1) Evolutionary classification of P-loop NTPases As a part of my ongoing research on the P-loop NTPases, which constitute the largest set of monophyletic protein domains in most proteomes, we completed the evolutionary classification of the AAA+ ATPases and the P-loop kinases. These studies helped us to identify the early diversification events in the history of these proteins. We provide evidence that the P-loop NTPases first differentiated into two classes, namely the KG class (Kinase, GTPase class) and the ASCE class (Additional strand conserved glutamate) that includes the AAA+, RecA-like, PilT/VirD4-like and ABC ATPases. These classes had several representatives that could be traced back to the last universal common ancestor of all life forms suggesting that they had undergone a vast radiation even before that stage. Hence they provide insights into some of the earliest aspects of protein evolution. 2) Analysis of the novel protease families A multi-pronged strategy including extensive sequence searches, structural modeling, and analysis of contextual information extracted from domain architectures, genetic screens, and large-scale protein-protein interaction analyses was employed to predict previously undetected components of the eukaryotic ubiquitin (Ub) signaling system. Two novel groups of proteins that are likely to function as de-ubiquitinating and de-SUMOylating peptidases (DUBs) were identified. The first group of putative DUBs, designated PPPDE superfamily (after Permuted Papain fold Peptidases of DsRNA viruses and Eukaryotes), consists of predicted thiol peptidases with a circularly permuted papain-like fold. In addition to eukaryotic proteins, the PPPDE superfamily includes predicted proteases from several groups of double-stranded RNA viruses and one single-stranded DNA virus. The apparent recruitment of DUBs for viral polyprotein processing seems to represent a common theme in evolution of viruses. The second group of putative DUBs identified in this study is the WLM (Wss1p-like metalloproteases) family of the Zincin-like superfamily of Zn-dependent peptidases, which are linked to the Ub-system by virtue of fusions with the UB-binding PUG (PUB), Ub-like, and Little Finger domains. More specifically, genetic evidence implicates the WLM family in de-SUMOylation. If validated experimentally, the WLM family proteins will represent the first case of a Zincin-like metalloprotease involvement in Ub-signaling. 3) Evolution of the nuclear membrane and nuclear pore complex The presence of a distinct nucleus, the compartment for confining the genome, transcription and RNA maturation, is a central (and eponymous) feature that distinguishes eukaryotes from prokaryotes. Structural integrity of the nucleus is maintained by the nuclear envelope (NE). A crucial element of this structure is the nuclear pore complex (NPC), a macromolecular machine with over 90 protein components, which mediates nucleo-cytoplasmic communication. Given the indispensability of these structures for nuclear function, the natural history of the nucleus can only be understood in terms of the origin and subsequent evolution of NE and NPC components. We investigated the provenance of the conserved domains found in these perinuclear proteins and reconstructed a parsimonious scenario for NE and NPC evolution by means of comparative-genomic analysis of their components from the available sequences of 28 sequenced eukaryotic genomes. We show that the NE and NPC proteins were tinkered together from diverse domains, which evolved from prokaryotic precursors at different points in eukaryotic evolution, divergence from pre-existing eukaryotic paralogs performing other functions, and de novo. It is shown that several central components of the NPC, in particular, the RanGDP import factor NTF2, the HEH domain of Src1p-Man1, and, probably, also the key domains of karyopherins and nucleoporins, the HEAT/ARM and WD40 repeats, have a bacterial, most likely, endosymbiotic origin. The specialized immunoglobulin (Ig) domain in the globular tail of the animal lamins, and the Ig domains in the nuclear membrane protein GP210 are shown to be related to distinct prokaryotic families of Ig domains. This suggests that independent, late horizontal gene transfer events from bacterial sources might have contributed to the evolution of perinuclear proteins in some of the major eukaryotic lineages. Snurportin 1, one of the highly conserved karyopherins, contains a cap-binding domain which is shown to be an inactive paralog of the guanylyl transferase domain of the mRNA-capping enzyme, exemplifying recruitment of paralogs of pre-exsiting proteins for perinuclear functions. We infer an autogenous scenario of nuclear evolution in which the nucleus emerged in the primitive eukaryotic ancestor (the ?prekaryote?) as part of cell compartmentalization triggered by archaeo-bacterial symbiosis. A pivotal event in this process was the radiation of Ras-superfamily GTPases yielding Ran, the key regulator of nuclear transport. A primitive NPC with approximately 20 proteins and a Src1p-Man1-like membrane protein with a DNA-tethering HEH domain are inferred to have been integral perinuclear components in the las common ancestor of modern eukaryotes.
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