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Frontotemporal lobar degeneration (FTLD) is the third most common neurodegenerative condition after Alzheimer's and Parkinson's diseases1. FTLD typically presents in 45 to 64 year olds with behavioural changes or progressive decline of language skills2. The subtype FTLD-TDP is characterized by certain clinical symptoms and pathological neuronal inclusions with TAR DNA-binding protein (TDP-43) immunoreactivity3. Here we extracted amyloid fibrils from brains of four patients representing four of the five FTLD-TDP subclasses, and determined their structures by cryo-electron microscopy. Unexpectedly, all amyloid fibrils examined were composed of a 135-residue carboxy-terminal fragment of transmembrane protein 106B (TMEM106B), a lysosomal membrane protein previously implicated as a genetic risk factor for FTLD-TDP4. In addition to TMEM106B fibrils, we detected abundant non-fibrillar aggregated TDP-43 by immunogold labelling. Our observations confirm that FTLD-TDP is associated with amyloid fibrils, and that the fibrils are formed by TMEM106B rather than TDP-43.

Different tauopathies are characterized by the isoform-specific composition of the aggregates found in the brain and by structurally distinct tau strains. Although tau oligomers have been implicated as important neurotoxic species, little is known about how the primary structures of the six human tau isoforms affect tau oligomerization because the oligomers are metastable and difficult to analyze. To address this knowledge gap, here, we analyzed the initial oligomers formed by the six tau isoforms in the absence of posttranslational modifications or other manipulations using dot blots probed by an oligomer-specific antibody, native-PAGE/western blots, photo-induced cross-linking of unmodified proteins, mass-spectrometry, and ion-mobility spectroscopy. We found that under these conditions, three-repeat (3R) isoforms are more prone than four-repeat (4R) isoforms to form oligomers. We also tested whether known inhibitors of tau aggregation affect its oligomerization using three small molecules representing different classes of tau aggregation inhibitors, Methylene Blue (MB), the molecular tweezer CLR01, and the all-D peptide TLKIVW, for their ability to inhibit or modulate the oligomerization of the six tau isoforms. Unlike their reported inhibitory effect on tau fibrillation, the inhibitors had little or no effect on the initial oligomerization. Our study provides novel insight into the primary-quaternary structure relationship of human tau and suggests that 3R-tau oligomers may be an important target for future development of compounds targeting pathological tau assemblies.

Acyl modifications vary greatly in terms of elemental composition and site of protein modification. Developing methods to identify acyl modifications more confidently can help to assess the scope of these modifications in large proteomic datasets. The utility of acyl-lysine immonium ions is analyzed for identifying the modifications in proteomic datasets. It is demonstrated that the cyclized immonium ion is a strong indicator of acyl-lysine presence when its rank or relative abundance compared to other ions within a spectrum is considered. Utilizing a stepped collision energy method in a shotgun experiment highlights the immonium ion. By implementing an analysis that accounted for features within each MS2 spectrum, the method clearly identifies peptides with short chain acyl-lysine modifications from complex lysates. Immonium ions can also be used to validate novel acyl modifications; in this study, the first examples of 3-hydroxylpimelyl-lysine modifications are reported and they are validated using immonium ions. Overall these results solidify the use of the immonium ion as a marker for acyl-lysine modifications in complex proteomic datasets.

Studies have successfully elucidated the mechanism of action of several effector domains that comprise the multifunctional-autoprocessing repeats-in-toxins (MARTX) toxins of Vibrio vulnificus. However, the biochemical linkage between the cysteine proteolytic activity of Makes Caterpillars Floppy (MCF)-like effector and its cellular effects remains unknown. In this study, we identify the host cell factors that activate in vivo and in vitro MCF autoprocessing as adenosine diphosphate (ADP)-Ribosylation Factor 1 (ARF1) and ADP-Ribosylation Factor 3 (ARF3). Autoprocessing activity is enhanced when ARF1 is in its active [guanosine triphosphate (GTP)-bound] form compared to the inactive [guanosine diphosphate (GDP)-bound] form. Subsequent to auto-cleavage, MCF is acetylated on its exposed N-terminal glycine residue. Acetylation apparently does not dictate subcellular localization as MCF is found localized throughout the cell. However, the cleaved form of MCF gains the ability to bind to the specialized lipid phosphatidylinositol 5-phosphate enriched in Golgi and other membranes necessary for endocytic trafficking, suggesting that a fraction of MCF may be subcellularly localized. Traditional thin-section electron microscopy, high-resolution cryoAPEX localization, and fluorescent microscopy show that MCF causes Golgi dispersal resulting in extensive vesiculation. In addition, host mitochondria are disrupted and fragmented. Mass spectrometry analysis found no reproducible modifications of ARF1 suggesting that ARF1 is not post-translationally modified by MCF. Further, catalytically active MCF does not stably associate with ARF1. Our data indicate not only that ARF1 is a cross-kingdom activator of MCF, but also that MCF may mediate cytotoxicity by directly targeting another yet to be identified protein. This study begins to elucidate the biochemical activity of this important domain and gives insight into how it may promote disease progression.

Improving the long-term stability of perovskite solar cells is critical to the deployment of this technology. Despite the great emphasis laid on stability-related investigations, publications lack consistency in experimental procedures and parameters reported. It is therefore challenging to reproduce and compare results and thereby develop a deep understanding of degradation mechanisms. Here, we report a consensus between researchers in the field on procedures for testing perovskite solar cell stability, which are based on the International Summit on Organic Photovoltaic Stability (ISOS) protocols. We propose additional procedures to account for properties specific to PSCs such as ion redistribution under electric fields, reversible degradation and to distinguish ambient-induced degradation from other stress factors. These protocols are not intended as a replacement of the existing qualification standards, but rather they aim to unify the stability assessment and to understand failure modes. Finally, we identify key procedural information which we suggest reporting in publications to improve reproducibility and enable large data set analysis.

Transparency in the lens is accomplished by the dense packing and short-range order interactions of the crystallin proteins in fiber cells lacking organelles. These features are accompanied by a lack of protein turnover, leaving lens proteins susceptible to a number of damaging modifications and aggregation. The loss of lens transparency is attributed in part to such aggregation during aging. Among the damaging post-translational modifications that accumulate in long-lived proteins, isomerization at aspartate residues has been shown to be extensive throughout the crystallins. In this study of the human lens, we localize the accumulation of l-isoaspartate within water-soluble protein extracts primarily to crystallin peptides in high-molecular weight aggregates and show with MS that these peptides are from a variety of crystallins. To investigate the consequences of aspartate isomerization, we investigated two αA crystallin peptides 52LFRTVLDSGISEVR65 and 89VQDDFVEIH98, identified within this study, with the l-isoaspartate modification introduced at Asp58 and Asp91, respectively. Importantly, whereas both peptides modestly increase protein precipitation, the native 52LFRTVLDSGISEVR65 peptide shows higher aggregation propensity. In contrast, the introduction of l-isoaspartate within a previously identified anti-chaperone peptide from water-insoluble aggregates, αA crystallin 66SDRDKFVIFL(isoAsp)VKHF80, results in enhanced amyloid formation in vitro The modification of this peptide also increases aggregation of the lens chaperone αB crystallin. These findings may represent multiple pathways within the lens wherein the isomerization of aspartate residues in crystallin peptides differentially results in peptides associating with water-soluble or water-insoluble aggregates. Here the eye lens serves as a model for the cleavage and modification of long-lived proteins within other aging tissues.

Structural characterization of intrinsically disordered proteins (IDPs) has been a major challenge in the field of protein science due to limited capabilities to obtain full-length high-resolution structures. Native ESI-MS with top-down MS was utilized to obtain structural features of protein-ligand binding for the Parkinson's disease-related protein, α-synuclein (αSyn), which is natively unstructured. Binding of heavy metals has been implicated in the accelerated formation of αSyn aggregation. Using high-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry, native top-down MS with various fragmentation methods, including electron capture dissociation (ECD), collisional activated dissociation (CAD), and multistage tandem MS (MS3), deduced the binding sites of cobalt and manganese to the C-terminal region of the protein. Ion mobility MS (IM-MS) revealed a collapse toward compacted states of αSyn upon metal binding. The combination of native top-down MS and IM-MS provides structural information of protein-ligand interactions for intrinsically disordered proteins. Graphical Abstract ᅟ.

Covalently cross-linked pilus polymers displayed on the cell surface of Gram-positive bacteria are assembled by class C sortase enzymes. These pilus-specific transpeptidases located on the bacterial membrane catalyze a two-step protein ligation reaction, first cleaving the LPXTG motif of one pilin protomer to form an acyl-enzyme intermediate and then joining the terminal Thr to the nucleophilic Lys residue residing within the pilin motif of another pilin protomer. To date, the determinants of class C enzymes that uniquely enable them to construct pili remain unknown. Here, informed by high-resolution crystal structures of corynebacterial pilus-specific sortase (SrtA) and utilizing a structural variant of the enzyme (SrtA2M), whose catalytic pocket has been unmasked by activating mutations, we successfully reconstituted in vitro polymerization of the cognate major pilin (SpaA). Mass spectrometry, electron microscopy, and biochemical experiments authenticated that SrtA2M synthesizes pilus fibers with correct Lys-Thr isopeptide bonds linking individual pilins via a thioacyl intermediate. Structural modeling of the SpaA-SrtA-SpaA polymerization intermediate depicts SrtA2M sandwiched between the N- and C-terminal domains of SpaA harboring the reactive pilin and LPXTG motifs, respectively. Remarkably, the model uncovered a conserved TP(Y/L)XIN(S/T)H signature sequence following the catalytic Cys, in which the alanine substitutions abrogated cross-linking activity but not cleavage of LPXTG. These insights and our evidence that SrtA2M can terminate pilus polymerization by joining the terminal pilin SpaB to SpaA and catalyze ligation of isolated SpaA domains in vitro provide a facile and versatile platform for protein engineering and bio-conjugation that has major implications for biotechnology.

Additional file 1: Table S1. Expression of actins, tubulin, myosin, fibroblast growth factor-16 and vimentin in control rats. Table S2. Expression of actins, tubulin and myosin in irradiated rats. Table S3. Membrane proteins present in murine endothelial cell cultures, and the rats model of AVM.

Site-specific chemical dimerization of fibroblast growth factor 2 (FGF2) with the optimal linker length resulted in a FGF2 homodimer with improved granulation tissue formation and blood vessel formation at exceptionally low concentrations. Homodimers of FGF2 were synthesized through site-specific linkages to both ends of different molecular weight poly(ethylene glycols) (PEGs). The optimal linker length was determined by screening dimer-induced metabolic activity of human dermal fibroblasts and found to be that closest to the inter-cysteine distance, 70Å, corresponding to 2kDa PEG. A straightforward analysis of the kinetics of second ligand binding as a function of tether length showed that, as the polymerization index (the number of monomer repeat units in the polymer, N) of the tether decreases, the mean time for second ligand capture decreases as ∼N(3/2), leading to an enhancement of the number of doubly bound ligands in steady-state for a given (tethered) ligand concentration. FGF2-PEG2k-FGF2 induced greater fibroblast metabolic activity than FGF2 alone, all other dimers, and all monoconjugates, at each concentration tested, with the greatest difference observed at low (0.1ng/mL) concentration. FGF2-PEG2k-FGF2 further exhibited superior activity compared to FGF2 for both metabolic activity and migration in human umbilical vein endothelial cells, as well as improved angiogenesis in a coculture model invitro. Efficacy in an invivo wound healing model was assessed in diabetic mice. FGF2-PEG2k-FGF2 increased granulation tissue and blood vessel density in the wound bed compared to FGF2. The results suggest that this rationally designed construct may be useful for improving the fibroblast matrix formation and angiogenesis in chronic wound healing.

Telomerase helps maintain telomeres by processive synthesis of telomere repeat DNA at their 3'-ends, using an integral telomerase RNA (TER) and telomerase reverse transcriptase (TERT). We report the cryo-electron microscopy structure of Tetrahymena telomerase at ~9 angstrom resolution. In addition to seven known holoenzyme proteins, we identify two additional proteins that form a complex (TEB) with single-stranded telomere DNA-binding protein Teb1, paralogous to heterotrimeric replication protein A (RPA). The p75-p45-p19 subcomplex is identified as another RPA-related complex, CST (CTC1-STN1-TEN1). This study reveals the paths of TER in the TERT-TER-p65 catalytic core and single-stranded DNA exit; extensive subunit interactions of the TERT essential N-terminal domain, p50, and TEB; and other subunit identities and structures, including p19 and p45C crystal structures. Our findings provide structural and mechanistic insights into telomerase holoenzyme function.

Drug affinity responsive target stability (DARTS) is a relatively quick and straightforward approach to identify potential protein targets for small molecules. It relies on the protection against proteolysis conferred on the target protein by interaction with a small molecule. The greatest advantage of this method is being able to use the native small molecule without having to immobilize or modify it (e.g., by incorporation of biotin, fluorescent, radioisotope, or photoaffinity labels). Here we describe in detail the protocol for performing unbiased DARTS with complex protein lysates to identify binding targets of small molecules and for using DARTS-Western blotting to test, screen, or validate potential small-molecule targets. Although the ideas have mainly been developed from studying molecules in areas of biology that are currently of interest to us and our collaborators, the general principles should be applicable to the analysis of all molecules in nature.

Methylation of various components of the translational machinery has been shown to globally affect protein synthesis. Little is currently known about the role of lysine methylation on elongation factors. Here we show that in Saccharomyces cerevisiae, the product of the EFM3/YJR129C gene is responsible for the trimethylation of lysine 509 on elongation factor 2. Deletion of EFM3 or of the previously described EFM2 increases sensitivity to antibiotics that target translation and decreases translational fidelity. Furthermore, the amino acid sequences of Efm3 and Efm2, as well as their respective methylation sites on EF2, are conserved in other eukaryotes. These results suggest the importance of lysine methylation modification of EF2 in fine tuning the translational apparatus.

We report a novel approach to a new class of bioengineered, monodispersed, self-assembling vault nanoparticles consisting of a protein shell exterior with a lipophilic core interior designed for drug and probe delivery. Recombinant vaults were engineered to contain a small amphipathic α-helix derived from the nonstructural protein 5A of hepatitis C virus, thereby creating within the vault lumen a lipophilic microenvironment into which lipophilic compounds could be reversibly encapsulated. Multiple types of electron microscopy showed that attachment of this peptide resulted in larger than expected additional mass internalized within the vault lumen attributable to incorporation of host lipid membrane constituents spanning the vault waist (>35 nm). These bioengineered lipophilic vaults reversibly associate with a sample set of therapeutic compounds, including all-trans retinoic acid, amphotericin B, and bryostatin 1, incorporating hundreds to thousands of drug molecules per vault nanoparticle. Bryostatin 1 is of particular therapeutic interest because of its ability to potently induce expression of latent HIV, thus representing a preclinical lead in efforts to eradicate HIV/AIDS. Vaults loaded with bryostatin 1 released free drug, resulting in activation of HIV from provirus latency in vitro and induction of CD69 biomarker expression following intravenous injection into mice. The ability to preferentially and reversibly encapsulate lipophilic compounds into these novel bioengineered vault nanoparticles greatly advances their potential use as drug delivery systems.

Mass spectrometry imaging (MSI) of tissue samples is a promising analytical tool that has quickly become associated with biomedical and pharmacokinetic studies. It eliminates several labor-intensive protocols associated with more classical imaging techniques and provides accurate histological data at a rapid pace. Because mass spectrometry is used as the readout, MSI can be applied to almost any molecule, especially those that are biologically relevant. Many examples of its utility in the study of peptides and proteins have been reported; here we discuss its value in the mass range of small molecules. We explore its success and potential in the analysis of lipids, medicinals, and metal-based compounds by featuring representative studies from MSI laboratories around the globe.

Single-seeded fruit of the sacred lotus Nelumbo nucifera Gaertn var. China Antique from NE China have viability as long as ~1300 years determined by direct radiocarbon-dating, having a germination rate of 84%. The pericarp, a fruit tissue that encloses the single seeds of Nelumbo, is considered one of the major factors that contribute to fruit longevity. Proteins that are heat stable and have protective function may be equally important to seed viability. We show proteins of Nelumbo fruit that are able to withstand heating, 31% of which remained soluble in the 110°C-treated embryo-axis of a 549-yr-old fruit and 76% retained fluidity in its cotyledons. Genome of Nelumbo is published. The amino-acid sequences of 11 "thermal proteins" (soluble at 100°C) of modern Nelumbo embryo-axes and cotyledons, identified by mass spectrometry, Western blot and bioassay, are assembled and aligned with those of an archaeal-hyperthermophile Methancaldococcus jannaschii (Mj; an anaerobic methanogen having a growth optimum of 85°C) and with five mesophile angiosperms. These thermal proteins have roles in protection and repair under stress. More than half of the Nelumbo thermal proteins (55%) are present in the archaean Mj, indicating their long-term durability and history. One Nelumbo protein-repair enzyme exhibits activity at 100°C, having a higher heat-tolerance than that of Arabidopsis. A list of 30 sequenced but unassembled thermal proteins of Nelumbo is supplemented.

Understanding the structural and assembly dynamics of the amyloid beta-protein (Abeta) has direct relevance to the development of therapeutic agents for Alzheimer disease. To elucidate these dynamics, we combined scanning amino acid substitution with a method for quantitative determination of the Abeta oligomer frequency distribution, photo-induced cross-linking of unmodified proteins (PICUP), to perform "scanning PICUP." Tyr, a reactive group in PICUP, was substituted at position 1, 10, 20, 30, or 40 (for Abeta40) or 42 (for Abeta42). The effects of these substitutions were probed using circular dichroism spectroscopy, thioflavin T binding, electron microscopy, PICUP, and mass spectrometry. All peptides displayed a random coil --> alpha/beta --> beta transition, but substitution-dependent alterations in assembly kinetics and conformer complexity were observed. Tyr(1)-substituted homologues of Abeta40 and Abeta42 assembled the slowest and yielded unusual patterns of oligomer bands in gel electrophoresis experiments, suggesting oligomer compaction had occurred. Consistent with this suggestion was the observation of relatively narrow [Tyr(1)]Abeta40 fibrils. Substitution of Abeta40 at the C terminus decreased the population conformational complexity and substantially extended the highest order of oligomers observed. This latter effect was observed in both Abeta40 and Abeta42 as the Tyr substitution position number increased. The ability of a single substitution (Tyr(1)) to alter Abeta assembly kinetics and the oligomer frequency distribution suggests that the N terminus is not a benign peptide segment, but rather that Abeta conformational dynamics and assembly are affected significantly by the competition between the N and C termini to form a stable complex with the central hydrophobic cluster.

Consortium for Top Down Proteomics members: Deborah Penque (INSA, IP) Support of the US National Institutes of Health (1R01GM067193 to N.L.K., 1P50HG004952 to L.M.S.) and a grant to N.L.K. from the Chicago Biomedical Consortium, which is supported by the Searle Funds at The Chicago Community Trust.