Global, site-specific analysis of neuronal protein S-acylation

Protein S-acylation (palmitoylation) is a reversible lipid modification that is an important regulator of dynamic membrane-protein interactions. Proteomic approaches have uncovered many putative palmitoylated proteins however, methods for comprehensive palmitoylation site characterization are lacking. We demonstrate a quantitative site-specific-Acyl-Biotin-Exchange (ssABE) method that allowed the identification of 906 putative palmitoylation sites on 641 proteins from mouse forebrain. 62% of sites map to known palmitoylated proteins and 102 individual palmitoylation sites are known from the literature. 54% of palmitoylation sites map to synaptic proteins including many GPCRs, receptors/ion channels and peripheral membrane proteins. Phosphorylation sites were also identified on a subset of peptides that were palmitoylated, demonstrating for the first time co-identification of these modifications by mass spectrometry. Palmitoylation sites were identified on over half of the family of palmitoyl-acyltransferases (PATs) that mediate protein palmitoylation, including active site thioester-linked palmitoyl intermediates. Distinct palmitoylation motifs and site topology were identified for integral membrane and soluble proteins, indicating potential differences in associated PAT specificity and palmitoylation function. ssABE allows the global identification of palmitoylation sites as well as measurement of the active site modification state of PATs, enabling palmitoylation to be studied at a systems level

Regulation and function of the palmitoyl‐acyltransferase ZDHHC5

Protein palmitoylation (S‐acylation) has emerged as an important player in a range of cellular processes, and as a result, the palmitoyl‐acyltransferase (PAT) enzymes which mediate this modification have entered into the spotlight. Palmitoyltransferase ZDHHC5 (ZDHHC5) is among the more unique members of the PAT family as it is mainly localised to the plasma membrane and contains an extended cytoplasmic domain with several regulatory features. ZDHHC5 plays a vital role in a wide range of processes in different cell types. In this review, we offer a summary of the functions of ZDHHC5 in synaptic plasticity, cardiac function, cell adhesion and fatty acid uptake, among other processes. We also explore recent work has revealed several mechanisms to control the activity, localisation and function of ZDHHC5

Batch fabrication of micro-coils for MR spectroscopy on silicon

Published versio

Cell‐type specific visualization and biochemical isolation of endogenous synaptic proteins in mice

In recent years, the remarkable molecular complexity of synapses has been revealed, with over 1000 proteins identified in the synapse proteome. Although it is known that different receptors and other synaptic proteins are present in different types of neurons, the extent of synapse diversity across the brain is largely unknown. This is mainly due to the limitations of current techniques. Here we report an efficient method for the purification of synaptic protein‐complexes, fusing a high‐affinity tag to endogenous PSD95 in specific cell types. We also developed a strategy which enables the visualization of endogenous PSD95 with fluorescent‐proteins tag in Cre‐recombinase expressing cells. We demonstrate the feasibility of proteomic analysis of synaptic protein‐complexes and visualization of these in specific cell types. We find that the composition of PSD95‐complexes purified from specific cell types differs from those extracted from tissues with diverse cellular composition. The results suggest that there might be differential interactions in the PSD95‐complexes in different brain regions. We have detected differentially interacting proteins by comparing datasets from the whole hippocampus and the CA3 subfield of the hippocampus. Therefore, these novel conditional PSD95 tagging lines will not only serve as powerful tools for precisely dissecting synapse diversity in specific brain regions and subsets of neuronal cells, but also provide an opportunity to better understand brain region‐ and cell type‐specific alterations associated with various psychiatric/neurological diseases. These newly developed conditional gene‐tagging methods can be applied to many different synaptic proteins and will facilitate research on the molecular complexity of synapses

Typhoid toxin hijacks Wnt5a to establish host senescence and Salmonella infection

Damage to our genome causes acute senescence in mammalian cells, which undergo growth arrest and release a senescence-associated secretory phenotype (SASP) that propagates the stress response to bystander cells. Thus, acute senescence is a powerful tumor suppressor. Salmonella enterica hijacks senescence through its typhoid toxin, which usurps unidentified factors in the stress secretome of senescent cells to mediate intracellular infections. Here, transcriptomics of toxin-induced senescent cells (TxSCs) and proteomics of their secretome identify the factors as Wnt5a, INHBA, and GDF15. Wnt5a establishes a positive feedback loop, driving INHBA and GDF15 expression. In fibroblasts, Wnt5a and INHBA mediate autocrine senescence in TxSCs and paracrine senescence in naive cells. Wnt5a synergizes with GDF15 to increase Salmonella invasion. Intestinal TxSCs undergo apoptosis without Wnt5a, which is required for establishing intestinal TxSCs. The study reveals how an innate defense against cancer is co-opted by a bacterial pathogen to cause widespread damage and mediate infections

Reproducibility of the lung anatomy under Active Breathing Coordinator control: Dosimetric consequences for scanned proton treatments.

Purpose/Objective The treatment of moving targets with scanning proton beams is challenging. By controlling lung volumes, Active Breathing Control (ABC) assists breath-holding for motion mitigation. The delivery of proton treatment fractions often exceeds feasible breath-hold durations, requiring high breath-hold reproducibility. Therefore, we investigated dosimetric consequences of anatomical reproducibility uncertainties in the lung under ABC, evaluating robustness of scanned proton treatments during breath-hold. Material/Methods T1-weighted MRIs of five volunteers were acquired during ABC, simulating image acquisition during four subsequent breath-holds within one treatment fraction. Deformation vector fields obtained from these MRIs were used to deform 95% inspiration phase CTs of 3 randomly selected non-small-cell lung cancer patients (Figure 1). Per patient, an intensity-modulated proton plan was recalculated on the 3 deformed CTs, to assess the dosimetric influence of anatomical breath-hold inconsistencies. Results Dosimetric consequences were negligible for patient 1 and 2 (Figure 1). Patient 3 showed a decreased volume (95.2%) receiving 95% of the prescribed dose for one deformed CT. The volume receiving 105% of the prescribed dose increased from 0.0% to 9.9%. Furthermore, the heart volume receiving 5 Gy varied by 2.3%. Figure 2 shows dose volume histograms for all relevant structures in patient 3. Conclusion Based on the studied patients, our findings suggest that variations in breath-hold have limited effect on the dose distribution for most lung patients. However, for one patient, a significant decrease in target coverage was found for one of the deformed CTs. Therefore, further investigation of dosimetric consequences from intra-fractional breath-hold uncertainties in the lung under ABC is needed

Proteomic approaches to study cysteine oxidation: applications in neurodegenerative diseases

Oxidative stress appears to be a key feature of many neurodegenerative diseases either as a cause or consequence of disease. A range of molecules are subject to oxidation, but in particular, proteins are an important target and measure of oxidative stress. Proteins are subject to a range of oxidative modifications at reactive cysteine residues, and depending on the level of oxidative stress, these modifications may be reversible or irreversible. A range of experimental approaches has been developed to characterize cysteine oxidation of proteins. In particular, mass spectrometry-based proteomic methods have emerged as a powerful means to identify and quantify cysteine oxidation sites on a proteome scale; however, their application to study neurodegenerative diseases is limited to date. Here we provide a guide to these approaches and highlight the under-exploited utility of these methods to measure oxidative stress in neurodegenerative diseases for biomarker discovery, target engagement and to understand disease mechanisms