, dual-function triad comprised of Arg69, Asp33, and His82 residues. The function of this triad is to both activate the phosphate group toward the nucleophilic attack and to protonate the leaving group. In addition, Asp33 and His82 mutants displayed much smaller degrees of activation by the fatty acid-containing leaving group as compared to the wild-type (WT) enzyme, and the level of activation was significantly reduced for substrates featuring the leaving group with low pK(a) values. These results strongly suggest that the assembly of the above three residues into the fully catalytically competent triad is controlled by the hydrophobic interactions of the enzyme with the substrate leaving group.

Mechanism of phosphatidylinositol-specific phospholipase C: origin of unusually high nonbridging thio effects.

Phosphatidylinositol-specific phospholipase C (PI-PLC) has been proposed previously to employ a catalytic mechanism highly reminiscent of that of ribonuclease A (RNase A). Both catalytic sites are comprised of two histidine side chains acting as a general base-general acid pair and a phosphate-activating residue: an arginine in the case of PI-PLC and a lysine in RNase A. Despite these structural similarities, the PI-PLC reaction is slowed 10(5)-fold upon substitution of one of the phosphate nonbridging oxygen atoms with sulfur, whereas a much smaller effect is observed in the analogous RNase A reaction. Here, we report a systematic study of this property in PI-PLC, conducted by means of site-directed chemical modification of a cysteine residue replacing the arginine at position 69. The results show that mutant enzymes featuring bidentate side chains at this position display significantly higher activity, higher thio effects, and greater stereoselectivity than do those with monodentate side chains. The results suggest that the bidentate nature of Arg69 is the origin of the large thio effects and stereoselectivity in PI-PLC. We propose that in addition to binding the phosphate, the function of arginine 69 is to bring the phosphate group and the 2-OH group of inositol into proximity and to induce proper alignment for nucleophilic attack, and possibly to lower the pK(a) of the 2-OH. The results presented here could be important to mechanisms of phosphoryl transfer enzymes in general, suggesting that a major part of thio effects observed in enzymatic phosphoryl transfer reactions can originate from factors other than direct interaction between a side chain and a phosphate group, and caution the use of the absolute magnitude of the thio effect as an indicator of the strength of such interactions.

Somatic INK4a-ARF locus mutations: a significant mechanism of gene inactivation in squamous cell carcinomas of the head and neck.

The INK4a-ARF locus is located on human chromosome 9p21 and is known to encode two functionally distinct tumor-suppressor genes. The p16(INK4a) (p16) tumor-suppressor gene product is a negative regulator of cyclin-dependent kinases 4 and 6, which in turn positively regulate progression of mammalian cells through the cell cycle. The p14(ARF) tumor-suppressor gene product specifically interacts with human double minute 2, leading to the subsequent stabilization of p53 and G(1) arrest. Previous investigations analyzing the p16 gene in squamous cell carcinomas of the head and neck (SCCHNs) have suggested the predominate inactivating events to be homozygous gene deletions and hypermethylation of the p16 promoter. Somatic mutational inactivation of p16 has been reported to be low (0-10%, with a combined incidence of 25 of 279, or 9%) and to play only a minor role in the development of SCCHN. The present study examined whether this particular mechanism of INK4a/ARF inactivation, specifically somatic mutation, has been underestimated in SCCHN by determining the mutational status of the p16 and p14(ARF) genes in 100 primary SCCHNs with the use of polymerase chain reaction technology and a highly sensitive, nonradioactive modification of single-stranded conformational polymorphism (SSCP) analysis termed "cold" SSCP. Exons 1alpha, 1beta, and 2 of INK4a/ARF were amplified using intron-based primers or a combination of intron- and exon-based primers. A total of 27 SCCHNs (27%) exhibited sequence alterations in this locus, 22 (22%) of which were somatic sequence alterations and five (5%) of which were a single polymorphism in codon 148. Of the 22 somatic alterations, 20 (91%) directly or indirectly involved exon 2, and two (9%) were located within exon 1alpha. No mutations were found in exon 1beta. All 22 somatic mutations would be expected to yield altered p16 proteins, but only 15 of them should affect p14(ARF) proteins. Specific somatic alterations included microdeletions or insertions (nine of 22, 41%), a microrearrangement (one of 22, 5%), and single nucleotide substitutions (12 of 22, 56%). In addition, we analyzed the functional characteristics of seven unique mutant p16 proteins identified in this study by assessing their ability to inhibit cyclin-dependent kinase 4 activity. Six of the seven mutant proteins tested exhibited reduced function compared with wild-type p16, ranging from minor decreases of function (twofold to eightfold) in four samples to total loss of function (29- to 38-fold decrease) in two other samples. Overall, somatic mutation of the INK4a/ARF tumor suppressor locus, resulting in functionally deficient p16 and possibly p14(ARF) proteins, seems to be a prevalent event in the development of SCCHN. Mol. Carcinog. 30:26-36, 2001.