Methods for Studying Interactions Between Atg8/LC3/GABARAP and LIR-Containing Proteins.

LC3/GABARAP proteins (LC3/GABARAPs) are mammalian orthologues of yeast Atg8, small ubiquitin (Ub)-like proteins (UBLs) whose covalent attachment to lipid membranes is crucial for the growth and closure of the double membrane vesicle called the autophagosome. In the past decade, it was demonstrated that Atg8/LC3/GABARAPs are also required for autophagic degradation of cargos in a selective fashion. Cargo selectivity is ensured by receptor proteins, such as p62/SQSTM1, NBR1, Cue5, Atg19, NIX, Atg32, NCOA4, and FAM134B, which simultaneously bind Atg8/LC3/GABARAPs and the cargo together, thereby linking the core autophagic machinery to the target structure: a protein, an organelle, or a pathogen. LC3-interacting regions (LIRs) are short linear motifs within selective autophagy receptors and some other structural and signaling proteins (e.g., ULK1, ATG13, FIP200, and Dvl2), which mediate binding to Atg8/LC3/GABARAPs. Identification and characterization of LIR-containing proteins have provided important insights into the biology of the autophagy pathway, and studying their interactions with the core autophagy machinery represents a growing area of autophagy research. Here, we present protocols for the identification of LIR-containing proteins, i.e., by yeast-two-hybrid screening, glutathione S-transferase (GST) pulldown experiments, and peptide arrays. The use of two-dimensional peptide arrays also represents a powerful method to identify the residues of the LIR motif that are critical for binding. We also describe a biophysical method for studying interactions between Atg8/LC3/GABARAP and LIR-containing proteins and a protocol for preparation and purification of LIR peptides.

The Fas ligand intracellular domain is released by ADAM10 and SPPL2a cleavage in T-cells.

Fas ligand (FasL) is a type II transmembrane protein belonging to the tumor necrosis factor family. Its binding to the cognate Fas receptor triggers the apoptosis that plays a pivotal role in the maintenance of immune system homeostasis. The cell death-inducing property of FasL has been associated with its extracellular domain, which can be cleaved off by metalloprotease activity to produce soluble FasL. The fate of the remaining membrane-anchored N-terminal part of the FasL molecule has not been determined. Here we show that post-translational processing of overexpressed and endogenous FasL in T-cells by the disintegrin and metalloprotease ADAM10 generates a 17-kDa N-terminal fragment, which lacks the receptor-binding extracellular domain. This FasL remnant is membrane anchored and further processed by SPPL2a, a member of the signal peptide peptidase-like family of intramembrane-cleaving proteases. SPPL2a cleavage liberates a smaller and highly unstable fragment mainly containing the intracellular FasL domain (FasL ICD). We show that this fragment translocates to the nucleus and is capable of inhibiting gene transcription. With ADAM10 and SPPL2a we have identified two proteases implicated in FasL processing and release of the FasL ICD, which has been shown to be important for retrograde FasL signaling.

Characterization of indolinones which preferentially inhibit VEGF-C- and VEGF-D-induced activation of VEGFR-3 rather than VEGFR-2.

VEGF-C and VEGF-D are lymphangiogenic factors that bind to and activate VEGFR-3, a fms-like tyrosine kinase receptor whose expression is limited almost exclusively to lymphatic endothelium in the adult. Processed forms of VEGF-C and VEGF-D can also activate VEGFR-2, a key player in the regulation of angiogenesis. There is increasing evidence to show that these receptor-ligand interactions play a pivotal role in a number of pathological situations. Inhibition of receptor activation by VEGF-C and VEGF-D could therefore be pharmaceutically useful. Furthermore, to understand the different roles of VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 in pathological situations it will be necessary to dissect the complex interactions of these ligands and their receptors. To facilitate such studies we cloned, sequenced and characterized the expression of rat VEGF-C and VEGF-D. We showed that Cys152-->Ser mutants of processed rat VEGF-C can activate VEGFR-3 but not VEGFR-2, while the corresponding mutation in rat VEGF-D inhibits its ability to activate both VEGFR-2 and VEGFR-3. We also synthesized and characterized indolinones that differentially block VEGF-C- and VEGF-D-induced VEGFR-3 kinase activity compared to that of VEGFR-2. These tools should be useful in analysing the different activities and roles of VEGF-C, VEGF-D and their ligands, and in blocking VEGFR-3-mediated lymphangiogenesis.

Markers for the lymphatic endothelium: in search of the holy grail?

The ability to discriminate reliably at the histological level between blood and lymphatic microcapillaries would greatly assist the study of a number of biological and pathological questions and may also be of clinical utility. A structure-function comparison of these types of microcapillary suggests that differences which could function as markers to allow discrimination between blood and lymphatic endothelium should exist. Indeed, to date a variety of such markers have been proposed, including basement membrane components, constituents of junctional complexes such as desmoplakin and enzymes such as 5'-nucleotidase. Additionally, a variety of cell surface molecules are thought to be differentially expressed, including PAL-E, VEGFR-3, podoplanin, and LYVE-1. Several of the lymphatic markers proposed in the literature require further characterization to demonstrate fully their lymphatic specificity and some have proven not to be reliable. The relative merits and drawbacks of each of the proposed markers is discussed.

Oral health and oral health behaviour in a population of diabetic outpatient clinic attenders.

The control of oral health in individuals suffering from diabetes mellitus may affect a diabetic individual's insulin requirements. This study examined the oral health status and behaviours of a group of diabetic patients and compared the results to those obtained in a recent UK national survey of oral health. The results showed that, despite reporting higher levels of oral self-care, the diabetic population suffered from higher rates of caries than "normal" individuals. These differences could not be accounted for by the treatment received from dentists. It is concluded that diabetic patients are more caries prone than the general population and that the cause of this difference should be sought, as the traditional aetiological agent for caries cannot account for the increased caries rate. If the aetiology of the findings of this study were determined, progress could be made in the search for indicators of increased caries risk.