The Physarum polycephalum Genome Reveals Extensive Use of Prokaryotic Two-Component and Metazoan-Type Tyrosine Kinase Signaling

Physarum polycephalum is a well-studied microbial eukaryote with unique experimental attributes relative to other experimental model organisms. It has a sophisticated life cycle with several distinct stages including amoebal, flagellated, and plasmodial cells. It is unusual in switching between open and closed mitosis according to specific life-cycle stages. Here we present the analysis of the genome of this enigmatic and important model organism and compare it with closely related species. The genome is littered with simple and complex repeats and the coding regions are frequently interrupted by introns with a mean size of 100 bases. Complemented with extensive transcriptome data, we define approximately 31,000 gene loci, providing unexpected insights into earlyeukaryoteevolution.Wedescribeextensiveuseofhistidinekinase-basedtwo-componentsystemsandtyrosinekinasesignaling, the presence of bacterial and plant type photoreceptors (phytochromes, cryptochrome, and phototropin) and of plant-type pentatricopeptide repeat proteins, as well as metabolic pathways, and a cell cycle control system typically found in more complex eukaryotes. Our analysis characterizes P. polycephalum as a prototypical eukaryote with features attributed to the last common ancestor of Amorphea, that is, the Amoebozoa and Opisthokonts. Specifically, the presence of tyrosine kinases inAcanthamoeba and Physarum as representatives of two distantly related subdivisions ofAmoebozoa argues against the later emergence of tyrosine kinase signaling in the opisthokont lineage and also against the acquisition by horizontal gene transfe

Genetically tagged TRE5-A retrotransposons reveal high amplification rates and authentic target site preference in the Dictyostelium discoideum genome

Retrotransposons contribute significantly to the evolution of eukaryotic genomes. They replicate by producing DNA copies of their own RNA, which are integrated at new locations in the host cell genome. In the gene-dense genome of the social amoeba Dictyostelium discoideum, retrotransposon TRE5-A avoids insertional mutagenesis by targeting the transcription factor (TF) IIIC/IIIB complex and integrating ∼50 bp upstream of tRNA genes. We generated synthetic TRE5-A retrotransposons (TRE5-Absr) that were tagged with a selection marker that conferred resistance to blasticidin after a complete retrotransposition cycle. We found that the TRE5-Absr elements were efficiently mobilized in trans by proteins expressed from the endogenous TRE5-A population found in D. discoideum cells. ORF1 protein translated from TRE5-Absr elements significantly enhanced retrotransposition. We observed that the 5′ untranslated region of TRE5-A could be replaced by an unrelated promoter, whereas the 3′ untranslated region of TRE5-A was essential for retrotransposition. A predicted secondary structure in the RNA of the 3′ untranslated region of TRE5-A may be involved in the retrotransposition process. The TRE5-Absr elements were capable of identifying authentic integration targets in vivo, including formerly unnoticed, putative binding sites for TFIIIC on the extrachromosomal DNA element that carries the ribosomal RNA genes

Binding Free Energy Landscape of Domain-Peptide Interactions

Peptide recognition domains (PRDs) are ubiquitous protein domains which mediate large numbers of protein interactions in the cell. How these PRDs are able to recognize peptide sequences in a rapid and specific manner is incompletely understood. We explore the peptide binding process of PDZ domains, a large PRD family, from an equilibrium perspective using an all-atom Monte Carlo (MC) approach. Our focus is two different PDZ domains representing two major PDZ classes, I and II. For both domains, a binding free energy surface with a strong bias toward the native bound state is found. Moreover, both domains exhibit a binding process in which the peptides are mostly either bound at the PDZ binding pocket or else interact little with the domain surface. Consistent with this, various binding observables show a temperature dependence well described by a simple two-state model. We also find important differences in the details between the two domains. While both domains exhibit well-defined binding free energy barriers, the class I barrier is significantly weaker than the one for class II. To probe this issue further, we apply our method to a PDZ domain with dual specificity for class I and II peptides, and find an analogous difference in their binding free energy barriers. Lastly, we perform a large number of fixed-temperature MC kinetics trajectories under binding conditions. These trajectories reveal significantly slower binding dynamics for the class II domain relative to class I. Our combined results are consistent with a binding mechanism in which the peptide C terminal residue binds in an initial, rate-limiting step

PDZ domains and their binding partners: structure, specificity, and modification

PDZ domains are abundant protein interaction modules that often recognize short amino acid motifs at the C-termini of target proteins. They regulate multiple biological processes such as transport, ion channel signaling, and other signal transduction systems. This review discusses the structural characterization of PDZ domains and the use of recently emerging technologies such as proteomic arrays and peptide libraries to study the binding properties of PDZ-mediated interactions. Regulatory mechanisms responsible for PDZ-mediated interactions, such as phosphorylation in the PDZ ligands or PDZ domains, are also discussed. A better understanding of PDZ protein-protein interaction networks and regulatory mechanisms will improve our knowledge of many cellular and biological processes

GAIP Interacting Protein C-Terminus Regulates Autophagy and Exosome Biogenesis of Pancreatic Cancer through Metabolic Pathways

<div><p>GAIP interacting protein C terminus (GIPC) is known to play an important role in a variety of physiological and disease states. In the present study, we have identified a novel role for GIPC as a master regulator of autophagy and the exocytotic pathways in cancer. We show that depletion of GIPC-induced autophagy in pancreatic cancer cells, as evident from the upregulation of the autophagy marker LC3II. We further report that GIPC regulates cellular trafficking pathways by modulating the secretion, biogenesis, and molecular composition of exosomes. We also identified the involvement of GIPC on metabolic stress pathways regulating autophagy and microvesicular shedding, and observed that GIPC status determines the loading of cellular cargo in the exosome. Furthermore, we have shown the overexpression of the drug resistance gene ABCG2 in exosomes from GIPC-depleted pancreatic cancer cells. We also demonstrated that depletion of GIPC from cancer cells sensitized them to gemcitabine treatment, an avenue that can be explored as a potential therapeutic strategy to overcome drug resistance in cancer.</p></div

GIPC modulates expression of drug resistance associated gene <i>ABCG2</i> and sensitizes pancreatic cancer cell lines to gemcitabine.

<p>A) ABCG2 expression was confirmed at protein level by western blot in GIPC knockdown and control cells as well as in corresponding exosomes. PLC γ is used as loading control for exosomes and β-Actin is used as loading control for cell lysates. B) GIPC +/- PANC-1 cells were treated with different concentration of the gemcitabine for 72 h. Effect of the drug treatment was evaluated using MTS cell viability assay. The horizontal bar represents the IC50 level.</p

GIPC regulates stress induced metabolic pathways.

<p>A) Immunoblot of the cell lysates from GIPC depleted and control AsPC-1 and PANC-1 cells are being probed with p-AMPK-α, total AMPK-α. β-Actin is used as loading control. B) Further, immunoblot of cell lysates from above condition were being probed with p–mTOR, total mTOR, p-p70S6K and total p70S6K. β-Actin is used as loading control.</p

GIPC regulates autophagy by interfering with glucose uptake.

<p>A) Quantitative PCR and B) Western blot analysis of Glut1 to analyze the effect of GIPC-depletion in Glut1 expression. β-Actin is used as loading control. Both Glut1 mRNA and protein levels decreased significantly upon GIPC depletion in AsPC-1 and PANC-1 cells. C) Glucose uptake was significantly decreased in GIPC depleted cells as compared to the control cells in AsPC-1 and PANC-1 cells (** denotes p<0.01). D) Intracellular glucose levels were also significantly decreased in the GIPC depleted AsPC-1 and PANC-1 cell lines confirming the role GIPC in glucose metabolism (** denotes p<0.01).</p

GIPC induce autophagy in the pancreatic cancer cells.

<p>A) AsPC-1 and PANC-1 cells were infected with lentiviruses expressing shRNAs to GIPC and scrambled control. An equal amount of whole-cell lysates from AsPC-1 and PANC-1 GIPC depleted cells were analyzed by immunoblotting (IB) with the antibodies for GIPC and LC3II. β-Actin is used as loading control. B) A representative immunofluorescence analysis of PANC-1 cells for expression of LC3 II (green) in GIPC depleted PANC-1 cells compared to the control cells. Cells were counterstained with DAPI (blue).</p