The Evolutionary History of Protein Domains Viewed by Species Phylogeny

Background

Protein structural domains are evolutionary units whose relationships can be detected over long evolutionary distances. The evolutionary history of protein domains, including the origin of protein domains, the identification of domain loss, transfer, duplication and combination with other domains to form new proteins, and the formation of the entire protein domain repertoire, are of great interest.

Methodology/Principal Findings

A methodology is presented for providing a parsimonious domain history based on gain, loss, vertical and horizontal transfer derived from the complete genomic domain assignments of 1015 organisms across the tree of life. When mapped to species trees the evolutionary history of domains and domain combinations is revealed, and the general evolutionary trend of domain and combination is analyzed.

Conclusions/Significance

We show that this approach provides a powerful tool to study how new proteins and functions emerged and to study such processes as horizontal gene transfer among more distant species.     

Protein Phosphatases and Signaling Cascades in Higher Plants

Protein kinases and phosphatases play a central role in cellular signaling. Although protein kinases have been widely studied in higher plants, protein phosphatases have been largely neglected until recently. The article focuses on the most recent studies that have placed protein phosphatases in the context of several signal cascades in higher plants. These pathways include stomatal regulation and abscisic acid signal transduction, pathogen and stress responses, and developmental control. Studies clearly have demonstrated that protein phosphatases function not only to counterbalance the protein kinases but also take a leading role in many signaling processes.     

高等植物体内酪氨酸蛋白磷酸酶及其功能

对高等植物体内酪氨酸蛋白磷酸酶及其功能的研究进展作了简要介绍.     

A Unique Protein Phosphatase with Kelch-Like Domains (PPKL) in Plasmodium Modulates Ookinete Differentiation,Motility and Invasion

Protein phosphorylation and dephosphorylation (catalysed by kinases and phosphatases, respectively) are post-translational modifications that play key roles in many eukaryotic signalling pathways, and are often deregulated in a number of pathological conditions in humans. In the malaria parasite Plasmodium, functional insights into its kinome have only recently been achieved, with over half being essential for blood stage development and another 14 kinases being essential for sexual development and mosquito transmission. However, functions for any of the plasmodial protein phosphatases are unknown. Here, we use reverse genetics in the rodent malaria model, Plasmodium berghei, to examine the role of a unique protein phosphatase containing kelch-like domains (termed PPKL) from a family related to Arabidopsis BSU1. Phylogenetic analysis confirmed that the family of BSU1-like proteins including PPKL is encoded in the genomes of land plants, green algae and alveolates, but not in other eukaryotic lineages. Furthermore, PPKL was observed in a distinct family, separate to the most closely-related phosphatase family, PP1. In our genetic approach, C-terminal GFP fusion with PPKL showed an active protein phosphatase preferentially expressed in female gametocytes and ookinetes. Deletion of the endogenous ppkl gene caused abnormal ookinete development and differentiation, and dissociated apical microtubules from the inner-membrane complex, generating an immotile phenotype and failure to invade the mosquito mid-gut epithelium. These observations were substantiated by changes in localisation of cytoskeletal tubulin and actin, and the micronemal protein CTRP in the knockout mutant as assessed by indirect immunofluorescence. Finally, increased mRNA expression of dozi, a RNA helicase vital to zygote development was observed in ppkl⁻ mutants, with global phosphorylation studies of ookinete differentiation from 1.5–24 h post-fertilisation indicating major changes in the first hours of zygote development. Our work demonstrates a stage-specific essentiality of the unique PPKL enzyme, which modulates parasite differentiation, motility and transmission.     

ECOD: An Evolutionary Classification of Protein Domains

Understanding the evolution of a protein, including both close and distant relationships, often reveals insight into its structure and function. Fast and easy access to such up-to-date information facilitates research. We have developed a hierarchical evolutionary classification of all proteins with experimentally determined spatial structures, and presented it as an interactive and updatable online database. ECOD (Evolutionary Classification of protein Domains) is distinct from other structural classifications in that it groups domains primarily by evolutionary relationships (homology), rather than topology (or “fold”). This distinction highlights cases of homology between domains of differing topology to aid in understanding of protein structure evolution. ECOD uniquely emphasizes distantly related homologs that are difficult to detect, and thus catalogs the largest number of evolutionary links among structural domain classifications. Placing distant homologs together underscores the ancestral similarities of these proteins and draws attention to the most important regions of sequence and structure, as well as conserved functional sites. ECOD also recognizes closer sequence-based relationships between protein domains. Currently, approximately 100,000 protein structures are classified in ECOD into 9,000 sequence families clustered into close to 2,000 evolutionary groups. The classification is assisted by an automated pipeline that quickly and consistently classifies weekly releases of PDB structures and allows for continual updates. This synchronization with PDB uniquely distinguishes ECOD among all protein classifications. Finally, we present several case studies of homologous proteins not recorded in other classifications, illustrating the potential of how ECOD can be used to further biological and evolutionary studies.     

PTP及其在植物MAPK途径中的作用

蛋白酪氨酸磷酸酶（protein tyrosine phosphatases,PTPs）是一个结构多样的磷酸酶家族,含有高度保守的催化结构域。在植物体内,PTP主要的靶蛋白是促细胞分裂剂激活性蛋白激酶（mitogen-activated protein kinase,MAPK）。MAPK级联途径参与有机体的发育、细胞增殖、激素调节以及逆境胁迫的信号转导,PTP在MAPK级联途径中主要起负调控作用。本文就PTP的结构和功能、MAPK在植物中的作用及PTP在MAPK级联途径中的功能进行综述,并着重介绍PTP在拟南芥中的研究进展。     

PTP 及其在植物MAPK 途径中的作用

蛋白酪氨酸磷酸酶(protein tyrosine phosphatases, PTPs)是一个结构多样的磷酸酶家族, 含有高度保守的催化结构域。在植物体内, PTP主要的靶蛋白是促细胞分裂剂激活性蛋白激酶(mitogen-activated protein kinase, MAPK)。MAPK级联途径参与有机体的发育、细胞增殖、激素调节以及逆境胁迫的信号转导, PTP在MAPK级联途径中主要起负调控作用。本文就PTP的结构和功能、MAPK在植物中的作用及PTP在MAPK级联途径中的功能进行综述, 并着重介绍PTP在拟南芥中的研究进展。     

Conserved Roles of the Prion Protein Domains on Subcellular Localization and Cell-Cell Adhesion

Analyses of cultured cells and transgenic mice expressing prion protein (PrP) deletion mutants have revealed that some properties of PrP -such as its ability to misfold, aggregate and trigger neurotoxicity- are controlled by discrete molecular determinants within its protein domains. Although the contributions of these determinants to PrP biosynthesis and turnover are relatively well characterized, it is still unclear how they modulate cellular functions of PrP. To address this question, we used two defined activities of PrP as functional readouts: 1) the recruitment of PrP to cell-cell contacts in Drosophila S2 and human MCF-7 epithelial cells, and 2) the induction of PrP embryonic loss- and gain-of-function phenotypes in zebrafish. Our results show that homologous mutations in mouse and zebrafish PrPs similarly affect their subcellular localization patterns as well as their in vitro and in vivo activities. Among PrP’s essential features, the N-terminal leader peptide was sufficient to drive targeting of our constructs to cell contact sites, whereas lack of GPI-anchoring and N-glycosylation rendered them inactive by blocking their cell surface expression. Importantly, our data suggest that the ability of PrP to homophilically trans-interact and elicit intracellular signaling is primarily encoded in its globular domain, and modulated by its repetitive domain. Thus, while the latter induces the local accumulation of PrPs at discrete punctae along cell contacts, the former counteracts this effect by promoting the continuous distribution of PrP. In early zebrafish embryos, deletion of either domain significantly impaired PrP’s ability to modulate E-cadherin cell adhesion. Altogether, these experiments relate structural features of PrP to its subcellular distribution and in vivo activity. Furthermore, they show that despite their large evolutionary history, the roles of PrP domains and posttranslational modifications are conserved between mouse and zebrafish.     

The Molecular Mechanism Underlying Morphine-Induced Akt Activation: Roles of Protein Phosphatases and Reactive Oxygen Species

Although Akt is reported to play a role in morphine’s cardioprotection, little is known about the mechanism underlying morphine-induced Akt activation. This study aimed to define the molecular mechanism underlying morphine-induced Akt activation and to determine if the mechanism contributes to the protective effect of morphine on ischemia/reperfusion injury. In cardiac H9c2 cells, morphine increased Akt phosphorylation at Ser⁴⁷³, indicating that morphine upregulates Akt activity. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a major regulator of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling, was not involved in the action of morphine on Akt activity. Morphine decreased the activity of PP2A, a major protein Ser/Thr phosphatase, and inhibition of PP2A with okadaic acid (OA) mimicked the effect of morphine on Akt activity. The effects of morphine on PP2A and Akt activities were inhibited by the reactive oxygen species (ROS) scavenger N-(2-mercaptopropionyl)glycine (MPG) and the mitochondrial K_ATP channel closer 5-hydroxydecanoate (5HD). In support, morphine could produce ROS and this was reversed by 5HD. Finally, the cardioprotective effect of morphine on ischemia–reperfusion injury was mimicked by OA but was suppressed by 5HD or MPG, indicating that protein phosphatases and ROS are involved in morphine’s protection. In conclusion, morphine upregulates Akt activity by inactivating protein Ser/Thr phosphatases via ROS, which may contribute to the cardioprotective effect of morphine.     

Vacuolar SNARE Protein Transmembrane Domains Serve as Nonspecific Membrane Anchors with Unequal Roles in Lipid Mixing

Membrane fusion is induced by SNARE complexes that are anchored in both fusion partners. SNAREs zipper up from the N to C terminus bringing the two membranes into close apposition. Their transmembrane domains (TMDs) might be mere anchoring devices, deforming bilayers by mechanical force. Structural studies suggested that TMDs might also perturb lipid structure by undergoing conformational transitions or by zipping up into the bilayer. Here, we tested this latter hypothesis, which predicts that the activity of SNAREs should depend on the primary sequence of their TMDs. We replaced the TMDs of all vacuolar SNAREs (Nyv1, Vam3, and Vti1) by a lipid anchor, by a TMD from a protein unrelated to the membrane fusion machinery, or by artificial leucine-valine sequences. Individual exchange of the native SNARE TMDs against an unrelated transmembrane anchor or an artificial leucine-valine sequence yielded normal fusion activities. Fusion activity was also preserved upon pairwise exchange of the TMDs against unrelated peptides, which eliminates the possibility for specific TMD-TMD interactions. Thus, a specific primary sequence or zippering beyond the SNARE domains is not a prerequisite for fusion. Lipid-anchored Vti1 was fully active, and lipid-anchored Nyv1 permitted the reaction to proceed up to hemifusion, and lipid-anchored Vam3 interfered already before hemifusion. The unequal contribution of proteinaceous TMDs on Vam3 and Nyv1 suggests that Q- and R-SNAREs might make different contributions to the hemifusion intermediate and the opening of the fusion pore. Furthermore, our data support the view that SNARE TMDs serve as nonspecific membrane anchors in vacuole fusion.     

Recent Evolutionary History of Tigers Highlights Contrasting Roles of Genetic Drift and Selection

Species conservation can be improved by knowledge of evolutionary and genetic history. Tigers are among the most charismatic of endangered species and garner significant conservation attention. However, their evolutionary history and genomic variation remain poorly known, especially for Indian tigers. With 70% of the world’s wild tigers living in India, such knowledge is critical. We re-sequenced 65 individual tiger genomes representing most extant subspecies with a specific focus on tigers from India. As suggested by earlier studies, we found strong genetic differentiation between the putative tiger subspecies. Despite high total genomic diversity in India, individual tigers host longer runs of homozygosity, potentially suggesting recent inbreeding or founding events, possibly due to small and fragmented protected areas. We suggest the impacts of ongoing connectivity loss on inbreeding and persistence of Indian tigers be closely monitored. Surprisingly, demographic models suggest recent divergence (within the last 20,000 years) between subspecies and strong population bottlenecks. Amur tiger genomes revealed the strongest signals of selection related to metabolic adaptation to cold, whereas Sumatran tigers show evidence of weak selection for genes involved in body size regulation. We recommend detailed investigation of local adaptation in Amur and Sumatran tigers prior to initiating genetic rescue.     

Coarse-Grained Simulations of the HIV-1 Matrix Protein Anchoring: Revisiting Its Assembly on Membrane Domains

In the accepted model for human immunodeficiency virus preassembly in infected host cells, the anchoring to the intracellular leaflet of the membrane of the matrix domain (MA) that lies at the N-terminus of the viral Gag protein precursor appears to be one of the crucial steps for particle assembly. In this study, we simulated the membrane anchoring of human immunodeficiency virus-1 myristoylated MA protein using a coarse-grained representation of both the protein and the membrane. Our calculations first suggest that the myristoyl group could spontaneously release from its initial hydrophobic pocket before MA protein interacts with the lipid membrane. All-atom simulations confirmed this possibility with a related energy cost estimated to be ∼5 kcal.mol⁻¹. The phosphatidylinositol (4,5) bisphosphate (PI(4,5)P₂) head binds preferentially to the MA highly basic region as described in available NMR data, but interestingly without flipping of its 2′ acyl chain into the MA protein. Moreover, MA was able to confine PI(4,5)P₂ lipids all around its molecular surface after having found a stable orientation at the membrane surface. Our results suggest that this orientation is dependent on Myr anchoring and that this confinement induces a lateral segregation of PI(4,5)P₂ in domains. This is consistent with a PI(4,5)P₂ enrichment of the virus envelope as compared to the host cell membrane.     

Endogenous Basic Protein Phosphatases in the Brain Myelin

Direct treatment of brain myelin with freezing/thawing in 0.2 M 2-mercaptoethanol stimulated the endogenous myelin phosphatase activity manyfold when 32P-labeled phosphorylase a was used as a substrate, a result indicating that an endogenous myelin phosphatase is a latent protein phosphatase. When myelin was treated with Triton X-100, this endogenous latent phosphatase activity was further stimulated 2.5-fold. Diethylaminoethyl-cellulose and Sephadex G-200 chromatography of solubilized myelin revealed a pronounced peak of protein phosphatase activity stimulated by freezing/thawing in 0.2 M 2-mercaptoethanol and with a molecular weight of 350,000, which is characteristic of latent phosphatase 2, as previously reported. Moreover, endogenous phosphorylation of myelin basic protein (MBP) in brain myelin was completely reversed by a homogeneous preparation of exogenous latent phosphatase 2. By contrast, under the same conditions, endogenous phosphorylation of brain myelin was entirely unaffected by ATP X Mg-dependent phosphatase and latent phosphatase 1, although both enzymes are potent MBP phosphatases. Together, these findings clearly indicate that a high-molecular-weight latent phosphatase, termed latent phosphatase 2, is the most predominant phosphatase responsible for dephosphorylation of brain myelin.     

Coarse-Grained Simulations of the HIV-1 Matrix Protein Anchoring: Revisiting Its Assembly on Membrane Domains

In the accepted model for human immunodeficiency virus preassembly in infected host cells, the anchoring to the intracellular leaflet of the membrane of the matrix domain (MA) that lies at the N-terminus of the viral Gag protein precursor appears to be one of the crucial steps for particle assembly. In this study, we simulated the membrane anchoring of human immunodeficiency virus-1 myristoylated MA protein using a coarse-grained representation of both the protein and the membrane. Our calculations first suggest that the myristoyl group could spontaneously release from its initial hydrophobic pocket before MA protein interacts with the lipid membrane. All-atom simulations confirmed this possibility with a related energy cost estimated to be ∼5 kcal.mol⁻¹. The phosphatidylinositol (4,5) bisphosphate (PI(4,5)P₂) head binds preferentially to the MA highly basic region as described in available NMR data, but interestingly without flipping of its 2′ acyl chain into the MA protein. Moreover, MA was able to confine PI(4,5)P₂ lipids all around its molecular surface after having found a stable orientation at the membrane surface. Our results suggest that this orientation is dependent on Myr anchoring and that this confinement induces a lateral segregation of PI(4,5)P₂ in domains. This is consistent with a PI(4,5)P₂ enrichment of the virus envelope as compared to the host cell membrane.     

Biological Roles of Prion Domains

In vivo amyloid formation is a widespread phenomenon in eukaryotes. Self-perpetuating amyloids provide a basis for the infectious or heritable protein isoforms (prions). At least for some proteins, amyloid-forming potential is conserved in evolution despite divergence of the amino acid (aa) sequences. In some cases, prion formation certainly represents a pathological process leading to a disease. However, there are several scenarios in which prions and other amyloids or amyloid-like aggregates are either shown or suspected to perform positive biological functions. Proven examples include self/nonself recognition, stress defense and scaffolding of other (functional) polymers. The role of prion-like phenomena in memory has been hypothesized. As an additional mechanism of heritable change, prion formation may in principle contribute to heritable variability at the population level. Moreover, it is possible that amyloid-based prions represent by-products of the transient feedback regulatory circuits, as normal cellular function of at least some prion proteins is decreased in the prion state.Key Words: amyloid, amyloidosis, epigenetic, evolution, inheritance, mammals, misfolding, protein, stress, yeast     

Disentangling the Roles of Functional Domains in the Aggregation and Adsorption of the Multimodular Sea Star Adhesive Protein Sfp1

Marine Biotechnology - Sea stars can adhere to various underwater substrata using an adhesive secretion of which Sfp1 is a major component. Sfp1 is a multimodular protein composed of four subunits...