Comprehensive Ubiquitin E2 Profiling of Ten Ubiquitin E3 Ligases

The ubiquitin pathway regulates diverse functions including protein localization and stability. The complexity of the pathway involving nearly 40 identified E2 conjugating enzymes and over 600 E3 ligases raises the issue of specificity. With the E2s and E3s fitting into a limited number of classes based on bioinformatics, structures, and proven activities, there is not a clear picture as to what would determine which E2/E3 enzyme pair would be functional. There have been many reports of limited E2/E3 activity profiling with a small number of E2s and E3s. We have expanded on this to investigate the activity of ubiquitin E2s covering the majority of the reported classes/families in concert with a number of E3s implicated in a variety of diseases. Using an ELISA-based assay we screened 10 E3 ligases against a panel of 11 E2s to determine which E2/E3 pairs exhibited E3 autoubiquitylation activity. In addition, the ubiquitin chain linkage preference by certain E2/E3 pairs was investigated. Finally, substrate ubiquitylation was assayed for the E3 ligase MuRF1 using various E2/MuRF1 pairs. These studies demonstrate the utility of identifying the correct E2/E3 pair to monitor specific substrate ubiquitylation.     

Suppression of cytokine signaling: The SOCS perspective

The discovery of the Suppressor of Cytokine Signaling (SOCS) family of proteins has resulted in a significant body of research dedicated to dissecting their biological functions and the molecular mechanisms by which they achieve potent and specific inhibition of cytokine and growth factor signaling. The Australian contribution to this field has been substantial, with the initial discovery of SOCS1 by Hilton, Starr and colleagues (discovered concurrently by two other groups) and the following work, providing a new perspective on the regulation of JAK/STAT signaling. In this review, we reflect on the critical discoveries that have lead to our current understanding of how SOCS proteins function and discuss what we see as important questions for future research.     

1H NMR spectroscopic investigations of tissue metabolite biomarker response to Cu II exposure in terrestrial invertebrates: identification of free histidine as a novel biomarker of exposure to copper in earthworms

High resolution 1H NMR spectroscopy of biofluids, cells and tissue extracts allows rapid, non destructive analysis for a wide range of metabolites and organic compounds with minimal sample pre treatment. We have applied high resolution 1H NMR spectroscopy to investigate the biochemical effects of Cu II in two earthworm species Eisenia andrei n =78 and Lumbricus rubellus n =45 exposed under laboratory and semi field conditions respectively. The most marked metabolic response was the elevation of endogenous whole body free histidine in animals which positively correlated with increasing copper exposure and total copper burden in the semi field experiment. Histidine forms thermodynamically stable copper complexes under a wide range of physico chemical conditions and we proposed that the elevation of free histidine in response to copper challenge provides an energetically low cost detoxification mechanism. Histidine elevation may also provide a novel molecular biomarker of Cu II exposure in environmental situations.     

TMEM106B p.T185S regulates TMEM106B protein levels: implications for frontotemporal dementia

Frontotemporal lobar degeneration (FTLD) is the second leading cause of dementia in individuals under age 65. In many patients, the predominant pathology includes neuronal cytoplasmic or intranuclear inclusions of ubiquitinated TAR DNA binding protein 43 (FTLD‐TDP). Recently, a genome‐wide association study identified the first FTLD‐TDP genetic risk factor, in which variants in and around the TMEM106B gene (top SNP rs1990622) were significantly associated with FTLD‐TDP risk. Intriguingly, the most significant association was in FTLD‐TDP patients carrying progranulin (GRN) mutations. Here, we investigated to what extent the coding variant, rs3173615 (p.T185S) in linkage disequilibrium with rs1990622, affects progranulin protein (PGRN) biology and transmembrane protein 106 B (TMEM106B) regulation. First, we confirmed the association of TMEM106B variants with FTLD‐TDP in a new cohort of GRN mutation carriers. We next generated and characterized a TMEM106B‐specific antibody for investigation of this protein. Enzyme‐linked immunoassay analysis of progranulin protein levels showed similar effects upon T185 and S185 TMEM106B over‐expression. However, over‐expression of T185 consistently led to higher TMEM106B protein levels than S185. Cycloheximide treatment experiments revealed that S185 degrades faster than T185 TMEM106B, potentially due to differences in N‐glycosylation at residue N183. Together, our results provide a potential mechanism by which TMEM106B variants lead to differences in FTLD‐TDP risk.

     

Linear fusigen as the major hydroxamate siderophore of the ectomycorrhizal Basidiomycota Laccaria laccata and Laccaria bicolor

A screening for siderophores produced by the ectomycorrhizal fungi Laccaria laccata and Laccaria bicolor in synthetic low iron medium revealed the release of several different hydroxamate siderophores of which four major siderophores could be identified by high resolution mass spectrometry. While ferricrocin, coprogen and triacetylfusarinine C were assigned as well as other known fungal siderophores, a major peak of the siderophore mixture revealed an average molecular mass of 797 for the iron-loaded compound. High resolution mass spectrometry indicated an absolute mass of m/z = 798.30973 ([M + H]⁺). With a relative error of Δ = 0.56 ppm this corresponds to linear fusigen (C₃₃H₅₂N₆O₁₃Fe; MW = 797.3). The production of large amounts of linear fusigen by these basidiomycetous mycorrhizal fungi may possibly explain the observed suppression of plant pathogenic Fusarium species. For comparative purposes Fusarium roseum was included in this study as a well known producer of cyclic and linear fusigen.     

Empty class II major histocompatibility complex created by peptide photolysis establishes the role of DM in peptide association

DM catalyzes the exchange of peptides bound to Class II major histocompatibility complex (MHC) molecules. Because the dissociation and association components of the overall reaction are difficult to separate, a detailed mechanism of DM catalysis has long resisted elucidation. UV irradiation of DR molecules loaded with a photocleavable peptide (caged Class II MHC molecules) enabled synchronous and verifiable evacuation of the peptide-binding groove and tracking of early binding events in real time by fluorescence polarization. Empty DR molecules generated by photocleavage rapidly bound peptide but quickly resolved into species with substantially slower binding kinetics. DM formed a complex with empty DR molecules that bound peptide with even faster kinetics than empty DR molecules just having lost their peptide cargo. Mathematical models demonstrate that the peptide association rate of DR molecules is substantially higher in the presence of DM. We therefore unequivocally establish that DM contributes directly to peptide association through formation of a peptide-loading complex between DM and empty Class II MHC. This complex rapidly acquires a peptide analogous to the MHC class I peptide-loading complex.