The microtubule-associated protein tau has been implicated in the pathogenesis of Alzheimer's disease (AD) and other neurodegenerative disorders. Reducing tau levels ameliorates AD-related synaptic, network, and behavioral abnormalities in transgenic mice expressing human amyloid precursor protein (hAPP). We used mass spectrometry to characterize the post-translational modification of endogenous tau isolated from wild-type and hAPP mice. We identified seven types of tau modifications at 63 sites in wild-type mice. Wild-type and hAPP mice had similar modifications, supporting the hypothesis that neuronal dysfunction in hAPP mice is enabled by physiological forms of tau. Our findings provide clear evidence for acetylation and ubiquitination of the same lysine residues; some sites were also targeted by lysine methylation. Our findings refute the hypothesis of extensive O-linked N-acetylglucosamine (O-GlcNAc) modification of endogenous tau. The complex post-translational modification of physiological tau suggests that tau is regulated by diverse mechanisms.

bThe activity of phosphatidylinositol 4-kinase class III beta (PI4KIII␤) has been shown to be required for the replication of multiple picornaviruses; however, it is unclear whether a physical association between PI4KIII␤ and the viral replication machinery exists and, if it does, whether association is necessary. We examined the ability of the 3A protein from 18 different picornaviruses to form a complex with PI4KIII␤ by affinity purification of Strep-Tagged transiently transfected constructs followed by mass spectrometry and Western blotting for putative interacting targets. We found that the 3A proteins of Aichi virus, bovine kobuvirus, poliovirus, coxsackievirus B3, and human rhinovirus 14 all copurify with PI4KIII␤. Furthermore, we found that multiple picornavirus 3A proteins copurify with the Golgi adaptor protein acyl coenzyme A (acyl-CoA) binding domain protein 3 (ACBD3/GPC60), including those from Aichi virus, bovine kobuvirus, human rhinovirus 14, poliovirus, and coxsackievirus B2, B3, and B5. Affinity purification of ACBD3 confirmed interaction with multiple picornaviral 3A proteins and revealed the ability to bind PI4KIII␤ in the absence of 3A. Mass-spectrometric analysis of transiently expressed Aichi virus, bovine kobuvirus, and human klassevirus 3A proteins demonstrated that the N-terminal glycines of these 3A proteins are myristoylated. Alanine-scanning mutagenesis along the entire length of Aichi virus 3A followed by transient expression and affinity purification revealed that copurification of PI4KIII␤ could be eliminated by mutation of specific residues, with little or no effect on recruitment of ACBD3. One mutation at the N terminus, I5A, significantly reduced copurification of both ACBD3 and PI4KIII␤. The dependence of Aichi virus replication on the activity of PI4KIII␤ was confirmed by both chemical and genetic inhibition. Knockdown of ACBD3 by small interfering RNA (siRNA) also prevented replication of both Aichi virus and poliovirus. Point mutations in 3A that eliminate PI4KIII␤ association sensitized Aichi virus to PIK93, suggesting that disruption of the 3A/ACBD3/ PI4KIII␤ complex may represent a novel target for therapeutic intervention that would be complementary to the inhibition of the kinase activity itself.

A simple and rapid multiplex substrate profiling method has been developed to reveal the substrate specificity of any endo- or exo-peptidase using LC-MS/MS sequencing. A physicochemically diverse library of peptides was generated by incorporating all combinations of neighbor and near-neighbor amino acid pairs into decapeptide sequences that are flanked by unique dipeptides at each terminus. Addition of a panel of evolutionarily diverse peptidases to a mixture of these tetradecapeptides generated prime and non-prime site information and substrate specificity matched or expanded upon previous substrate motifs. This method biochemically confirmed the activity of the klassevirus 3C gene responsible for polypeptide processing and allowed Granzyme B substrates to be ranked by enzymatic turnover efficiency using label-free quantitation of precursor ion abundance. Furthermore, the proteolytic secretions from a parasitic flatworm larvae and a pancreatic cancer cell line were deconvoluted in a subtractive strategy using class-specific peptidase inhibitors.

Schistosomiasis is a global health problem caused by several species of schistosome blood flukes. The initial stage of infection is invasion of human skin by a multicellular larva, the cercaria. We identified proteins released by cercariae when they are experimentally induced to exhibit invasive behavior. Comparison of the proteome obtained from skin lipid-induced cercariae (the natural activator), a cleaner mechanical induction procedure, and an uninduced proteomic control allowed identification of protein groups contained in cercarial acetabular gland secretion versus other sources. These included a group of proteins involved in calcium binding, calcium regulation, and calcium-activated functions; two proteins (paramyosin and SPO-1) implicated in immune evasion; and protease isoforms implicated in degradation of host skin barriers. Several other protein families, traditionally found as cytosolic proteins, appeared concentrated in secretory cells. These included proteins with chaperone activity such as HSP70, -86, and -60. Comparison of the three experimental proteomes also allowed identification of protein contaminants from the environment that were identified because of the high sensitivity of the MS/MS system used. These included proteins from the intermediate host snail in which cercariae develop, the investigator, and the laboratory environment. Identification of proteins secreted by invasive larvae provides important new information for validation of models of skin invasion and immune evasion and aids in rational development of an anti-schistosome vaccine.

Neutrophil extracellular traps (NETs) consist of antimicrobial molecules embedded in a web of extracellular DNA. Formation of NETs is considered to be a defense mechanism utilized by neutrophils to ensnare and kill invading pathogens, and has been recently termed NETosis. Neutrophils can be stimulated to undergo NETosis ex vivo, and are predicted to contain high levels of serine proteases, such as neutrophil elastase (NE), cathepsin G (CG) and proteinase 3 (PR3). Serine proteases are important effectors of neutrophil-mediated immunity, which function directly by degrading pathogenic virulent factors and indirectly via proteolytic activation or deactivation of cytokines, chemokines and receptors. In this study, we utilized a diverse and unbiased peptide library to detect and profile protease activity associated with NETs induced by phorbol-12-myristate-13-acetate (PMA). We obtained a “proteolytic signature” from NETs derived from healthy donor neutrophils and used proteomics to assist in the identification of the source of this proteolytic activity. In addition, we profiled each neutrophil serine protease and included the newly identified enzyme, neutrophil serine protease 4 (NSP4). Each enzyme had overlapping yet distinct endopeptidase activities and often cleaved at unique sites within the same peptide substrate. The dominant proteolytic activity in NETs was attributed to NE; however, cleavage sites corresponding to CG and PR3 activity were evident. When NE was immunodepleted, the remaining activity was attributed to CG and to a lesser extent PR3 and NSP4. Our results suggest that blocking NE activity would abrogate the major protease activity associated with NETs. In addition, the newly identified substrate specificity signatures will guide the design of more specific probes and inhibitors that target NET-associated proteases.