Systematic simulation of the interactions of Pleckstrin homology domains with membranes

Pleckstrin homology (PH) domains can recruit proteins to membranes by recognition of phosphatidylinositol phosphates (PIPs). Here we report the systematic simulation of the interactions of 100 mammalian PH domains with PIP containing model membranes. Comparison with crystal structures of PH domains bound to PIP analogues demonstrates that our method correctly identifies interactions at known canonical and non-canonical sites, while revealing additional functionally important sites for interaction not observed in the crystal structure, such as for P-Rex1 and Akt1. At the family level, we find that the β1 and β2 strands and their connecting loop constitute the primary PIP interaction site for the majority of PH domains, but we highlight interesting exceptional cases. Simultaneous interaction with multiple PIPs and clustering of PIPs induced by PH domain binding are also observed. Our findings support a general paradigm for PH domain membrane association involving multivalent interactions with anionic lipids.

Evidence for the Involvement of Pleckstrin Homology Domain-Containing Proteins in the Transport of Enterocin DD14 (EntDD14); a Leaderless Two-Peptide Bacteriocin

Bacteriocins synthesis is initiated from an inactive precursor, which is composed of an N-terminal leader peptide attached to a C-terminal pro-peptide. However, leaderless bacteriocins (LLB) do not possess this N-terminal leader peptide nor undergo post-translational modifications. These atypical bacteriocins are observed to be immediately active after their translation in the cytoplasm. However, although considered to be simple, the biosynthetic pathway of LLB remains to be fully understood. Enterocin DD14 (EntDD14) is a two-peptide LLB produced by Enterococcus faecalis 14, which is a strain isolated from meconium. In silico analysis of DNA encoding EntDD14 located a cluster of 10 genes ddABCDEFGHIJ, where ddE and ddF encode the peculiar DdE and DdF proteins, carrying pleckstrin homology (PH) domains. These modules are quite common in Eucarya proteins and are known to be involved in intracellular signaling or cytoskeleton organization. To elucidate their role within the EntDD14 genetic determinants, we constructed deletion mutants of the ddE and ddF genes. As a result, the mutants were unable to export EntDD14 outside of the cytoplasm even though there was a clear expression of structural genes ddAB encoding EntDD14, and genes ddHIJ encoding an ABC transporter. Importantly, in these mutant strains (ΔddE and ΔddF), EntDD14 was detected by mass spectrometry in the intracellular soluble fraction exerting, upon its accumulation, a toxic effect on the producing strain as revealed by cell-counting and confocal microscopy analysis. Taken together, these results clearly indicate that PH domain-containing proteins, such as DdE and DdF, are involved in the transport of the leaderless two-peptide EntDD14.

Trypanosome morphogenesis involves recruitment of a pleckstrin homology domain protein by an orphan kinesin to the microtubule quartet

ABSTRACTKinesins are motor proteins found in all eukaryotic lineages that move along microtubule tracks to mediate numerous cellular processes such as mitosis and intracellular transport of cargo. In trypanosomatids, the kinesin protein superfamily has undergone a prominent expansion, giving these protists one of the most diverse kinesin repertoires. This has led to the emergence of two trypanosomatid-restricted groups of kinesins. Here, we characterize in Trypanosoma brucei TbKifX2, a hitherto orphaned kinesin that belongs to one of these groups. Representing a rare instance, TbKifX2 tightly interacts with TbPH1, a kinesin-like protein with an inactive motor domain. TbPH1 is named after a pleckstrin homology (PH) domain present within its carboxy-terminal tail. TbKifX2 recruits TbPH1 to the microtubule quartet (MtQ), a characteristic but poorly understood cytoskeletal structure that is part of the multipartite flagellum attachment zone (FAZ) and extends from the basal body to the anterior of the cell body. The proximal proteome of TbPH1 is comprised of four proteins that localize to the FAZ, consistent with the notion that the TbKifX2/TbPH1 complex are the first identified proteins to bind the MtQ along its whole length. Simultaneous ablation of both TbKifX2 and TbPH1 leads to the formation of prominent protrusions from the cell posterior. Thus, these two trypanosomatid-restricted proteins, which specifically localize to the MtQ in a microtubule-rich cell, appear to be contributors to morphogenesis in T. brucei.IMPORTANCETrypanosomatids are a group of unicellular parasites that infect a wide range of hosts from land plants to animals. They are also eukaryotes that have been shaped by prolonged independent evolution since this domain of life has radiated from a common ancestor almost 2 billion years ago. Thus, any resulting unique biological properties can be potentially exploited for treatment of infectious diseases caused by trypanosomatids. The cytoskeleton of trypanosomatids represents an ancient organelle that has undergone such modification. Here, we show that two trypanosomatid-specific proteins named TbPH1 and TbKifX2 form a complex that localizes to the microtubule quartet, a cytoskeletal structure characteristic to trypanosomatids. Ablation of these proteins in Trypansoma brucei leads to distinct morphological defects, making them not only intrinsically interesting topics of study, but potential therapeutic targets as well.

Evidence for a Pleckstrin Homology Domain-containing Proteins as Novel Class of Leaderless Bacteriocins Transporters

Bacteriocins biosynthetic pathway is initiated from an inactive precursor, which is composed of an N-terminal leader peptide attached to a C-terminal pro-peptide. However, leaderless bacteriocins (LLB) do not possess this N-terminal leader peptide, nor undergo post-translational modifications. These atypical bacteriocins are observed to be immediately active after their translation in the cytoplasm. However, although considered to be simple, the biosynthetic pathway of LLB remains to be fully understood. Enterocin DD14 (EntDD14) is a two-peptide LLB produced by Enterococcus faecalis 14, a strain isolated from meconium. In silico analysis of DNA encoding EntDD14 located a cluster of 10 genes ddABCDEFGHIJ, where ddE and ddF encode the peculiar DdE and DdF proteins, carrying pleckstrin homology (PH) domains. These modules are quite common in Eucarya proteins and are known to be involved in intracellular signalling or cytoskeleton organization. To elucidate their role within the EntDD14 genetic determinant, we constructed deletion mutants of the 2 corresponding genes. As a result, the ddE or ddF mutants are unable to externalize EntDD14 outside of the cytoplasm, even though there was clear expression of structural genes ddAB encoding EntDD14, and genes ddHIJ encoding an ABC transporter. Importantly, in these mutant strains (DddE and DddF), EntDD14 was detected by mass spectrometry in the intracellular soluble fraction exerting, upon its accumulation, a toxic effect on the producing strain as revealed by cell-counting and confocal microscopy analysis. Taken together, these results clearly indicate that PH domain-containing proteins, like DdE and DdF, are acting as transporters of the leaderless two-peptide EntDD14.

ANÁLISE IN SÍLICO DETECTOU MEMBROS DA FAMÍLIA PHLDB (PLECKSTRIN HOMOLOGY‐LIKE DOMAIN FAMILY B) COMO POTENCIAIS BIOMARCADORES PROGNÓSTICOS EM PACIENTES COM CÂNCER DE MAMA

Introdução: A expressão alterada dos membros da família PHLDB (Pleckstrin homology‐like domain family b) vem sendo frequentemente relatada em diferentes neoplasias malignas, embora as funções bioquímicas exatas não estejam totalmente elucidadas. Objetivo: Neste estudo, buscamos avaliar a expressão gênica da família PHLDB em amostras normais e tumorais da mama e estimar o valor prognóstico desses potenciais biomarcadores. Material e métodos: A expressão da família PHLDB foi avaliada usando o banco de dados UALCAN e GEPIA2. A relevância entre o nível de expressão dos integrantes da família PHLDB e os parâmetros clínico-patológicos, bem como os dados de sobrevida de pacientes com câncer de mama, foram analisados usando as plataformas de web KM Plotter, PrognoScan, bc-GenExMiner. Resultados: Identificamos que a expressão de PHLDB1 e PHLDB2 é reduzida em amostrais tumorais quando comparado às amostras normais da mama. Contrariamente, PHLDB3 apresentouse mais expresso nos tumores vs tecido normal adjacente. As evidencias amostrais permitiram inferir que existe associação estatisticamente significante entre o status da expressão da família PHLDB com diversas variáveis testadas, incluindo status linfonodal, classificação SBR, status mutacional de TP53 e subtipo molecular. Por fim, notamos que níveis reduzidos da expressão dos integrantes da família PHLDB foram significativamente associados com redução nas taxas cumulativas de sobrevida global e sobrevida livre de doença. Conclusão: A expressão alterada dos membros da família PHLDB foi observada como um evento frequente nos tumores de mama. Além disso, nossas descobertas fornecem novas compreensões sobre o papel potencial da família PHLDB como potenciais biomarcadores prognósticos em câncer de mama.