Preferred in vivo ubiquitination sites

MOTIVATION: The conjugation of ubiquitin to target molecules involves several enzymatic steps. Little is known about the specificity of ubiquitination. How E3 ligases select their substrate and which lysines are targeted for ubiquitin conjugation is largely an enigma. The object of this study is to identify preferred ubiquitination sites. Genetic approaches to study this question have proven difficult, because of the redundancy of ligases and the lack of strictly required motifs. However, a better understanding of acceptor site selection could help to predict ubiquitination sites and clarify yet unsolved structure-function relationships of the transfer reaction. RESULTS: In an effort to define preferences for ubiquitination, we systematically analyzed structure and sequence of 135 known ubiquitination sites in 95 proteins in Saccharomyces cerevisiae. The results show clear structural preferences for ubiquitin ligation to target proteins, and compartment-specific amino acid patterns in close proximity to the modified side chain. SUPPLEMENTARY INFORMATION: http://www.people.fas.harvard.edu/~catic.     

The origins and ongoing evolution of viruses

Genome analyses of double strand DNA tailed bacteriophages argue that they evolve by recombinational reassortment of genes and by the acquisition of novel genes as simple genetic elements termed morons. These processes suggest a model for early virus evolution, wherein viruses can be regarded less as having derived from cells and more as being partners in their mutual co-evolution.     

The evolution of milk secretion and its ancient origins

Lactation represents an important element of the life history strategies of all mammals, whether monotreme, marsupial, or eutherian. Milk originated as a glandular skin secretion in synapsids (the lineage ancestral to mammals), perhaps as early as the Pennsylvanian period, that is, approximately 310 million years ago (mya). Early synapsids laid eggs with parchment-like shells intolerant of desiccation and apparently dependent on glandular skin secretions for moisture. Mammary glands probably evolved from apocrine-like glands that combined multiple modes of secretion and developed in association with hair follicles. Comparative analyses of the evolutionary origin of milk constituents support a scenario in which these secretions evolved into a nutrient-rich milk long before mammals arose. A variety of antimicrobial and secretory constituents were co-opted into novel roles related to nutrition of the young. Secretory calcium-binding phosphoproteins may originally have had a role in calcium delivery to eggs; however, by evolving into large, complex casein micelles, they took on an important role in transport of amino acids, calcium and phosphorus. Several proteins involved in immunity, including an ancestral butyrophilin and xanthine oxidoreductase, were incorporated into a novel membrane-bound lipid droplet (the milk fat globule) that became a primary mode of energy transfer. An ancestral c-lysozyme lost its lytic functions in favor of a role as α-lactalbumin, which modifies a galactosyltransferase to recognize glucose as an acceptor, leading to the synthesis of novel milk sugars, of which free oligosaccharides may have predated free lactose. An ancestral lipocalin and an ancestral whey acidic protein four-disulphide core protein apparently lost their original transport and antimicrobial functions when they became the whey proteins β-lactoglobulin and whey acidic protein, which with α-lactalbumin provide limiting sulfur amino acids to the young. By the late Triassic period (ca 210 mya), mammaliaforms (mammalian ancestors) were endothermic (requiring fluid to replace incubatory water losses of eggs), very small in size (making large eggs impossible), and had rapid growth and limited tooth replacement (indicating delayed onset of feeding and reliance on milk). Thus, milk had already supplanted egg yolk as the primary nutrient source, and by the Jurassic period (ca 170 mya) vitellogenin genes were being lost. All primary milk constituents evolved before the appearance of mammals, and some constituents may have origins that predate the split of the synapsids from sauropsids (the lineage leading to 'reptiles' and birds). Thus, the modern dairy industry is built upon a very old foundation, the cornerstones of which were laid even before dinosaurs ruled the earth in the Jurassic and Cretaceous periods.     

The Gibbs conference on biothermodynamics: origins and evolution

The Gibbs conference on biothermodynamics arose in the late 1980's as a 'self-organized' endeavor by researchers at eleven institutions of the US. Over a period of 10 years these annual conferences have grown steadily in size. They have fostered the development of new thermodynamic approaches and their applications in biochemistry. By emphasizing participation by students and postdoctoral fellows they have contributed significantly to the career development of young scientists in this field.     

Triggering ubiquitination of a CDK inhibitor at origins of DNA replication

To ensure proper timing of the G1-S transition in the cell cycle, the cyclin E-Cdk2 complex, which is responsible for the initiation of DNA replication, is restrained by the p21(Cip1)/p27(Kip1)/p57(Kip2) family of CDK (cyclin-dependent kinase) inhibitors in humans and by the related p27(Xic1) protein in Xenopus. Activation of cyclin E-Cdk2 is linked to the ubiquitination of human p27(Kip1) or Xenopus p27(Xic1) by SCF (for Skp1-Cullin-F-box protein) ubiquitin ligases. For human p27(Kip1), ubiquitination requires direct phosphorylation by cyclin E-Cdk2. We show here that Xic1 ubiquitination does not require phosphorylation by cyclin E-Cdk2, but it does require nuclear accumulation of the Xic1-cyclin E-Cdk2 complex and recruitment of this complex to chromatin by the origin-recognition complex together with Cdc6 replication preinitiation factors; it also requires an activation step necessitating cyclin E-Cdk2-kinase and SCF ubiquitin-ligase activity, and additional factors associated with mini-chromosome maintenance proteins, including the inactivation of geminin. Components of the SCF ubiquitin-ligase complex, including Skp1 and Cul1, are also recruited to chromatin through cyclin E-Cdk2 and the preinitiation complex. Thus, activation of the cyclin E-Cdk2 kinase and ubiquitin-dependent destruction of its inhibitor are spatially constrained to the site of a properly assembled preinitiation complex.     

Replication origins, factors and attachment sites

The initiation of eukaryotic DNA synthesis occurs at specific sites determined by both cis- and trans-acting elements. Here I review advances in the characterization of yeast origins, origin-binding proteins and the relationship of DNA replication to nuclear substructure in yeast.     

Molecular evolution and modern human origins

While molecular evolutionists may be fascinated by the features and history of a particular gene or DNA segment, evolutionary anthropologists are often more interested in the activities and history of groups of people. We may want to know, for instance, when and where humans have migrated, how much exchange between groups has taken place, and how population sizes have changed. Population genetic theory provides the hope that through analyses of genetic data we will gain insight into the history of populations. Genetic data from extant human populations are now accruing at a remarkable rate. We might, therefore, expect to have answers in hand. There remains, however, a wide gap between the available theory and data; too often we fail to draw firm conclusions because our interpretation of analytic results requires that we make myriad assumptions about our data. In any one instance, these assumptions might include estimates of mutation rate, mutational mechanism, population sizes, the role that natural selection has played, and the rate of migration among groups. Often these assumptions are implicit, invisible to most. How, then, are we to make any progress? © 1998 Wiley-Liss, Inc.     

Mapping ubiquitination sites of S. cerevisiae Mcm10

Minichromosome maintenance protein (Mcm) 10 is a part of the eukaryotic replication machinery and highly conserved throughout evolution. As a multivalent DNA scaffold, Mcm10 coordinates the action of proteins that are indispensable for lagging strand synthesis, such as the replication clamp, proliferating cell nuclear antigen (PCNA). The binding between Mcm10 and PCNA serves an essential function during DNA elongation and is mediated by the ubiquitination of Mcm10. Here we map lysine 372 as the primary attachment site for ubiquitin on S. cerevisiae Mcm10. Moreover, we identify five additional lysines that can be ubiquitinated. Mutation of lysine 372 to arginine ablates ubiquitination of overexpressed protein and causes sensitivity to the replication inhibitor hydroxyurea in cells that are S-phase checkpoint compromised. Together, these findings reveal the high selectivity of the ubiquitination machinery that targets Mcm10 and that ubiquitination has a role in suppressing replication stress.     

A data set of human endogenous protein ubiquitination sites

Lysine ubiquitination is an important and versatile protein post-translational modification. Numerous cellular functions are regulated by ubiquitination, suggesting that extensive numbers of proteins, if not all, are modified with ubiquitin at certain times. However, proteome-wide profiling of ubiquitination sites in the mammalian system is technically challenging. We report the design and characterization of an engineered protein affinity reagent for the isolation of ubiquitinated proteins and the identification of ubiquitination sites with mass spectrometry. This recombinant protein consists of four tandem repeats of ubiquitin-associated domain from UBQLN1 fused to a GST tag. We used this GST-qUBA reagent to isolate polyubiquitinated proteins and identified 294 endogenous ubiquitination sites on 223 proteins from human 293T cells without proteasome inhibitors or overexpression of ubiquitin. Mitochondrial proteins constitute 14.7% of this data set, implicating ubiquitination in a wide range of mitochondrial functions.     

Primate origins and the evolution of angiosperms

Traditionally, the morphological traits of primates were assumed to be adaptations to an arboreal way of life. However, Cartmill [1972] pointed out that a number of morphological traits characteristic of primates are not found in many other arboreal mammals. He contends that orbital convergence and grasping extremities indicate that the initial divergence of primates involved visual predation on insects in the lower canopy and undergrowth of the tropical forest. However, recent research on nocturnal primates does not support the visually-oriented predation theory. Although insects were most likely important components of the diets of the earliest euprimates, it is argued here that visual predation was not the major impetus for the evolution of the adaptive traits of primates. Recent paleobotanical research has yielded evidence that a major evolutionary event occurred during the Eocene, involving the angiosperms and their dispersal agents. As a result of long-term diffuse coevolutionary interactions with flowering plants, modern primates, bats, and plant-feeding birds all first arose around the Paleocene-Eocene boundary and became the major seed dispersers of modern tropical flora during the Eocene. Thus, it is suggested here that the multitude of resources available on the terminal branches of the newly evolved angiosperm, rain forest trees led to the morphological adaptations of primates of modern aspect.     

Evolutionary origins and directed evolution of RNA

In vitro selection experiments show first and foremost that it is possible that functional nucleic acids can arise from random sequence libraries. Indeed, even simple sequence and structural motifs can prove to be robust binding species and catalysts, indicating that it may have been possible to transition from even the earliest self-replicators to a nascent, RNA-catalyzed metabolism. Because of the diversity of aptamers and ribozymes that can be selected, it is possible to construct a 'fossil record' of the evolution of the RNA world, with in vitro selected catalysts filling in as doppelgangers for molecules long gone. In this way a plausible pathway from simple oligonucleotide replicators to genomic polymerases can be imagined, as can a pathway from basal ribozyme activities to the ribosome. Most importantly, though, in vitro selection experiments can give a true and quantitative idea of the likelihood that these scenarios could have played out in the RNA world. Simple binding species and catalysts could have evolved into other structures and functions. As replicating sequences grew longer, new, more complex functions or faster catalytic activities could have been accessed. Some activities may have been isolated in sequence space, but others could have been approached along large, interconnected neutral networks. As the number, type, and length of ribozymes increased, RNA genomes would have evolved and eventually there would have been no area in a fitness landscape that would have been inaccessible. Self-replication would have inexorably led to life.     

iUbiq-Lys: prediction of lysine ubiquitination sites in proteins by extracting sequence evolution information via a gray system model

As one of the most important posttranslational modifications (PTMs), ubiquitination plays an important role in regulating varieties of biological processes, such as signal transduction, cell division, apoptosis, and immune response. Ubiquitination is also named “lysine ubiquitination” because it occurs when an ubiquitin is covalently attached to lysine (K) residues of targeting proteins. Given an uncharacterized protein sequence that contains many lysine residues, which one of them is the ubiquitination site, and which one is of non-ubiquitination site? With the avalanche of protein sequences generated in the postgenomic age, it is highly desired for both basic research and drug development to develop an automated method for rapidly and accurately annotating the ubiquitination sites in proteins. In view of this, a new predictor called “iUbiq-Lys” was developed based on the evolutionary information, gray system model, as well as the general form of pseudo-amino acid composition. It was demonstrated via the rigorous cross-validations that the new predictor remarkably outperformed all its counterparts. As a web-server, iUbiq-Lys is accessible to the public at http://www.jci-bioinfo.cn/iUbiq-Lys. For the convenience of most experimental scientists, we have further provided a protocol of step-by-step guide, by which users can easily get their desired results without the need to follow the complicated mathematics that were presented in this paper just for the integrity of its development process.