Structural insights into piRNA biogenesis

Discovered two decades ago, Piwi-interacting RNAs (piRNAs) play critical roles in gene regulation, transposon element repression, and antiviral defense. Dysregulation of piRNAs has been noted in diverse human diseases including cancers. Recently, extensive studies have revealed that many more proteins are involved in piRNA biogenesis. This review will summarize the recent progress in piRNA biogenesis and functions, especially the molecular mechanisms by which piRNA biogenesis-related proteins contribute to piRNA processing.     

Co-evolutionary analysis reveals insights into protein-protein interactions

Protein-protein interactions play crucial roles in biological processes. Experimental methods have been developed to survey the proteome for interacting partners and some computational approaches have been developed to extend the impact of these experimental methods. Computational methods are routinely applied to newly discovered genes to infer protein function and plausible protein-protein interactions. Here, we develop and extend a quantitative method that identifies interacting proteins based upon the correlated behavior of the evolutionary histories of protein ligands and their receptors. We have studied six families of ligand-receptor pairs including: the syntaxin/Unc-18 family, the GPCR/G-alpha's, the TGF-beta/TGF-beta receptor system, the immunity/colicin domain collection from bacteria, the chemokine/chemokine receptors, and the VEGF/VEGF receptor family. For correlation scores above a defined threshold, we were able to find an average of 79% of all known binding partners. We then applied this method to find plausible binding partners for proteins with uncharacterized binding specificities in the syntaxin/Unc-18 protein and TGF-beta/TGF-beta receptor families. Analysis of the results shows that co-evolutionary analysis of interacting protein families can reduce the search space for identifying binding partners by not only finding binding partners for uncharacterized proteins but also recognizing potentially new binding partners for previously characterized proteins. We believe that correlated evolutionary histories provide a route to exploit the wealth of whole genome sequences and recent systematic proteomic results to extend the impact of these studies and focus experimental efforts to categorize physiologically or pathologically relevant protein-protein interactions.     

Lipoproteins in Gram-negative bacteria: new insights into their biogenesis,subcellular targeting and functional roles

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Structural insights into methyltransferase KsgA function in 30S ribosomal subunit biogenesis

The assembly of the ribosomal subunits is facilitated by ribosome biogenesis factors. The universally conserved methyltransferase KsgA modifies two adjacent adenosine residues in the 3'-terminal helix 45 of the 16 S ribosomal RNA (rRNA). KsgA recognizes its substrate adenosine residues only in the context of a near mature 30S subunit and is required for the efficient processing of the rRNA termini during ribosome biogenesis. Here, we present the cryo-EM structure of KsgA bound to a nonmethylated 30S ribosomal subunit. The structure reveals that KsgA binds to the 30S platform with the catalytic N-terminal domain interacting with substrate adenosine residues in helix 45 and the C-terminal domain making extensive contacts to helix 27 and helix 24. KsgA excludes the penultimate rRNA helix 44 from adopting its position in the mature 30S subunit, blocking the formation of the decoding site and subunit joining. We suggest that the activation of methyltransferase activity and subsequent dissociation of KsgA control conformational changes in helix 44 required for final rRNA processing and translation initiation.     

Atomic resolution insights into curli fiber biogenesis

Bacteria produce functional amyloid fibers called curli in a controlled, noncytotoxic manner. These extracellular fimbriae enable biofilm formation and promote pathogenicity. Understanding curli biogenesis is important for appreciating microbial lifestyles and will offer clues as to how disease-associated human amyloid formation might be ameliorated. Proteins encoded by the curli specific genes (csgA-G) are required for curli production. We have determined the structure of CsgC and derived the first structural model of the outer-membrane subunit translocator CsgG. Unexpectedly, CsgC is related to the N-terminal domain of DsbD, both in structure and oxido-reductase capability. Furthermore, we show that CsgG belongs to the nascent class of helical outer-membrane macromolecular exporters. A cysteine in a CsgG transmembrane helix is a potential target of CsgC, and mutation of this residue influences curli assembly. Our study provides the first high-resolution structural insights into curli biogenesis.     

Systems biology analysis reveals new insights into invasive lung cancer

Background

Adenocarcinoma in situ (AIS) is a pre-invasive lesion in the lung and a subtype of lung adenocarcinoma. The patients with AIS can be cured by resecting the lesion completely. In contrast, the patients with invasive lung adenocarcinoma have very poor 5-year survival rate. AIS can develop into invasive lung adenocarcinoma. The investigation and comparison of AIS and invasive lung adenocarcinoma at the genomic level can deepen our understanding of the mechanisms underlying lung cancer development.

Results

In this study, we identified 61 lung adenocarcinoma (LUAD) invasive-specific differentially expressed genes, including nine long non-coding RNAs (lncRNAs) based on RNA sequencing techniques (RNA-seq) data from normal, AIS, and invasive tissue samples. These genes displayed concordant differential expression (DE) patterns in the independent stage III LUAD tissues obtained from The Cancer Genome Atlas (TCGA) RNA-seq dataset. For individual invasive-specific genes, we constructed subnetworks using the Genetic Algorithm (GA) based on protein-protein interactions, protein-DNA interactions and lncRNA regulations. A total of 19 core subnetworks that consisted of invasive-specific genes and at least one putative lung cancer driver gene were identified by our study. Functional analysis of the core subnetworks revealed their enrichment in known pathways and biological progresses responsible for tumor growth and invasion, including the VEGF signaling pathway and the negative regulation of cell growth.

Conclusions

Our comparison analysis of invasive cases, normal and AIS uncovered critical genes that involved in the LUAD invasion progression. Furthermore, the GA-based network method revealed gene clusters that may function in the pathways contributing to tumor invasion. The interactions between differentially expressed genes and putative driver genes identified through the network analysis can offer new targets for preventing the cancer invasion and potentially increase the survival rate for cancer patients.

     

Genome-wide genetic diversity of Holstein Friesian cattle reveals new insights into Australian and global population variability, including impact of selection

Past breeding strategies for dairy cattle have been very effective in producing rapid genetic gain to achieve industry targets and raise profitability. Such gains have been largely facilitated by intense selection of sires combined with the use of artificial insemination. However, this practice can potentially limit the level of genetic diversity through inbreeding and selection plateaus. The rate of inbreeding in Australia is increasing, primarily as a result of semen importation from a small number of prominent bulls from the USA. The effect of this genetic influx in the Australian dairy cattle population is poorly understood both in terms of diversity and local adaptation/divergence. This study uses 845 genome-wide SNP genetic markers and 431 bulls to characterize the level of genetic diversity and genetic divergence within the Australian and international Holstein Friesian dairy population. No significant differences in genetic diversity (as measured by heterozygosity [H(o)] and allelic richness [A]) were observed over the 25-year time period (1975-1999) for bulls used in Australia. The importation of foreign semen into Australia has increased the effective population size until it was in effect a sub-sample of the global population. Our data indicate that most individuals are equally closely related to one another, regardless of country of origin and year of birth. In effect, the global population can be considered as one single population unit. These results indicate that inbreeding, genetic drift and selection has had little effect at reducing genetic diversity and differentiating the Australian Holstein Friesian population at a genome-wide level.     

Molecular insights into peroxisome homeostasis and peroxisome biogenesis disorders

Peroxisomes are single-membrane organelles essential for cell metabolism including the β-oxidation of fatty acids, synthesis of etherlipid plasmalogens, and redox homeostasis. Investigations into peroxisome biogenesis and the human peroxisome biogenesis disorders (PBDs) have identified 14 PEX genes encoding peroxins involved in peroxisome biogenesis and the mutation of PEX genes is responsible for the PBDs. Many recent findings have further advanced our understanding of the biology, physiology, and consequences of a functional deficit of peroxisomes. In this Review, we discuss cell defense mechanisms that counteract oxidative stress by 1) a proapoptotic Bcl-2 factor BAK-mediated release to the cytosol of H₂O₂-degrading catalase from peroxisomes and 2) peroxisomal import suppression of catalase by Ser232-phosphorylation of Pex14, a docking protein for the Pex5–PTS1 complex. With respect to peroxisome division, the important issue of how the energy-rich GTP is produced and supplied for the division process was recently addressed by the discovery of a nucleoside diphosphate kinase-like protein, termed DYNAMO1 in a lower eukaryote, which has a mammalian homologue NME3. In regard to the mechanisms underlying the pathogenesis of PBDs, a new PBD model mouse defective in Pex14 manifests a dysregulated brain-derived neurotrophic factor (BDNF)-TrkB pathway, an important signaling pathway for cerebellar morphogenesis. Communications between peroxisomes and other organelles are also addressed.     

Genome-wide BAC-end sequencing of Musa acuminata DH Pahang reveals further insights into the genome organization of banana

Banana and plantain (Musa spp.) are grown in more than 120 countries in tropical and subtropical regions and constitute an important staple food for millions of people. A Musa acuminata ssp. malaccencis DH Pahang bacterial artificial chromosome (BAC) library (MAMB) was submitted for BAC-end sequencing. MAMB consists of 23,040 clones, with a 140-kbp average insert size, accounting for a five times coverage of the banana genome. A total of 46,080 reads were generated, and 42,750 (92.8%) high-quality sequences were obtained after trimming for vector and quality. Analysis of these data shows a GC content of 41.39%, whereas interspersed repeats comprise 32.3%. The most common repeated sequences found show homology to ribosomal RNA genes, particularly 18S rRNA, while the Ty3/gypsy type monkey retrotransposon is the most common retro element. The sequence data were used to generate a banana-specific repeat library containing 54 new repetitive elements which accounted for 11.86% of the total nucleotides. Simple sequence repeats represent 0.7% of the sequence data and allowed the identification of 2,455 potentially useful marker sites. Functional annotation identified 2,705 sequences that could code for proteins of known function. Microsynteny analysis shows a higher number of co-linear matches to Oryza sativa, in contrast to Arabidopsis thaliana. This database of BAC-end sequences is useful for the assembly of the complete banana genome sequence and is important for identification in functional genomics experiments.     

Sirtuins in mammals: insights into their biological function

Vitamin B6 is well known in its biochemically active form as pyridoxal 5'-phosphate, an essential cofactor of numerous metabolic enzymes. The vitamin is also implicated in numerous human body functions ranging from modulation of hormone function to its recent discovery as a potent antioxidant. Its de novo biosynthesis occurs only in bacteria, fungi and plants, making it an essential nutrient in the human diet. Despite its paramount importance, its biosynthesis was predominantly investigated in Escherichia coli, where it is synthesized from the condensation of deoxyxylulose 5-phosphate and 4-phosphohydroxy-L-threonine catalysed by the concerted action of PdxA and PdxJ. However, it has now become clear that the majority of organisms capable of producing this vitamin do so via a different route, involving precursors from glycolysis and the pentose phosphate pathway. This alternative pathway is characterized by the presence of two genes, Pdx1 and Pdx2. Their discovery has sparked renewed interest in vitamin B6, and numerous studies have been conducted over the last few years to characterize the new biosynthesis pathway. Indeed, enormous progress has been made in defining the nature of the enzymes involved in both pathways, and important insights have been provided into their mechanisms of action. In the present review, we summarize the recent advances in our knowledge of the biosynthesis of this versatile molecule and compare the two independent routes to the biosynthesis of vitamin B6. Surprisingly, this comparison reveals that the key biosynthetic enzymes of both pathways are, in fact, very similar both structurally and mechanistically.     

Biochemical and structural insights into bacterial organelle form and biogenesis

Many heterotrophic bacteria have the ability to make polyhedral structures containing metabolic enzymes that are bounded by a unilamellar protein shell (metabolosomes or enterosomes). These bacterial organelles contain enzymes associated with a specific metabolic process (e.g. 1,2-propanediol or ethanolamine utilization). We show that the 21 gene regulon specifying the pdu organelle and propanediol utilization enzymes from Citrobacter freundii is fully functional when cloned in Escherichia coli, both producing metabolosomes and allowing propanediol utilization. Genetic manipulation of the level of specific shell proteins resulted in the formation of aberrantly shaped metabolosomes, providing evidence for their involvement as delimiting entities in the organelle. This is the first demonstration of complete recombinant metabolosome activity transferred in a single step and supports phylogenetic evidence that the pdu genes are readily horizontally transmissible. One of the predicted shell proteins (PduT) was found to have a novel Fe-S center formed between four protein subunits. The recombinant model will facilitate future experiments establishing the structure and assembly of these multiprotein assemblages and their fate when the specific metabolic function is no longer required.     

New insights into the biogenesis of human high-density lipoproteins

PURPOSE OF REVIEW: The interest for the human HDL system was recently revived by the identification of the ABCA1 as a critical component in the formation and maintenance of plasma HDL levels. The present review focuses on recent progress in our understanding of the basic mechanisms underlying HDL biogenesis pathways. RECENT FINDINGS: Several novel mechanisms governing ABCA1/apoA-I interactions have recently been identified: apolipoprotein A-I activates ABCA1 phosphorylation through the cAMP/protein kinase A-dependent pathway; the majority of ABCA1 exists as a tetramer in human living cell, supporting the concept that the homotetrameric ABCA1 complex constitutes the minimum functional unit for the formation of nascent HDL particles; apolipoprotein A-I has been shown to have a recycling retroendocytic pathway with uptake and resecretion of the lipidated nascent HDL particles by the cell, most likely through the ABCA1 transporter pathway; there is evidence that the speciation of nascent HDL into pre-beta and alpha-HDL is linked to specific cell lines, and occurs by both ABCA1-dependent and independent pathways. SUMMARY: The fundamental mechanisms underlying the biogenesis, speciation and maturation of HDL remain complex and not well understood. Understanding the mechanisms governing HDL genesis at the cellular level could provide novel insights into the human atheroprotective system in health and disease.     

Mutational analysis of endoxylanases XylA and XylB from the phytopathogen Fusarium graminearum reveals comprehensive insights into their inhibitor insensitivity

Endo-beta-1,4-xylanases (EC 3.2.1.8; endoxylanases), key enzymes in the degradation of xylan, are considered to play an important role in phytopathogenesis, as they occupy a prominent position in the arsenal of hydrolytic enzymes secreted by phytopathogens to breach the cell wall and invade the plant tissue. Plant endoxylanase inhibitors are increasingly being pinpointed as part of a counterattack mechanism. To understand the surprising XIP-type endoxylanase inhibitor insensitivity of endoxylanases XylA and XylB from the phytopathogen Fusarium graminearum, an extensive mutational study of these enzymes was performed. Using combinatorial and site-directed mutagenesis, the XIP insensitivity of XylA as well as XylB was proven to be solely due to amino acid sequence adaptations in the "thumb" structural region. While XylB residues Cys141, Asp148, and Cys149 were shown to prevent XIP interaction, the XIP insensitivity of XylA could be ascribed to the occurrence of only one aberrant residue, i.e., Val151. This study, in addition to providing a thorough explanation for the XIP insensitivity of both F. graminearum endoxylanases at the molecular level, generated XylA and XylB mutants with altered inhibition specificities and pH optima. As this is the first experimental elucidation of the molecular determinants dictating the specificity of the interaction between endoxylanases of phytopathogenic origin and a plant inhibitor, this work sheds more light on the ongoing evolutionary arms race between plants and phytopathogenic fungi involving recognition of endoxylanases.