Universal sample preparation method for proteome analysis

Autophagy and Neurodegeneration: Pathogenic Mechanisms and Therapeutic Opportunities

Autophagy and the ubiquitin-proteasome system are the two major quality control pathways responsible for cellular homeostasis. As such, they provide protection against age-associated changes and a plethora of human diseases. Ubiquitination is utilized as a degradation signal by both systems, albeit in different ways, to mark cargoes for proteasomal and lysosomal degradation. Both systems intersect and communicate at multiple points to coordinate their actions in proteostasis and organelle homeostasis. This review summarizes molecular details of how proteasome and autophagy pathways are functionally interconnected in cells and indicates common principles and nodes of communication that can be therapeutically exploited.

Proteasomal and Autophagic Degradation Systems.

Amyotrophic lateral sclerosis

The Ubiquitin Code in the Ubiquitin-Proteasome System and Autophagy

Growing evidence implicates impairment of autophagy as a candidate pathogenic mechanism in the spectrum of neurodegenerative disorders which includes amyotrophic lateral sclerosis and frontotemporal lobar degeneration (ALS-FTLD). SQSTM1, which encodes the autophagy receptor SQSTM1/p62, is genetically associated with ALS-FTLD, although to date autophagy-relevant functional defects in disease-associated variants have not been described. A key protein-protein interaction in autophagy is the recognition of a lipid-anchored form of LC3 (LC3-II) within the phagophore membrane by SQSTM1, mediated through its LC3-interacting region (LIR), and notably some ALS-FTLD mutations map to this region. Here we show that although representing a conservative substitution and predicted to be benign, the ALS-associated L341V mutation of SQSTM1 is defective in recognition of LC3B. We place our observations on a firm quantitative footing by showing the L341V-mutant LIR is associated with a ∼3-fold reduction in LC3B binding affinity and using protein NMR we rationalize the structural basis for the effect. This functional deficit is realized in motor neuron-like cells, with the L341V mutant EGFP-mCherry-SQSTM1 less readily incorporated into acidic autophagic vesicles than the wild type. Our data supports a model in which the L341V mutation limits the critical step of SQSTM1 recruitment to the phagophore. The oligomeric nature of SQSTM1, which presents multiple LIRs to template growth of the phagophore, potentially gives rise to avidity effects which amplify the relatively modest impact of any single mutation on LC3B binding. Over the lifetime of a neuron, impaired autophagy could expose a vulnerability, which ultimately tips the balance from cell survival toward cell death.

https://www.tandfonline.com/doi/pdf/10.1080/15548627.2016.1170257

Defective recognition of LC3B by mutant SQSTM1/p62 implicates impairment of autophagy as a pathogenic mechanism in ALS-FTLD.

Specific interactions between proteins and their binding partners are fundamental to life processes. The ability to detect protein complexes, and map their sites of binding, is crucial to understanding basic biology at the molecular level. Methods that employ sensitive analytical techniques such as mass spectrometry have the potential to provide valuable insights with very little material and on short time scales. Here we present a differential protein footprinting technique employing an efficient photo-activated probe for use with mass spectrometry. Using this methodology the location of a carbohydrate substrate was accurately mapped to the binding cleft of lysozyme, and in a more complex example, the interactions between a 100 kDa, multi-domain deubiquitinating enzyme, USP5 and a diubiquitin substrate were located to different functional domains. The much improved properties of this probe make carbene footprinting a viable method for rapid and accurate identification of protein binding sites utilizing benign, near-UV photoactivation.

/pdf/carbene-footprinting-accurately-maps-binding-sites-in-3srvaaxcuu.pdf

Carbene footprinting accurately maps binding sites in protein-ligand and protein-protein interactions.

The importance of offspring value : maternal defence in parasitoid contests

Ubiquitin-binding domains (UBDs) are modular units found within ubiquitin-binding proteins that mediate the non-covalent recognition of (poly)ubiquitin modifications. A variety of mechanisms are employed in vivo to achieve polyubiquitin linkage and chain length selectivity by UBDs, the structural basis of which have in some instances been determined. Here, we review current knowledge related to ubiquitin recognition mechanisms at the molecular level and explore how such information has been exploited in the design and application of UBDs in isolation or artificially arranged in tandem as tools to investigate ubiquitin-modified proteomes. Specifically, we focus on the use of UBDs to directly purify or detect (poly)ubiquitin-modified proteins and more broadly for the targeted manipulation of ubiquitin-mediated processes, highlighting insights into ubiquitin signalling that have been provided.

Ubiquitin-binding domains: mechanisms of ubiquitin recognition and use as tools to investigate ubiquitin-modified proteomes.

Excerpt To the editor: Bronchoscopy with bronchoalveolar lavage and transbronchial lung biopsy are now readily available for detection of opportunistic lung infections in patients with the acquired...

Daniel Scott

Papers

Defective recognition of LC3B by mutant SQSTM1/p62 implicates impairment of autophagy as a pathogenic mechanism in ALS-FTLD.

Carbene footprinting accurately maps binding sites in protein-ligand and protein-protein interactions.

The importance of offspring value : maternal defence in parasitoid contests

Ubiquitin-binding domains: mechanisms of ubiquitin recognition and use as tools to investigate ubiquitin-modified proteomes.

Cytomegalovirus Infection, Ascending Myelitis, and Pulmonary Embolus