Andrew F. Neuwald

A superfamily of conserved domains in DNA damage-responsive cell cycle checkpoint proteins.

Computer analysis of a conserved domain, BRCT, first described at the carboxyl ter‐minus of the breast cancer protein BRCA1, a p53 binding protein (53BP1), and the yeast cell cycle checkpoint protein RAD9 revealed a large super‐ family of domains that occur predominantly in proteins involved in cell cycle checkpoint functions responsive to DNA damage. The BRCT domain consists of ~95 amino acid residues and occurs as a tandem repeat at the carboxyl terminus of numerous proteins, but has been observed also as a tandem repeat at the amino terminus or as a single copy. The BRCT superfamily presently includes ~40 nonorthologous proteins, namely, BRCA1, 53BP1, and RAD9; a protein family that consists of the fission yeast replication checkpoint protein Rad4, the oncoprotein ECT2, the DNA repair protein XRCC1, and yeast DNA polymerase subunit DPB11; DNA binding enzymes such as terminal deoxynucleotidyltransferases, deoxycy‐ tidyl transferase involved in DNA repair, and DNA‐ligases III and IV; yeast multifunctional transcription factor RAP1; and several uncharacterized gene products. Another previously described domain that is shared by bacterial NAD‐dependent DNA‐ligases, the large subunits of eukaryotic replication factor C, and poly(ADP‐ri‐ bose) polymerases appears to be a distinct version of the BRCT domain. The retinoblastoma protein (a universal tumor suppressor) and related proteins may contain a distant relative of the BRCT domain. Despite the functional diversity of all these proteins, participation in DNA damage‐re‐ sponsive checkpoints appears to be a unifying theme. Thus, the BRCT domain is likely to perform critical, yet uncharacterized, functions in the cell cycle control of organisms from bacteria to humans. The car boxyterminal BRCT domain of BRCA1 corresponds precisely to the recently identified minimal transcription activation domain of this protein, indicating one such function.— Bork, P., Hofmann, K., Bucher, P., Neuwald, A. F., Altschul, S. F., Koonin, E. V. A superfamily of conserved domains in DNA damage‐responsive cell cycle checkpoint proteins. FASEB J. 11, 68‐ 76 (1997)

Natural Mutagenesis of Human Genomes by Endogenous Retrotransposons

Two abundant classes of mobile elements, namely Alu and L1 elements, continue to generate new retrotransposon insertions in human genomes. Estimates suggest that these elements have generated millions of new germline insertions in individual human genomes worldwide. Unfortunately, current technologies are not capable of detecting most of these young insertions, and the true extent of germline mutagenesis by endogenous human retrotransposons has been difficult to examine. Here, we describe technologies for detecting these young retrotransposon insertions and demonstrate that such insertions indeed are abundant in human populations. We also found that new somatic L1 insertions occur at high frequencies in human lung cancer genomes. Genome-wide analysis suggests that altered DNA methylation may be responsible for the high levels of L1 mobilization observed in these tumors. Our data indicate that transposon-mediated mutagenesis is extensive in human genomes and is likely to have a major impact on human biology and diseases.

Differential Contributions of Condensin I and Condensin II to Mitotic Chromosome Architecture in Vertebrate Cells

The canonical condensin complex (henceforth condensin I) plays an essential role in mitotic chromosome assembly and segregation from yeast to humans. We report here the identification of a second condensin complex (condensin II) from vertebrate cells. Condensins I and II share the same pair of structural maintenance of chromosomes (SMC) subunits but contain different sets of non-SMC subunits. siRNA-mediated depletion of condensin I- or condensin II-specific subunits in HeLa cells produces a distinct, highly characteristic defect in chromosome morphology. Simultaneous depletion of both complexes causes the severest defect. In Xenopus egg extracts, condensin I function is predominant, but lack of condensin II results in the formation of irregularly shaped chromosomes. Condensins I and II show different distributions along the axis of chromosomes assembled in vivo and in vitro. We propose that the two condensin complexes make distinct mechanistic contributions to mitotic chromosome architecture in vertebrate cells.

Bayesian Models for Multiple Local Sequence Alignment and Gibbs Sampling Strategies

Abstract A wealth of data concerning lifes basic molecules, proteins and nucleic acids, has emerged from the biotechnology revolution. The human genome project has accelerated the growth of these data. Multiple observations of homologous protein or nucleic acid sequences from different organisms are often available. But because mutations and sequence errors misalign these data, multiple sequence alignment has become an essential and valuable tool for understanding structures and functions of these molecules. A recently developed Gibbs sampling algorithm has been applied with substantial advantage in this setting. In this article we develop a full Bayesian foundation for this algorithm and present extensions that permit relaxation of two important restrictions. We also present a rank test for the assessment of the significance of multiple sequence alignment. As an example, we study the set of dinucleotide binding proteins and predict binding segments for dozens of its members.

Purification and biochemical characterization of interchromatin granule clusters

Components of the pre‐mRNA splicing machinery are localized in interchromatin granule clusters (IGCs) and perichromatin fibrils (PFs). Here we report the biochemical purification of IGCs. Approximately 75 enriched proteins were present in the IGC fraction. Protein identification employing a novel mass spectrometry strategy and peptide microsequencing identified 33 known proteins, many of which have been linked to pre‐mRNA splicing, as well as numerous uncharacterized proteins. Thus far, three new protein constituents of the IGCs have been identified. One of these, a 137 kDa protein, has a striking sequence similarity over its entire length to UV‐damaged DNA‐binding protein, a protein associated with the hereditary disease xeroderma pigmentosum group E, and to the 160 kDa subunit of cleavage polyadenylation specificity factor. Overall, these results provide a key framework that will enable the biological functions associated with the IGCs to be elucidated.

The hallmark of AGC kinase functional divergence is its C-terminal tail, a cis-acting regulatory module

The catalytic activities of eukaryotic protein kinases (EPKs) are regulated by movement of the C-helix, movement of the N and C lobes upon ATP binding, and movement of the activation loop upon phosphorylation. Statistical analysis of the selective constraints associated with AGC kinase functional divergence reveals conserved interactions between these regulatory regions and three regions of the C-terminal tail (C-tail): the N-lobe tether (NLT), the active-site tether (AST), and the C-lobe tether (CLT). The NLT serves as a docking site for an upstream kinase PDK1 and, upon activation, positions the C-helix within the ATP binding pocket. The AST directly interacts with the ATP binding pocket, and the CLT interacts with the interlobe linker and the αC–β4 loop, which appears to serve as a hinge for C-helix movement. The C-tail is a hallmark of AGC functional divergence inasmuch as most of the conserved core residues that distinguish AGC kinases from other EPKs are associated with the NLT, AST, or CLT. Moreover, several AGC catalytic core conserved residues that interact with the C-tail strikingly diverge from the canonical residues observed at corresponding positions in nearly all other EPKs, suggesting that the catalytic core may have coevolved with the C-tail in AGC kinases. These observations, along with the fact that the C-tail is needed for catalytic activity suggests that the C-tail is a cis-acting regulatory module that can also serve as a regulatory “handle,” to which trans-acting cellular components can bind to modulate activity.

Barth syndrome may be due to an acyltransferase deficiency

Barth syndrome is an X-linked inherited disorder characterized by short stature, cardioskeletal myopathy, neutropenia, abnormal mitochondria, and respiratory-chain dysfunction [[1]xX-linked cardioskeletal myopathy and neutropenia (Barth syndrome): respiratory-chain abnormalities in cultured fibroblasts. Barth, PG, Van den Bogert, C, Bolhuis, PA, Scholte, HR, van Gennip, AH, Schutgens, RB, and Ketel, AG. J Inherit Metab Dis. 1996; 19: 157–160Crossref | PubMed | Scopus (57)See all References, [2]xAn X-linked mitochondrial disease affecting cardiac muscle, skeletal muscle and neutrophil leucocytes. Barth, PG, Scholte, HR, Berden, JA, Van der Klei-Van Moorsel, JM, Luyt-Houwen, IE, Van’t Veer-Korthof, ET et al. J Neurol Sci. 1983; 62: 327–355Abstract | Full Text PDF | PubMed | Scopus (365)See all References]. It is often fatal in childhood due to cardiac failure or sepsis arising from agranulocytosis. The phenotype associated with this disorder is quite variable, however, and other X-linked cardiomyopathies [[3]xEndocardial fibroelastosis: possible X-linked inheritance. Hodgson, S, Child, A, and Dyson, M. J Med Genet. 1987; 24: 210–214Crossref | PubMedSee all References, [4]xPossible X-linked congenital mitochondrial cardiomyopathy in three families. Orstavik, KH, Skjorten, F, Hellebostad, M, Haga, P, and Langslet, A. J Med Genet. 1993; 30: 269–272Crossref | PubMedSee all References, [5]xX-linked fatal infantile cardiomyopathy maps to Xq28 and is possibly allelic to Barth syndrome. Gedeon, AK, Wilson, MJ, Colley, AC, Sillence, DO, and Mulley, JC. J Med Genet. 1995; 32: 383–388Crossref | PubMedSee all References] may be allelic to Barth syndrome, which maps to a gene-rich region of Xq28 [6xMapping of the locus for X-linked cardioskeletal myopathy with neutropenia and abnormal mitochondria (Barth syndrome) to Xq28. Bolhuis, PA, Hensels, GW, Hulsebos, TJ, Baas, F, and Barth, PG. Am J Hum Genet. 1991; 48: 481–485PubMedSee all References[6]. Recently, a gene mutated in patients afflicted with Barth syndrome (G4.5) was cloned and sequenced [7xA novel X-linked gene, G4.5, is responsible for Barth syndrome. Bione, SP, D’Adamo, P, Maestrini, E, Gedeon, AK, Bolhuis, PA, and Toniolo, D. Nat Genet. 1996; 12: 385–389Crossref | PubMed | Scopus (429)See all References[7]. It encodes several proteins (designated tafazzins) by means of alternate splicing. The biological function of tafazzins is unclear; a BLAST search [8xBasic local alignment search tool. Altschul, SF, Gish, W, Miller, W, Myers, EW, and Lipman, DJ. J Mol Biol. 1990; 215: 403–410PubMed | Scopus (0)See all References[8] finds significant pairwise similarity only to two hypothetical proteins: one from worm (g1130664), and one from yeast (g1066481).Here, I report that human tafazzins belong to a superfamily consisting of acyltransferases involved in phospholipid biosynthesis and other proteins of unknown function. This superfamily was found using PROBE [9xExtracting protein alignment models from the sequence database. Neuwald, AF, Liu, JS, Lipman, DJ, and Lawrence, CE. Nucleic Acids Res. 1997; 25: 1665–1677Crossref | PubMed | Scopus (184)See all References[9], an automated search and multiple alignment program based on iterative database searches. Starting with a plant 1-acylglycerol-3-phosphate acyltransferase (EC 2.3.1.51), g1197334, PROBE returned a superfamily that includes known and putative acyltransferases from bacteria, fungi, plants, and vertebrate and invertebrate metazoans. Characterized enzymes in this superfamily all function in phospholipid biosynthesis and have either glycerolphosphate, 1-acylglycerolphosphate, or 2-acylglycerolphosphoethanolamine acyltransferase activity.The sequence alignment contains five conserved regions that presumably reflect similar structural and functional features shared by these proteins (Figure 1Figure 1). As all of the characterized proteins are acyltransferases involved in phospholipid biosynthesis, the uncharacterized proteins are likely to have similar catalytic activity. Notably, motif A contains a fully conserved residue position that may correspond to a catalytic histidine, as has been found at the active site of other CoA-dependent hydrolases [10xStructure of chloramphenicol acetyltransferase at 1.75-A resolution. Leslie, AG, Moody, PC, and Shaw, WV. Proc Natl Acad Sci USA. 1988; 85: 4133–4137Crossref | PubMedSee all References[10]. Of course, it is possible that tafazzins perform some other hydrolytic function. Indeed, hydrolytic activity was previously predicted for tafazzins based on weak similarity to the Escherichia coli radC gene [11xPositionally cloned human disease genes: patterns of evolutionary conservation and new functional motifs. Mushegian, AR, Bassett, DE, Boguski, MS, Bork, P, and Koonin, EV. Proc Natl Acad Sci USA. 1997; 94: 5831–5836Crossref | PubMed | Scopus (202)See all References[11], which may possess hydrolytic activity needed for DNA repair. Nevertheless, the more extensive similarity of tafazzins to these acyltransferases implies a closer similarity in function.Figure 1Alignment of representative sequences in the tafazzins, or acyltransferase superfamily. A total of 53 proteins in the NCBI non-redundant database were detected by the PROBE search [9xExtracting protein alignment models from the sequence database. Neuwald, AF, Liu, JS, Lipman, DJ, and Lawrence, CE. Nucleic Acids Res. 1997; 25: 1665–1677Crossref | PubMed | Scopus (184)See all References[9], which used default parameter settings. Conserved residues are highlighted in red (for the most conserved positions) or black. Numbers in parentheses are gap lengths. Human tafazzin is detected at the p<0.00001 level of significance; this is based on a database search using an alignment lacking sequences with statistically significant pairwise similarity to tafazzins (that is, lacking sequences g1263110, g1130664, g1066481, g1841552, g1403001 and g1673483). (The database search and the p-value calculation were done as previously described [13xGibbs motif sampling: detection of bacterial outer membrane protein repeats. Neuwald, AF, Liu, JS, and Lawrence, CE. Protein Sci. 1995; 4: 1618–1632Crossref | PubMedSee all References[13].) Protein identifiers are highlighted according to the following color scheme: black, tafazzin and close homologs; red, 1-acylglycerol-phosphate acyltransferases (agpat); blue, glycerolphosphate acyltransferases (gpat); green, 2-acylglycerolphosphoethanolamine acyltransferase (agpeat); unhighlighted, proteins of unknown function.View Large Image | View Hi-Res Image | Download PowerPoint SlideThe potential acyltransferase activity of tafazzins suggests a possible disease mechanism underlying Barth syndrome. Differential splicing of tafazzins [7xA novel X-linked gene, G4.5, is responsible for Barth syndrome. Bione, SP, D’Adamo, P, Maestrini, E, Gedeon, AK, Bolhuis, PA, and Toniolo, D. Nat Genet. 1996; 12: 385–389Crossref | PubMed | Scopus (429)See all References[7] and the existence of at least nine of these putative acyltransferases in roundworm — by contrast with the four detected in the E. coli genome — suggests that a variety of substrate-specific or tissue-and organelle-specific forms of these acyltransferases exist in eukaryotes. If so, then the mitochondrial structural and respiratory-chain abnormalities associated with Barth syndrome may be due to alterations in mitochondrial membrane phospholipid composition. Consistent with this notion, a temperature-sensitive Chinese hamster ovary cell mutant deficient in an enzyme needed for cardiolipin biosynthesis showed alterations in mitochondrial morphology and respiration [12xMitochondrial dysfunction of a cultured Chinese hamster ovary cell mutant deficient in cardiolipin. Ohtsuka, T, Nishijima, M, Suzuki, K, and Akamatsu, Y. J Biol Chem. 1993; 268: 22914–22919PubMedSee all References[12].It is important to note that the roundworm ZK809.2 gene may be an ortholog of the human G4.5 (tafazzins) gene. It shares several splice sites with the human gene and the predicted product is more closely related to tafazzins than to any other protein in the superfamily. Notably, the worm protein is missing exon 5, which appears to be removed from many of the tafazzin splice variants [7xA novel X-linked gene, G4.5, is responsible for Barth syndrome. Bione, SP, D’Adamo, P, Maestrini, E, Gedeon, AK, Bolhuis, PA, and Toniolo, D. Nat Genet. 1996; 12: 385–389Crossref | PubMed | Scopus (429)See all References[7]. Furthermore, the worm homolog shares several conserved regions with human tafazzins that are unconserved in the superfamily as a whole. Thus, ZK809.2 mutants may serve as a useful model to explore the molecular mechanisms underlying Barth syndrome.

Evolutionary constraints associated with functional specificity of the CMGC protein kinases MAPK, CDK, GSK, SRPK, DYRK, and CK2α

Amino acid residues associated with functional specificity of cyclin‐dependent kinases (CDKs), mitogen‐activated protein kinases (MAPKs), glycogen synthase kinases (GSKs), and CDK‐like kinases (CLKs), which are collectively termed the CMGC group, were identified by categorizing and quantifying the selective constraints acting upon these proteins during evolution. Many constraints specific to CMGC kinases correspond to residues between the N‐terminal end of the activation segment and a CMGC‐conserved insert segment associated with coprotein binding. The strongest such constraint is imposed on a “CMGC‐arginine” near the substrate phosphorylation site with a side chain that plays a role both in substrate recognition and in kinase activation. Two nearby buried waters, which are also present in non‐CMGC kinases, typically position the main chain of this arginine relative to the catalytic loop. These and other CMGC‐specific features suggest a structural linkage between coprotein binding, substrate recognition, and kinase activation. Constraints specific to individual subfamilies point to mechanisms for CMGC kinase specialization. Within casein kinase 2α (CK2α), for example, the binding of one of the buried waters appears prohibited by the side chain of a leucine that is highly conserved within CK2α and that, along with substitution of lysine for the CMGC‐arginine, may contribute to the broad substrate specificity of CK2α by relaxing characteristically conserved, precise interactions near the active site. This leucine is replaced by a conserved isoleucine or valine in other CMGC kinases, thereby illustrating the potential functional significance of subtle amino acid substitutions. Analysis of other CMGC kinases similarly suggests candidate family‐specific residues for experimental follow‐up.

Markovian Structures in Biological Sequence Alignments

Abstract The alignment of multiple homologous biopolymer sequences is crucial in research on protein modeling and engineering, molecular evolution, and prediction in terms of both gene function and gene product structure. In this article we provide a coherent view of the two recent models used for multiple sequence alignment—the hidden Markov model (HMM) and the block-based motif model—to develop a set of new algorithms that have both the sensitivity of the block-based model and the flexibility of the HMM. In particular, we decompose the standard HMM into two components: the insertion component, which is captured by the so-called “propagation model,” and the deletion component, which is described by a deletion vector. Such a decomposition serves as a basis for rational compromise between biological specificity and model flexibility. Furthermore, we introduce a Bayesian model selection criterion that—in combination with the propagation model, genetic algorithm, and other computational aspects—forms the cor...

Evolution of allostery in the cyclic nucleotide binding module

BackgroundThe cyclic nucleotide binding (CNB) domain regulates signaling pathways in both eukaryotes and prokaryotes. In this study, we analyze the evolutionary information embedded in genomic sequences to explore the diversity of signaling through the CNB domain and also how the CNB domain elicits a cellular response upon binding to cAMP.ResultsIdentification and classification of CNB domains in Global Ocean Sampling and other protein sequences reveals that they typically are fused to a wide variety of functional domains. CNB domains have undergone major sequence variation during evolution. In particular, the sequence motif that anchors the cAMP phosphate (termed the PBC motif) is strikingly different in some families. This variation may contribute to ligand specificity inasmuch as members of the prokaryotic cooA family, for example, harbor a CNB domain that contains a non-canonical PBC motif and that binds a heme ligand in the cAMP binding pocket. Statistical comparison of the functional constraints imposed on the canonical and non-canonical PBC containing sequences reveals that a key arginine, which coordinates with the cAMP phosphate, has co-evolved with a glycine in a distal β2-β3 loop that allosterically couples cAMP binding to distal regulatory sites.ConclusionOur analysis suggests that CNB domains have evolved as a scaffold to sense a wide variety of second messenger signals. Based on sequence, structural and biochemical data, we propose a mechanism for allosteric regulation by CNB domains.