A Role for Phosphatidic Acid in the Formation of “Supersized” Lipid Droplets

Lipid droplets (LDs) are important cellular organelles that govern the storage and turnover of lipids. Little is known about how the size of LDs is controlled, although LDs of diverse sizes have been observed in different tissues and under different (patho)physiological conditions. Recent studies have indicated that the size of LDs may influence adipogenesis, the rate of lipolysis and the oxidation of fatty acids. Here, a genome-wide screen identifies ten yeast mutants producing “supersized” LDs that are up to 50 times the volume of those in wild-type cells. The mutated genes include: FLD1, which encodes a homologue of mammalian seipin; five genes (CDS1, INO2, INO4, CHO2, and OPI3) that are known to regulate phospholipid metabolism; two genes (CKB1 and CKB2) encoding subunits of the casein kinase 2; and two genes (MRPS35 and RTC2) of unknown function. Biochemical and genetic analyses reveal that a common feature of these mutants is an increase in the level of cellular phosphatidic acid (PA). Results from in vivo and in vitro analyses indicate that PA may facilitate the coalescence of contacting LDs, resulting in the formation of “supersized” LDs. In summary, our results provide important insights into how the size of LDs is determined and identify novel gene products that regulate phospholipid metabolism

Characterization of Trapped Lignin-Degrading Microbes in Tropical Forest Soil

Lignin is often the most difficult portion of plant biomass to degrade, with fungi generally thought to dominate during late stage decomposition. Lignin in feedstock plant material represents a barrier to more efficient plant biomass conversion and can also hinder enzymatic access to cellulose, which is critical for biofuels production. Tropical rain forest soils in Puerto Rico are characterized by frequent anoxic conditions and fluctuating redox, suggesting the presence of lignin-degrading organisms and mechanisms that are different from known fungal decomposers and oxygen-dependent enzyme activities. We explored microbial lignin-degraders by burying bio-traps containing lignin-amended and unamended biosep beads in the soil for 1, 4, 13 and 30 weeks. At each time point, phenol oxidase and peroxidase enzyme activity was found to be elevated in the lignin-amended versus the unamended beads, while cellulolytic enzyme activities were significantly depressed in lignin-amended beads. Quantitative PCR of bacterial communities showed more bacterial colonization in the lignin-amended compared to the unamended beads after one and four weeks, suggesting that the lignin supported increased bacterial abundance. The microbial community was analyzed by small subunit 16S ribosomal RNA genes using microarray (PhyloChip) and by high-throughput amplicon pyrosequencing based on universal primers targeting bacterial, archaeal, and eukaryotic communities. Community trends were significantly affected by time and the presence of lignin on the beads. Lignin-amended beads have higher relative abundances of representatives from the phyla Actinobacteria, Firmicutes, Acidobacteria and Proteobacteria compared to unamended beads. This study suggests that in low and fluctuating redox soils, bacteria could play a role in anaerobic lignin decomposition

High incidence of Noonan syndrome features including short stature and pulmonic stenosis in patients carrying NF1 missense mutations affecting p.Arg1809: genotype-phenotype correlation

Neurofibromatosis type 1 (NF1) is one of the most frequent genetic disorders, affecting 1:3,000 worldwide. Identification of genotype-phenotype correlations is challenging because of the wide range clinical variability, the progressive nature of the disorder, and extreme diversity of the mutational spectrum. We report 136 individuals with a distinct phenotype carrying one of five different NF1 missense mutations affecting p.Arg1809. Patients presented with multiple cafe-au-lait macules (CALM) with or without freckling and Lisch nodules, but no externally visible plexiform neurofibromas or clear cutaneous neurofibromas were found. About 25% of the individuals had Noonan-like features. Pulmonic stenosis and short stature were significantly more prevalent compared with classic cohorts (P<0.0001). Developmental delays and/or learning disabilities were reported in over 50% of patients. Melanocytes cultured from a CALM in a segmental NF1-patient showed two different somatic NF1 mutations, p.Arg1809Cys and a multi-exon deletion, providing genetic evidence that p.Arg1809Cys is a loss-of-function mutation in the melanocytes and causes a pigmentary phenotype. Constitutional missense mutations at p.Arg1809 affect 1.23% of unrelated NF1 probands in the UAB cohort, therefore this specific NF1 genotype-phenotype correlation will affect counseling and management of a significant number of patients

Mechanisms Underlying the Confined Diffusion of Cholera Toxin B-Subunit in Intact Cell Membranes

Multivalent glycolipid binding toxins such as cholera toxin have the capacity to cluster glycolipids, a process thought to be important for their functional uptake into cells. In contrast to the highly dynamic properties of lipid probes and many lipid-anchored proteins, the B-subunit of cholera toxin (CTxB) diffuses extremely slowly when bound to its glycolipid receptor GM1 in the plasma membrane of living cells. In the current study, we used confocal FRAP to examine the origins of this slow diffusion of the CTxB/GM1 complex at the cell surface, relative to the behavior of a representative GPI-anchored protein, transmembrane protein, and fluorescent lipid analog. We show that the diffusion of CTxB is impeded by actin- and ATP-dependent processes, but is unaffected by caveolae. At physiological temperature, the diffusion of several cell surface markers is unchanged in the presence of CTxB, suggesting that binding of CTxB to membranes does not alter the organization of the plasma membrane in a way that influences the diffusion of other molecules. Furthermore, diffusion of the B-subunit of another glycolipid-binding toxin, Shiga toxin, is significantly faster than that of CTxB, indicating that the confined diffusion of CTxB is not a simple function of its ability to cluster glycolipids. By identifying underlying mechanisms that control CTxB dynamics at the cell surface, these findings help to delineate the fundamental properties of toxin-receptor complexes in intact cell membranes

BMQ

BMQ: Boston Medical Quarterly was published from 1950-1966 by the Boston University School of Medicine and the Massachusetts Memorial Hospitals

Rab18 Binds to Hepatitis C Virus NS5A and Promotes Interaction between Sites of Viral Replication and Lipid Droplets

Hepatitis C virus (HCV) is a single-stranded RNA virus that replicates on endoplasmic reticulum-derived membranes. HCV particle assembly is dependent on the association of core protein with cellular lipid droplets (LDs). However, it remains uncertain whether HCV assembly occurs at the LD membrane itself or at closely associated ER membranes. Furthermore, it is not known how the HCV replication complex and progeny genomes physically associate with the presumed sites of virion assembly at or near LDs. Using an unbiased proteomic strategy, we have found that Rab18 interacts with the HCV nonstructural protein NS5A. Rab18 associates with LDs and is believed to promote physical interaction between LDs and ER membranes. Active (GTP-bound) forms of Rab18 bind more strongly to NS5A than a constitutively GDP-bound mutant. NS5A colocalizes with Rab18-positive LDs in HCV-infected cells, and Rab18 appears to promote the physical association of NS5A and other replicase components with LDs. Modulation of Rab18 affects genome replication and possibly also the production of infectious virions. Our results support a model in which specific interactions between viral and cellular proteins may promote the physical interaction between membranous HCV replication foci and lipid droplets

Host proteins already known to interact with NS5A and/or the HCV life cycle identified by SILAC and GeLC-MS.

<p>Proteins that specifically bind to NS5A(SF) will have incorporated heavy amino acids and thus will have a high heavy∶light (H/L) peptide ratio. PEP (Posterior error probability) refers to the probability that a peptide-spectrum match is incorrect. Intensity refers to total summed peptide intensity for the identified protein group.</p

Rab18 silencing reduces the production of infectious HCV particles.

<p><b>A.</b> Schematic of viral assembly/secretion assay. <b>B.</b> Huh 7.5.1 cell lines stably expressing shRab18-A, -B, or NTshRNA were infected with Jc1/Gluc2A at an MOI of 1. Cell culture medium was collected at day 2 postinfection, then used to infect naïve Huh7.5.1 cells. <i>Gaussia</i> luciferase activity was measured at 72 hr post-infection. These values were normalized to Huh7.5.1 cells infected with supernatant from NTshRNA-expressing cells. Values represent means ± SD of three independent experiments. <b>C.</b> Huh7.5.1 cells lines stably expressing shRab18-A, -B, or NTshRNA were transfected with <i>in vitro</i> transcribed Jc1/Gluc2A RNA. Relative quantitation of infectious particle release was performed as described above. <b>D.</b> Effect of Rab18 silencing on wild type JFH-1 secretion. Stable shRNA-expressing cell lines were infected with the JFH-1 strain of HCV at an MOI of 3. Five days post-infection, the secreted virus titer was determined using a focus-forming assay in naive Huh7.5.1 cells. Values represent means ± SD of three independent experiments. <b>E.</b> Huh 7.5.1 cell lines stably expressing shRab18-A, -B, or NTshRNA were infected with Jc1/Gluc2A at an MOI of 1. At 72 hr post-infection, the cell culture supernatant was collected for quantitation of released extracellular virus as described above. The infected cell monolayer was washed, trypsinized, and then the pellet was subjected to three rounds of freeze-thawing to release intracellular virus particles, which were then quantitated by infection of naïve Huh7.5.1 cells as described for extracellular infectious virus.</p

Identification of NS5A-binding host proteins using a proteomic strategy.

<p><b>A.</b> Diagram of Jc1(SF) showing location of FLAG and tandem Strep-Tags in domain III of NS5A. <b>B.</b> Detection of proteins specifically associated with NS5A(SF). Huh7.5.1 cells were infected with Jc1(SF) (left lane) and untagged wild-type Jc1 (right lane). Cell lysates were incubated with Streptactin-Sepharose and the affinity matrix was washed extensively followed by elution with biotin. Eluted proteins were separated by SDS-PAGE and visualized by colloidal Coomassie Blue staining. The position of NS5A(SF) is indicated by the gray arrowhead. Proteins that specifically associate with NS5A(SF) are indicated by black arrows. <b>C.</b> Schematic showing strategy of affinity purification combined with Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC). Huh 7.5.1 cells were metabolically labeled with medium containing normal “light” arginine and lysine amino acids (LAA) or “heavy” L-arg-¹³C₆, ¹⁵N₄ and L-lys-¹³C₆, ¹⁵N₂ (HAA) for 6 days before infecting LAA-labeled cells with Jc1 and HAA-labeled cells with Jc1(SF) virus. Cells were harvested at day 6 post infection. Equal amounts of protein from HAA and LAA-labeled lysates were mixed and then subjected to Streptactin affinity purification as described above. <b>D.</b> Lysates from LAA-labeled, Jc1-infected cells and HAA-labeled, Jc1(SF)-infected cells were mixed and subjected to Streptactin affinity purification. The entire eluate was concentrated by precipitation, separated by SDS-PAGE and stained with Coomassie Blue prior to gel slice excision for mass spectrometry. The expected position of Rab18 is indicated with an arrow, although no discrete band is visible on this gel by Coomassie Blue staining.</p