Greg Gloor

A Peptide-Based Target Screen Implicates the Protein Kinase CK2 in the Global Regulation of Caspase Signaling

Phosphorylation of caspase substrates by the protein kinase CK2 may underlie its role in tumorigenesis. Protection from Death Caspases are cysteine-dependent proteases that cleave target proteins—including other caspases—at aspartate residues within caspase recognition motifs. Caspase-3, which operates at the point of convergence of extrinsic and intrinsic cell death pathways, is generated as a proenzyme that must be cleaved by upstream caspases to become activated. Noting that phosphorylation of caspase substrates at residues near the caspase recognition motif protects them from cleavage (see the Perspective by Filhol and Cochet), Duncan et al. scanned the human proteome for sequences that contained overlapping target sites for caspases and any of 10 kinases implicated in promoting cell survival or tumorigenesis. The protein kinase CK2 emerged as the kinase with the greatest number of such overlapping sequences, and potential targets of CK2 and caspases were evaluated by a newly developed technique for identifying caspase substrates. In addition to phosphorylating targets of caspase-3, CK2 also phosphorylated procaspase-3, thereby blocking its activation by upstream caspases and thus protecting cells from apoptosis. Together, these data suggest a global role for CK2 in preventing apoptosis, which may underlie its role in tumorigenesis. The convergence of caspase and protein kinase signaling pathways has become increasingly evident, as illustrated by the protection of caspase substrates from cleavage upon undergoing phosphorylation at or near to their caspase recognition motifs. To investigate the global role of phosphorylation in the regulation of caspase signaling, we designed a peptide match program to identify sequences from the human proteome that contained overlapping recognition motifs for caspases and kinases. We identified the protein kinase CK2 as the most prominent kinase with a consensus site for phosphorylation that overlapped with caspase recognition motifs. We then evaluated potential targets of CK2 and caspases by combining peptide array target screens with identification of caspase substrates. We identified numerous shared candidate targets of CK2 and caspases, including procaspase-3, which functions at a level at which both intrinsic and extrinsic apoptotic signals converge. Together, these data support a role for CK2-dependent phosphorylation as a global mechanism for inhibiting caspase signaling pathways.

A truncated form of the bacteriophage Mu B protein promotes conservative integration, but not replicative transposition, of Mu DNA.

The phage-encoded proteins required for conservative integration of infecting bacteriophage Mu DNA were investigated. Our findings show that functional gpA, an essential component of the phage transposition system, is required for integration. The Mu B protein, which greatly enhances replicative transposition of Mu DNA, is also required. Furthermore, a truncated form of gpB lacking 18 amino acids from the carboxy terminus is blocked in replicative transposition, but not conservative integration. Our results point to a more prominent role for gpB than simply a replication enhancer in Mu DNA transposition. The ability of a truncated form of B to function in conservative integration, but not replicative transposition, also suggests a key role for the carboxy-terminal domain of the protein in the replicative reaction. The existence of a shortened form of gpB, which uncouples conservative integration from replicative transposition, should be invaluable for future dissection of Mu DNA transposition.

Circular contextual insertions/deletions with applications to biomolecular computation

Insertions and deletions of small circular DNA strands into long linear DNA strands are phenomena that happen frequently in nature and thus constitute an attractive paradigm for biomolecular computing. The paper presents a new model for DNA-based computation that involves circular as well as linear molecules, and that uses the operations of insertion and deletion. After introducing the formal model, we investigate its properties and prove in particular that the circular insertion/deletion systems are capable of universal computation. We also give the results of an experimental laboratory implementation of our model. This shows that rewriting systems of the circular insertion/deletion type are viable alternatives in DNA computation.

Using DNA to solve the bounded Post correspondence problem

Theoretical research in DNA computing includes designing practical experiments for solving various computational problems by means of DNA manipulation. This paper proposes a DNA algorithm for an NP-complete problem, The Bounded Post Correspondence Problem. The proposed experiment can be used to test several standard molecular biology laboratory procedures for their usability as bio-operations in DNA computing. c 2000 Elsevier Science B.V. All rights reserved.

Regulation of caspase pathways by protein kinase CK2: identification of proteins with overlapping CK2 and caspase consensus motifs

Apoptosis, or programmed cell death, is a vital cellular process often impaired in diseases such as cancer. Aspartic acid-directed proteases known as caspases cleave a broad spectrum of cellular proteins and are central constituents of the apoptotic machinery. Caspases are regulated by a variety of mechanisms including protein phosphorylation. One intriguing mechanism by which protein kinases can modulate caspase pathways is by blocking substrate cleavage through phosphorylation of residues adjacent to caspase cleavage sites. To explore this mechanism in detail, we recently undertook a systematic investigation using a combination of bioinformatics, peptide arrays, and peptide cleavage assays to identify proteins with overlapping protein kinase and caspase recognition motifs (Duncan et al., Sci Signal 4:ra30, 2011). These studies implicated protein kinase CK2 as a global regulator of apoptotic pathways. In this article, we extend the analysis of proteins with overlapping CK2 and caspase consensus motifs to examine the convergence of CK2 with specific caspases and to identify CK2/caspase substrates known to be phosphorylated or cleaved in cells. Given its constitutive activity and elevated expression in cancer, these observations suggest that the ability of CK2 to modulate caspase pathways may contribute to a role in promoting cancer cell survival and raise interesting prospects for therapeutic targeting of CK2.

ATP(GTP)-dependent conversion of MVM parvovirus single-stranded DNA to its replicative form by a purified 10 S species of mouse DNA polymerase α

A species of DNA polymerase alpha that is active in the ATP(GTP)-dependent conversion of MVM parvovirus single-stranded linear DNA to the duplex replicative form has been purified 4300-fold from Ehrlich ascites mouse tumour cells. The single-stranded----replicative form activity is maintained throughout ammonium sulfate precipitation, DEAE-cellulose, phosphocellulose and hydroxyapatite column chromatography and glycerol gradient sedimentation. Polypeptides with Mr = 230 000, 220 000, 183 000, 157 000, 125 000, 70 000, 65 000, 62 000, 57 000, 53 000 and 48 000 copurify with the single-stranded----replicative form activity, which sediments at approx. 10 S. The Mr = 183 000, 157 000 and 125 000 polypeptides exhibit catalytic activity when assayed in situ following SDS-polyacrylamide gel electrophoresis. The 10 S form of DNA polymerase alpha is functionally distinguishable from an 8.4 S form of the enzyme obtained from the same cells on the basis of single-stranded----replicative form activity. The single-stranded----replicative form activity of the 10 S enzyme is stable at 22 degrees C for up to 3 h, but exhibits a half life of only 5 min at 45 degrees C.

TOWARDS A DNA SOLUTION TO THE SHORTEST COMMON SUPERSTRING PROBLEM

This paper proposes a DNA algorithm for solving an NP-complete problem (The Shortest Common Superstring Problem) by manipulation of biomolecules, and presents partial results of the experiment that implements our algorithm. We also discuss practical constraints that have to be taken into account when implementing the algorithm, propose a coding system as a solution to these practical restrictions, and discuss the control experiments performed for establishing the parameters controlling the specificity of the assay.

A computer scientist's guide to molecular biology

Abstract In this paper, we explain the basic structure and properties of both single- and double-stranded DNA in vivo (in living organisms). We also review the first in vitro (test tube) experiment that solved a mathematical problem, The Directed Hamiltonian Path Problem, by manipulating DNA strands. Lastly, we give a list of bio-operations that have so far been used in DNA computation.

Towards a DNA solution to the shortest common superstring problem

This paper proposes a DNA algorithm for solving an NP-complete problem (the shortest common superstring problem) by manipulation of biomolecules, and presents partial results of the experiment that implements our algorithm. We also discuss practical constraints that have to be taken into account when implementing the algorithm, propose a coding system as a solution to these practical restrictions, and discuss the control experiments performed for establishing the parameters controlling the specificity of the assay.

How to Compute with DNA

This paper addresses two main aspects of DNA computing research: DNA computing in vitro and in vivo. We first present a model of DNA computation developed in [5]: the circular insertion/deletion system. We review the result obtained in [5] stating that this system has the computational power of a Turing machine, and present the outcome of a molecular biologylab oratoryex periment from [5] that implements a small instance of such a system. This shows that rewriting systems of the circular insertion/deletion type are viable alternatives in DNA computation in vitro. In the second half of the paper we address DNA computing in vivo byp resenting a model proposed in [17] and developed in [18] for the homologous recombinations that take place during gene rearrangement in ciliates. Such a model has universal computational power which indicates that, in principle, some unicellular organisms mayha ve the capacity to perform anycom putation carried out byan electronic computer.