Unusual N-glycan structures required for trafficking Toxoplasma gondii GAP50 to the inner membrane complex regulate host cell entry through parasite motility

Toxoplasma gondii motility, which is essential for host cell entry, migration through host tissues, and invasion, is a unique form of actin-dependent gliding. It is powered by a motor complex mainly composed of myosin heavy chain A, myosin light chain 1, gliding associated proteins GAP45, and GAP50, the only integral membrane anchor so far described. In the present study, we have combined glycomic and proteomic approaches to demonstrate that all three potential N-glycosylated sites of GAP50 are occupied by unusual N-glycan structures that are rarely found on mature mammalian glycoproteins. Using site-directed mutagenesis, we show that N-glycosylation is a prerequisite for GAP50 transport from the endoplasmic reticulum to the Golgi apparatus and for its subsequent delivery into the inner complex membrane. Assembly of key partners into the gliding complex, and parasite motility are severely impaired in the unglycosylated GAP50 mutants. Furthermore, comparative affinity purification using N-glycosylated and unglycosylated GAP50 as bait identified three novel hypothetical proteins including the recently described gliding associated protein GAP40, and we demonstrate that N-glycans are required for efficient binding to gliding partners. Collectively, these results provide the first detailed analyses of T. gondii N-glycosylation functions that are vital for parasite motility and host cell entry.     

Toxoplasma sortilin-like receptor regulates protein transport and is essential for apical secretory organelle biogenesis and host infection

Apicomplexan parasites have an assortment of unique apical secretory organelles (rhoptries and micronemes), which have crucial functions in host infection. Here, we show that a Toxoplasma gondii sortilin-like receptor (TgSORTLR) is required for the subcellular localization and formation of apical secretory organelles. TgSORTLR is a transmembrane protein that resides within Golgi-endosomal related compartments. The lumenal domain specifically interacts with rhoptry and microneme proteins, while the cytoplasmic tail of TgSORTLR recruits cytosolic sorting machinery involved in anterograde and retrograde protein transport. Ectopic expression of the N-terminal TgSORTLR lumenal domain results in dominant negative effects with the mislocalization of both endogenous TgSORTLR as well as rhoptry and microneme proteins. Conditional ablation of TgSORTLR disrupts rhoptry and microneme biogenesis, inhibits parasite motility, and blocks both invasion into and egress from host cells. Thus, the sortilin-like receptor is essential for protein trafficking and the biogenesis of key secretory organelles in Toxoplasma.     

Physiological changes in green stems of Vitis vinifera L. cv. Chardonnay in response to esca proper and apoplexy revealed by proteomic and transcriptomic analyses

Among grapevine trunk diseases, esca proper and apoplexy commonly represent a threat for viticulture worldwide. To retrieve further information about the mechanisms activated in apoplectic and esca proper-affected plants, a two-dimensional gel electrophoresis (2-DE) based analysis was conducted on green stems from 26-year-old standing vines. Symptomatic and asymptomatic stems from both apoplectic (A) and esca proper-affected (E) plants compared to control (without visual symptom since 10 years) stems were studied. Thirty-three differentially expressed proteins were identified by nanoLC-MS/MS and included into three groups conceptually defined as proteins involved in (i) metabolism and energy, (ii) stress tolerance, and (iii) defense response. For nine of them, expression of the relative mRNA's was also monitored by qRT-PCR. Proteome variations were specifically related to apoplexy and esca proper but were more similar in asymptomatic stems than in the symptomatic ones. Remarkable quantitative differences were noted for several proteins in symptomatic stems according to the expressed form, A and E. Results further indicate that similar responses are likely activated in asymptomatic stems but a various quantitative expression is triggered upon onset of apoplexy or esca proper symptoms while both kind of plants are infected by the same pathogenic fungi.     

Increased level of phosphorylated desmin and its degradation products in heart failure

Although several risk factors such as infarct size have been identified, the progression/severity of heart failure (HF) remains difficult to predict in clinical practice. Using an experimental rat model of ischemic HF and phosphoproteomic technology, we found an increased level of phosphorylated desmin in the left ventricle (LV) of HF-rats. The purpose of the present work is to assess whether desmin is a circulating or only a tissue biomarker of HF. We used several antibodies in order to detect desmin, its proteolytic fragments and its phosphorylated form in LV and plasma by western blot, phosphate affinity electrophoresis, mass spectrometry and immunofluorescence. Plasma was treated with combinatorial peptide ligand library or depleted for albumin and immunoglobulins to increase the sensitivity of detection. We found a 2-fold increased serine-desmin phosphorylation in the LV of HF-rats, mainly in the insoluble fraction, suggesting the formation of desmin aggregates. Desmin cleavage products were also detected in the LV of HF rats, indicating that the increased phosphorylation of desmin results in more susceptibility to proteolytic activity, likely mediated by calpain activity. The native desmin and its degradation products were undetectable in the plasma of rat, mouse or human. These data suggest the potential of serine-phosphorylated form of desmin and its degradation products, but not of desmin itself, as tissue but not circulating biomarkers of HF.