TBC1D14 regulates autophagy via the TRAPP complex and ATG9 traffic

Macroautophagy requires membrane trafficking and remodelling to form the autophagosome and deliver its contents to lysosomes for degradation. We have previously identified the TBC domain‐containing protein, TBC1D14, as a negative regulator of autophagy that controls delivery of membranes from RAB11‐positive recycling endosomes to forming autophagosomes. In this study, we identify the TRAPP complex, a multi‐subunit tethering complex and GEF for RAB1, as an interactor of TBC1D14. TBC1D14 binds to the TRAPP complex via an N‐terminal 103 amino acid region, and overexpression of this region inhibits both autophagy and secretory traffic. TRAPPC8, the mammalian orthologue of a yeast autophagy‐specific TRAPP subunit, forms part of a mammalian TRAPPIII‐like complex and both this complex and TBC1D14 are needed for RAB1 activation. TRAPPC8 modulates autophagy and secretory trafficking and is required for TBC1D14 to bind TRAPPIII. Importantly, TBC1D14 and TRAPPIII regulate ATG9 trafficking independently of ULK1. We propose a model whereby TBC1D14 and TRAPPIII regulate a constitutive trafficking step from peripheral recycling endosomes to the early Golgi, maintaining the cycling pool of ATG9 required for initiation of autophagy.     

Sphingolipid trafficking and protein sorting in epithelial cells

Sphingolipids represent a minor, but highly dynamic subclass of lipids in all eukaryotic cells. They are involved in functions that range from structural protection to signal transduction and protein sorting, and participate in lipid raft assembly. In polarized epithelial cells, which display an asymmetric apical and basolateral membrane surface, rafts have been proposed as a sorting principle for apical resident proteins, following their biosynthesis. However, raft-mediated trafficking is ubiquitous in cells. Also, sphingolipids per se, which are strongly enriched in the apical domain, are subject to sorting in polarity development. Next to the trans Golgi network, a subapical compartment called SAC or common endosome appears instrumental in regulating these sorting events.     

It’s all in your gut and mind

Obesity has become a global problem affecting adults and children alike. Lifestyle choices both personal and industry driven can be blamed for the rise in obesity. One must distinguish between the possibly reversible overweight condition and the almost intractable actual morbid obesity where predisposing genetic factors may come into play. Both however exhibit consequences to health with a severity that cannot be underestimated. Deleterious changes to metabolism can lead to type II diabetes and atherosclerosis and other organ dysfunctions. It has long been recognized that there are two main types of fatty tissue in the body, white adipose tissue (WAT) serving a storage function and brown adipose tissue (BAT) serving a thermogenic function. The new discovery has been that WAT cells can be induced to undergo conversion (browning) to BAT to yield what is called beige adipose tissue, acquiring the thermogenic function. The clinical dream is to be able to promote browning and to induce, what may be called, burning off the fat. In this B&B article I entice the reader with a recent study that shows how two key hormones insulin and leptin operate cooperatively in the brain to monitor and regulate energy balance and the downstream effect of browning. I present other studies to add additional perspectives to the understanding of the mechanisms in peripheral tissues and other hormones that play additional key roles. Whether obesity can be conquered therapeutically by manipulating the regulatory systems is still an open question.     

Localization and function of cytosolic phospholipase A2α at the Golgi

Cytosolic phospholipase A₂α (cPLA₂α, Group IVA phospholipase A₂) is a central mediator of arachidonate release from cellular phospholipids for the biosynthesis of eicosanoids. cPLA₂α translocates to intracellular membranes including the Golgi in response to a rise in intracellular calcium level. The enzyme’s calcium-dependent phospholipid-binding C2 domain provides the targeting specificity for cPLA₂α translocation to the Golgi. However, other features of cPLA₂α regulation are incompletely understood such as the role of phosphorylation of serine residues in the catalytic domain and the function of basic residues in the cPLA₂α C2 and catalytic domains that are proposed to interact with anionic phospholipids in the membrane to which cPLA₂α is targeted. Increasing evidence strongly suggests that cPLA₂α plays a role in regulating Golgi structure, tubule formation and intra-Golgi transport. For example, recent data suggests that cPLA₂α regulates the transport of tight junction and adherens junction proteins through the Golgi to cell–cell contacts in confluent endothelial cells. However, there are now examples where data based on knockdown using siRNA or pharmacological inhibition of enzymatic activity of cPLA₂α affects fundamental cellular processes yet these phenotypes are not observed in cells from cPLA₂α deficient mice. These results suggest that in some cases there may be compensation for the lack of cPLA₂α. Thus, there is continued need for studies employing highly specific cPLA₂α antagonists in addition to genetic deletion of cPLA₂α in mice.     

Quantitative analysis of biomarkers,drugs and toxins in biological samples by immunoaffinity chromatography coupled to mass spectrometry or tandem mass spectrometry: A focused review of recent applications

Immunoaffinity chromatography (IAC), mass spectrometry and especially tandem mass spectrometry (MS/MS) represent the most efficient and reliable analytical techniques for specific isolation, unequivocal identification and accurate quantification of numerous natural and synthetic substances in biological samples. This review article focuses on the combined use of these outstanding methodologies in basic and clinical research and in life sciences for the quantitative analysis of low- and high-molecular mass biomarkers, drugs and toxins in urine, plasma or serum samples, in tissue and other biologicals systems published in the last decade. The analytes discussed in some detail include the biomarkers of oxidative stress 15(S)-8-iso-prostaglandin F_2α {15(S)-8-iso-PGF_2α} and 3-nitrotyrosine, the major urinary metabolite of the lipid mediators cysteinyl leukotrienes, i.e., the leukotriene E₄ (LTE₄), melatonin, and the major collagen type II neoepitope peptide in human urine.     

A novel statin-mediated “prenylation block-and-release” assay provides insight into the membrane targeting mechanisms of small GTPases

Ras super-family small GTPases regulate diverse cellular processes such as vesicular transport and signal transduction. Critical to these activities is the ability of these proteins to target to specific intracellular membranes. To allow association with membranes Ras-related GTPases are post-translationally modified by covalent attachment of prenyl groups to conserved cysteine residues at or near their C-terminus. Here we used the HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase (HMGCR) inhibitor mevastatin to develop a ‘prenylation block-and-release’ assay that allows membrane targeting of prenylated proteins to be visualized in living cells. Using this assay we investigated the cytosol to membrane targeting of several small GTPases to compartments of the secretory and endocytic pathways. We found that all Rabs tested were targeted directly to the membrane on which they reside at steady-state and not via an intermediate location as reported for Ras and Rho proteins. However, we observed that the kinetics of cytosol to membrane targeting differed for each Rab tested. Comparison of the mevastatin sensitivity and kinetics of membrane targeting of Rab23, Rab23 prenylation motif mutants and H-Ras revealed that these parameters are strongly dependent upon the prenyl transferase with Rab geranylgeranyl transferase substrates exhibiting higher sensitivity and requiring greater time to recover from mevastatin inhibition than farnesyl transferase substrates. We propose that this assay is a useful tool to investigate the kinetics, biological functions and the mechanisms of membrane targeting of prenylated proteins.     

Examination of Sec22 Homodimer Formation and Role in SNARE-dependent Membrane Fusion

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein complexes play essential roles in catalyzing intracellular membrane fusion events although the assembly pathway and molecular arrangement of SNARE complexes in membrane fusion reactions are not well understood. Here we monitored interactions of the R-SNARE protein Sec22 through a cysteine scanning approach and detected efficient formation of cross-linked Sec22 homodimers in cellular membranes when cysteine residues were positioned in the SNARE motif or C terminus of the transmembrane domain. When specific Sec22 cysteine derivatives are present on both donor COPII vesicles and acceptor Golgi membranes, the formation of disulfide cross-links provide clear readouts on trans- and cis-SNARE arrangements during this fusion event. The Sec22 transmembrane domain was required for efficient homodimer formation and for membrane fusion suggesting a functional role for Sec22 homodimers. We propose that Sec22 homodimers promote assembly of higher-order SNARE complexes to catalyze membrane fusion. Sec22 is also reported to function in macroautophagy and in formation of endoplasmic reticulum-plasma membrane contact sites therefore homodimer assembly may regulate Sec22 activity across a range of cellular processes.