Showing papers by "L. Aravind published in 2004"

De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-κB signalling

Abstract: NF-kappaB transcription factors mediate the effects of pro-inflammatory cytokines such as tumour necrosis factor-alpha and interleukin-1beta. Failure to downregulate NF-kappaB transcriptional activity results in chronic inflammation and cell death, as observed in A20-deficient mice. A20 is a potent inhibitor of NF-kappaB signalling, but its mechanism of action is unknown. Here we show that A20 downregulates NF-kappaB signalling through the cooperative activity of its two ubiquitin-editing domains. The amino-terminal domain of A20, which is a de-ubiquitinating (DUB) enzyme of the OTU (ovarian tumour) family, removes lysine-63 (K63)-linked ubiquitin chains from receptor interacting protein (RIP), an essential mediator of the proximal TNF receptor 1 (TNFR1) signalling complex. The carboxy-terminal domain of A20, composed of seven C2/C2 zinc fingers, then functions as a ubiquitin ligase by polyubiquitinating RIP with K48-linked ubiquitin chains, thereby targeting RIP for proteasomal degradation. Here we define a novel ubiquitin ligase domain and identify two sequential mechanisms by which A20 downregulates NF-kappaB signalling. We also provide an example of a protein containing separate ubiquitin ligase and DUB domains, both of which participate in mediating a distinct regulatory effect.

Complete genome sequence of the apicomplexan, Cryptosporidium parvum.

Abstract: The apicomplexan Cryptosporidium parvum is an intestinal parasite that affects healthy humans and animals, and causes an unrelenting infection in immunocompromised individuals such as AIDS patients. We report the complete genome sequence of C. parvum, type II isolate. Genome analysis identifies extremely streamlined metabolic pathways and a reliance on the host for nutrients. In contrast to Plasmodium and Toxoplasma, the parasite lacks an apicoplast and its genome, and possesses a degenerate mitochondrion that has lost its genome. Several novel classes of cell-surface and secreted proteins with a potential role in host interactions and pathogenesis were also detected. Elucidation of the core metabolism, including enzymes with high similarities to bacterial and plant counterparts, opens new avenues for drug development.

Comparative genomics of the FtsK–HerA superfamily of pumping ATPases: implications for the origins of chromosome segregation, cell division and viral capsid packaging

Abstract: Recently, it has been shown that a predicted P-loop ATPase (the HerA or MlaA protein), which is highly conserved in archaea and also present in many bacteria but absent in eukaryotes, has a bidirectional helicase activity and forms hexameric rings similar to those described for the TrwB ATPase. In this study, the FtsK-HerA superfamily of P-loop ATPases, in which the HerA clade comprises one of the major branches, is analyzed in detail. We show that, in addition to the FtsK and HerA clades, this superfamily includes several families of characterized or predicted ATPases which are predominantly involved in extrusion of DNA and peptides through membrane pores. The DNA-packaging ATPases of various bacteriophages and eukaryotic double-stranded DNA viruses also belong to the FtsK-HerA superfamily. The FtsK protein is the essential bacterial ATPase that is responsible for the correct segregation of daughter chromosomes during cell division. The structural and evolutionary relationship between HerA and FtsK and the nearly perfect complementarity of their phyletic distributions suggest that HerA similarly mediates DNA pumping into the progeny cells during archaeal cell division. It appears likely that the HerA and FtsK families diverged concomitantly with the archaeal-bacterial division and that the last universal common ancestor of modern life forms had an ancestral DNA-pumping ATPase that gave rise to these families. Furthermore, the relationship of these cellular proteins with the packaging ATPases of diverse DNA viruses suggests that a common DNA pumping mechanism might be operational in both cellular and viral genome segregation. The herA gene forms a highly conserved operon with the gene for the NurA nuclease and, in many archaea, also with the orthologs of eukaryotic double-strand break repair proteins MRE11 and Rad50. HerA is predicted to function in a complex with these proteins in DNA pumping and repair of double-stranded breaks introduced during this process and, possibly, also during DNA replication. Extensive comparative analysis of the 'genomic context' combined with in-depth sequence analysis led to the prediction of numerous previously unnoticed nucleases of the NurA superfamily, including a specific version that is likely to be the endonuclease component of a novel restriction-modification system. This analysis also led to the identification of previously uncharacterized nucleases, such as a novel predicted nuclease of the Sir2-type Rossmann fold, and phosphatases of the HAD superfamily that are likely to function as partners of the FtsK-HerA superfamily ATPases.

Comparative Analysis of Apicomplexa and Genomic Diversity in Eukaryotes

Abstract: The apicomplexans Plasmodium and Cryptosporidium have developed distinctive adaptations via lineage-specific gene loss and gene innovation in the process of diverging from a common parasitic ancestor. The two lineages have acquired distinct but overlapping sets of surface protein adhesion domains typical of animal proteins, but in no case do they share multidomain architectures identical to animals. Cryptosporidium, but not Plasmodium, possesses an animal-type O-linked glycosylation pathway, along with >30 predicted surface proteins having mucin-like segments. The two parasites have notable qualitative differences in conserved protein architectures associated with chromatin dynamics and transcription. Cryptosporidium shows considerable reduction in the number of introns and a concomitant loss of spliceosomal machinery components. We also describe additional molecular characteristics distinguishing Apicomplexa from other eukaryotes for which complete genome sequences are available.

A Multidomain Adhesion Protein Family Expressed in Plasmodium falciparum Is Essential for Transmission to the Mosquito

Abstract: The recent sequencing of several apicomplexan genomes has provided the opportunity to characterize novel antigens essential for the parasite life cycle that might lead to the development of new diagnostic and therapeutic markers. Here we have screened the Plasmodium falciparum genome sequence for genes encoding extracellular multidomain putative adhesive proteins. Three of these identified genes, named PfCCp1, PfCCp2, and PfCCp3, have multiple adhesive modules including a common Limulus coagulation factor C domain also found in two additional Plasmodium genes. Orthologues were identified in the Cryptosporidium parvum genome sequence, indicating an evolutionary conserved function. Transcript and protein expression analysis shows sexual stage-specific expression of PfCCp1, PfCCp2, and PfCCp3, and cellular localization studies revealed plasma membrane-associated expression in mature gametocytes. During gametogenesis, PfCCps are released and localize surrounding complexes of newly emerged microgametes and macrogametes. PfCCp expression markedly decreased after formation of zygotes. To begin to address PfCCp function, the PfCCp2 and PfCCp3 gene loci were disrupted by homologous recombination, resulting in parasites capable of forming oocyst sporozoites but blocked in the salivary gland transition. Our results describe members of a conserved apicomplexan protein family expressed in sexual stage Plasmodium parasites that may represent candidates for subunits of a transmission-blocking vaccine.

A bipolar DNA helicase gene, herA, clusters with rad50, mre11 and nurA genes in thermophilic archaea

Abstract: We showed previously that rad50 and mre11 genes of thermophilic archaea are organized in an operon-like structure with a third gene (nurA) encoding a 5′ to 3′ exonuclease. Here, we show that the rad50, mre11 and nurA genes from the hyperthermo philic archaeon Sulfolobus acidocaldarius are co-transcribed with a fourth gene encoding a DNA helicase. This enzyme (HerA) is the prototype of a new class of DNA helicases able to utilize either 3′ or 5′ single-stranded DNA extensions for loading and subsequent DNA duplex unwinding. To our knowledge, DNA helicases capable of translocating along the DNA in both directions have not been identified previously. Sequence analysis of HerA shows that it is a member of the TrwB, FtsK and VirB4/VirD4 families of the PilT class NTPases. HerA homologs are found in all thermophilic archaeal species and, in all cases except one, the rad50, mre11, nurA and herA genes are grouped together. These results suggest that the archaeal Rad50–Mre11 complex might act in association with a 5′ to 3′ exonuclease (NurA) and a bipolar DNA helicase (HerA) indicating a probable involvement in the initiation step of homologous recombination.

Novel predicted peptidases with a potential role in the ubiquitin signaling pathway.

Abstract: A multi-pronged strategy including extensive sequence searches, structuralmodeling, and analysis of contextual information extracted from domainarchitectures, genetic screens, and large-scale protein-protein interaction analyseswas employed to predict previously undetected components of the eukaryoticubiquitin signaling system. Two novel groups of proteins that are likely to function asde-ubiquitinating and de-SUMOylating peptidases (DUBs) were identified. The firstgroup of putative DUBs, designated PPPDE superfamily (after Permuted Papain foldPeptidases of DsRNA viruses and Eukaryotes), consists of predicted thiol peptidaseswith a circularly permuted papain-like fold. The inference of the likely DUB functionof the PPPDE superfamily proteins is based on the fusions of the catalytic domain toUb-binding PUG (PUB)/UBA domains and a novel alpha-helical Ub-associated domain(the PLAP, Ufd3p and Lub1p or PUL domain) amongst different members of thePPPDE supefamily. The presence of the PPPDE superfamily proteins ...

Novel conserved domains in proteins with predicted roles in eukaryotic cell-cycle regulation, decapping and RNA stability.

Abstract: The emergence of eukaryotes was characterized by the expansion and diversification of several ancient RNA-binding domains and the apparent de novo innovation of new RNA-binding domains The identification of these RNA-binding domains may throw light on the emergence of eukaryote-specific systems of RNA metabolism Using sensitive sequence profile searches, homology-based fold recognition and sequence-structure superpositions, we identified novel, divergent versions of the Sm domain in the Scd6p family of proteins This family of Sm-related domains shares certain features of conventional Sm domains, which are required for binding RNA, in addition to possessing some unique conserved features We also show that these proteins contain a second previously uncharacterized C-terminal domain, termed the FDF domain (after a conserved sequence motif in this domain) The FDF domain is also found in the fungal Dcp3p-like and the animal FLJ22128-like proteins, where it fused to a C-terminal domain of the YjeF-N domain family In addition to the FDF domains, the FLJ22128-like proteins contain yet another divergent version of the Sm domain at their extreme N-terminus We show that the YjeF-N domains represent a novel version of the Rossmann fold that has acquired a set of catalytic residues and structural features that distinguish them from the conventional dehydrogenases Several lines of contextual information suggest that the Scd6p family and the Dcp3p-like proteins are conserved components of the eukaryotic RNA metabolism system We propose that the novel domains reported here, namely the divergent versions of the Sm domain and the FDF domain may mediate specific RNA-protein and protein-protein interactions in cytoplasmic ribonucleoprotein complexes More specifically, the protein complexes containing Sm-like domains of the Scd6p family are predicted to regulate the stability of mRNA encoding proteins involved in cell cycle progression and vesicular assembly The Dcp3p and FLJ22128 proteins may localize to the cytoplasmic processing bodies and possibly catalyze a specific processing step in the decapping pathway The explosive diversification of Sm domains appears to have played a role in the emergence of several uniquely eukaryotic ribonucleoprotein complexes, including those involved in decapping and mRNA stability

A novel family of P-loop NTPases with an unusual phyletic distribution and transmembrane segments inserted within the NTPase domain

Abstract: Background Recent sequence-structure studies on P-loop-fold NTPases have substantially advanced the existing understanding of their evolution and functional diversity. These studies provide a framework for characterization of novel lineages within this fold and prediction of their functional properties.

The SHS2 module is a common structural theme in functionally diverse protein groups, like Rpb7p, FtsA, GyrI, and MTH1598/TM1083 superfamilies

Abstract: Using structural comparisons, we identified a novel domain with a simple fold in the bacterial cell division ATPase FtsA, the archaeo-eukaryotic RNA polymerase subunit Rpb7p, the GyrI superfamily, and the uncharacterized MTH1598/Tm1083-like proteins. The fold contains a core of 3 strands, forming a curved sheet, and a single helix in a strand-helix-strand-strand (SHS2) configuration. The SHS2 domain may exist either in single or duplicate copies within the same polypeptide. The single-copy versions of the domain in FtsA and Rbp7p are most closely related, and appear to mediate protein-protein interactions by means of strand 1, and the loop between strand 2 and strand 3 of the domain. We predict that the interactions between FtsA and its functional partners in bacterial cell division are likely to be similar to the interactions of Rbp7p in the archaeo-eukaryotic RNA polymerase complex. The dimeric versions typified by the GyrI superfamily appear to have been adapted for small-molecule binding. Sequence profiles searches helped us to identify several new versions of the GyrI superfamily, including a family of secreted forms that is found only in animals and the bacterial pathogen Leptospira. Through sequence-structure comparisons, we predict the positions that are likely to be important for ligand specificity in the GyrI superfamily. In the MTH1598/Tm1083-like proteins, a SHS2 domain is inserted into the loop between strand 1 and helix 1 of another SHS2 domain. This has resulted in a structure that has convergent similarities with the Hsp33 and green fluorescent protein folds. The sequence conservation pattern and its phyletic profile suggest that it might function as an enzyme in some conserved aspect of nucleic acid metabolism. Thus, the SHS2 domain is an example of a simple module that has been adapted to perform an entire spectrum of functions ranging from protein-protein interactions to small-molecule recognition and catalysis.

The emergence of catalytic and structural diversity within the beta-clip fold.

Abstract: The beta-clip fold includes a diverse group of protein domains that are unified by the presence of two characteristic waist-like constrictions, which bound a central extended region Members of this fold include enzymes like deoxyuridine triphosphatase and the SET methylase, carbohydrate-binding domains like the fish antifreeze proteins/Sialate synthase C-terminal domains, and functionally enigmatic accessory subunits of urease and molybdopterin biosynthesis protein MoeA In this study, we reconstruct the evolutionary history of this fold using sensitive sequence and structure comparisons methods Using sequence profile searches, we identified novel versions of the beta-clip fold in the bacterial flagellar chaperone FlgA and the related pilus protein CpaB, the StrU-like dehydrogenases, and the UxaA/GarD-like hexuronate dehydratases (SAF superfamily) We present evidence that these versions of the beta-clip domain, like the related type III anti-freeze proteins and C-terminal domains of sialic acid synthases, are involved in interactions with carbohydrates We propose that the FlgA and CpaB-like proteins mediate the assembly of bacterial flagella and Flp pili by means of their interactions with the carbohydrate moieties of peptidoglycan The N-terminal beta-clip domain of the hexuronate dehydratases appears to have evolved a novel metal-binding site, while their C-terminal domain is likely to adopt a metal-binding TIM barrel-like fold Using structural comparisons, we show that the beta-clip fold can be further classified into two major groups, one that includes the SAF, SET, dUTPase superfamilies, and the other that includes the phage lambda head decoration protein, the beta subunit of urease and the C-terminal domain of the molybdenum cofactor biosynthesis protein MoeA Structural comparisons also suggest the beta-clip fold was assembled through the duplication of a three-stranded unit Though the three-stranded units are likely to have had a common origin, we present evidence that complete beta-clip domains were assembled through such duplications, independently on multiple occasions There is also evidence for circular permutation of the basic three-stranded unit on different occasions in the evolution of the beta-clip unit We also describe how assembly of this fold from a basic three-stranded unit has been utilized to accommodate a variety of activities in its different versions