TSWIFT, a novel method for iterative staining of embedded and mounted human brain sections.

Comprehensive characterization of protein networks in mounted brain tissue represents a major challenge in brain and neurodegenerative disease research. In this study, we develop a simple staining method, called TSWIFT, to iteratively stain pre-mounted formalin fixed, paraffin embedded (FFPE) brain sections, thus enabling high-dimensional sample phenotyping. We show that TSWIFT conserves tissue architecture and allows for relabeling a single mounted FFPE sample more than 10 times, even after prolonged storage at 4 °C. Our results establish TSWIFT as an efficient method to obtain integrated high-dimensional knowledge of cellular proteomes by analyzing mounted FFPE human brain tissue.

TSWIFT, a novel method for iterative staining of embedded and mounted human brain sections.

Comprehensive characterization of protein networks in mounted brain tissue represents a major challenge in brain and neurodegenerative disease research. In this study, we develop a simple staining method, called TSWIFT, to iteratively stain pre-mounted formalin fixed, paraffin embedded (FFPE) brain sections, thus enabling high-dimensional sample phenotyping. We show that TSWIFT conserves tissue architecture and allows for relabeling a single mounted FFPE sample more than 10 times, even after prolonged storage at 4 °C. Using TSWIFT, we profile the abundance and localization of the HSP70 family chaperones HSC70 (HSPA8) and BiP (HSPA5) in mounted human brain tissue. Our results establish TSWIFT as an efficient method to obtain integrated high-dimensional knowledge of cellular proteomes by analyzing mounted FFPE human brain tissue.

A meta-analysis of RNA-Seq studies to identify novel genes that regulate aging.

Aging is a ubiquitous biological process that limits the maximal lifespan of most organisms. Significant efforts by many groups have identified mechanisms that, when triggered by natural or artificial stimuli, are sufficient to either enhance or decrease maximal lifespan. Previous aging studies using the nematode Caenorhabditis elegans (C. elegans) generated a wealth of publicly available transcriptomics datasets linking changes in gene expression to lifespan regulation. However, a comprehensive comparison of these datasets across studies in the context of aging biology is missing. Here, we carry out a systematic meta-analysis of over 1200 bulk RNA sequencing (RNASeq) samples obtained from 74 peer-reviewed publications on aging-related transcriptomic changes in C. elegans. Using both differential expression analyses and machine learning approaches, we mine the pooled data for novel pro-longevity genes. We find that both approaches identify known and propose novel pro-longevity genes. Further, we find that inter-lab experimental variance complicates the application of machine learning algorithms, a limitation that was not solved using bulk RNA-Seq batch correction and normalization techniques. Taken as a whole, our results indicate that machine learning approaches may hold promise for the identification of genes that regulate aging but will require more sophisticated batch correction strategies or standardized input data to reliably identify novel pro-longevity genes.

The AMPylase FIC-1 modulates TGF-ß signaling in Caenorhabditis elegans.

Post-translational protein modifications are essential for the spatio-temporal regulation of protein function. In this study, we examine how the activity of the Caenorhabditis elegans AMPylase FIC-1 modulates physiological processes in vivo. We find that over-expression (OE) of the constitutive AMPylase FIC-1(E274G) impairs C. elegans development, fertility, and stress resilience. We also show that FIC-1(E274G) OE inhibits pathogen avoidance behavior by selectively suppressing production of the Transforming Growth Factor-ß (TGF-ß) ligands DAF-7 and DBL-1 in ASI sensory neurons. Finally, we demonstrate that FIC-1 contributes to the regulation of adult body growth, cholinergic neuron function, and larval entry into dauer stage; all processes controlled by TGF-ß signaling. Together, our results suggest a role for FIC-1 in regulating TGF-ß signaling in C. elegans.

Explaining inter-lab variance in C. elegans N2 lifespan: Making a case for standardized reporting to enhance reproducibility.

The nematode Caenorhabditis elegans is a powerful model organism for studying cell development, apoptosis, neuronal circuits, and aging. The isolate N2 is recognized by the C. elegans community as the reference wild-type strain. Interestingly, the lifespan of presumably isogenic C. elegans N2 worms-even when grown under comparable conditions-varies significantly amongst distinct laboratories. This hinders the inter-laboratory comparability of C. elegans lifespan data and raises questions regarding data interpretation and reproducibility. Here, we hypothesized slight alterations in experimental design and worm handling could explain the observed discrepancies. To test this hypothesis, we collected and assessed data from over 1000 published C. elegans N2 lifespan assays as well as corresponding methodological meta-data. We find that mean N2 lifespans range from approximately 7 days to upwards of 35 days, despite laboratories disclosing seemingly comparable experimental conditions. We further demonstrate that, in addition to temperature, the use of the chemical sterilizer 5-fluoro-2'-deoxyuridine (FUDR) may change N2 lifespan. Additionally, we observed differences in average N2 lifespan from experiments originating from distinct geographic locations, indicating a potential effect of location-specific factors on experimental outcomes. Taken as a whole, our work indicates the sum of many small, rather than a few critical, differences in experimental conditions may account for the observed variance in N2 lifespan. We also find that the absence of standardized experimental methods and the insufficient disclosure of experiment details in the peer-reviewed literature limits the inter-lab comparability of published results. We thus propose the establishment of a succinct reporting standard for C. elegans lifespan experiments to increase the reliability and reproducibility, and thus scientific value, of these studies.

Deletion of mFICD AMPylase alters cytokine secretion and affects visual short-term learning in vivo.

Fic domain-containing AMP transferases (fic AMPylases) are conserved enzymes that catalyze the covalent transfer of AMP to proteins. This posttranslational modification regulates the function of several proteins, including the ER-resident chaperone Grp78/BiP. Here we introduce a mouse FICD (mFICD) AMPylase knockout mouse model to study fic AMPylase function in vertebrates. We find that mFICD deficiency is well tolerated in unstressed mice. We also show that mFICD-deficient mouse embryonic fibroblasts are depleted of AMPylated proteins. mFICD deletion alters protein synthesis and secretion in splenocytes, including that of IgM, an antibody secreted early during infections, and the proinflammatory cytokine IL-1ß, without affecting the unfolded protein response. Finally, we demonstrate that visual nonspatial short-term learning is stronger in old mFICD-/- mice than in wild-type controls while other measures of cognition, memory, and learning are unaffected. Together, our results suggest a role for mFICD in adaptive immunity and neuronal plasticity in vivo.

Over-expression of the constitutive AMPylase FIC-1(E274G) does not deplete cellular ATP pools in C. elegans.

Protein AMPylation has emerged as a posttranslational protein modification regulating cellular proteostasis. AMPylation is conferred by Fic AMPylases, which catalyze the covalent attachment of AMP to target proteins at the expense of ATP. Over-expression of constitutive-active Fic AMPylases is toxic. Here, we test the hypothesis that excessive Fic AMPylase activity could deplete cellular ATP pools, leading to cell death. We find that increased/decreased Fic AMPylase activity only alters cellular ATP concentrations by approximately 15%. This suggests that hyper-AMPylation-mediated cell death is likely not the consequence of cellular ATP depletion.

Fic and non-Fic AMPylases: protein AMPylation in metazoans.

Protein AMPylation refers to the covalent attachment of an AMP moiety to the amino acid side chains of target proteins using ATP as nucleotide donor. This process is catalysed by dedicated AMP transferases, called AMPylases. Since this initial discovery, several research groups have identified AMPylation as a critical post-translational modification relevant to normal and pathological cell signalling in both bacteria and metazoans. Bacterial AMPylases are abundant enzymes that either regulate the function of endogenous bacterial proteins or are translocated into host cells to hijack host cell signalling processes. By contrast, only two classes of metazoan AMPylases have been identified so far: enzymes containing a conserved filamentation induced by cAMP (Fic) domain (Fic AMPylases), which primarily modify the ER-resident chaperone BiP, and SelO, a mitochondrial AMPylase involved in redox signalling. In this review, we compare and contrast bacterial and metazoan Fic and non-Fic AMPylases, and summarize recent technological and conceptual developments in the emerging field of AMPylation.

Post-translational modifications of Hsp70 family proteins: Expanding the chaperone code.

Cells must be able to cope with the challenge of folding newly synthesized proteins and refolding those that have become misfolded in the context of a crowded cytosol. One such coping mechanism that has appeared during evolution is the expression of well-conserved molecular chaperones, such as those that are part of the heat shock protein 70 (Hsp70) family of proteins that bind and fold a large proportion of the proteome. Although Hsp70 family chaperones have been extensively examined for the last 50 years, most studies have focused on regulation of Hsp70 activities by altered transcription, co-chaperone "helper" proteins, and ATP binding and hydrolysis. The rise of modern proteomics has uncovered a vast array of post-translational modifications (PTMs) on Hsp70 family proteins that include phosphorylation, acetylation, ubiquitination, AMPylation, and ADP-ribosylation. Similarly to the pattern of histone modifications, the histone code, this complex pattern of chaperone PTMs is now known as the "chaperone code." In this review, we discuss the history of the Hsp70 chaperone code, its currently understood regulation and functions, and thoughts on what the future of research into the chaperone code may entail.

Caenorhabditis elegans as a model system for studying aging-associated neurodegenerative diseases.

Neurodegenerative diseases (NDs) are a heterogeneous group of aging-associated disorders characterized by the disruption of cellular proteostasis machinery and the misfolding of distinct protein species to form toxic aggregates in neurons. The increasing prevalence of NDs represents a growing healthcare burden worldwide, a concern compounded by the fact that few, if any, treatments exist to target the underlying cause of these diseases. Consequently, the application of a high-throughput, physiologically relevant model system to studies dissecting the molecular mechanisms governing ND pathology is crucial for identifying novel avenues for the development of targeted therapeutics. The nematode Caenorhabditis elegans (C. elegans) has emerged as a powerful tool for the study of disease mechanisms due to its ease of genetic manipulation and swift cultivation, while providing a whole-animal system amendable to numerous molecular and biochemical techniques. To date, numerous C. elegans models have been generated for a variety of NDs, allowing for the large-scale in vivo study of protein-conformation disorders. Furthermore, the comparatively low barriers to entry in the development of transgenic worm models have facilitated the modeling of rare or "orphan" NDs, thereby providing unparalleled insight into the shared mechanisms underlying these pathologies. In this review, we summarize findings from a comprehensive collection of C. elegans neurodegenerative disease models of varying prevalence to emphasize shared mechanisms of proteotoxicity, and highlight the utility of these models in elucidating the molecular basis of ND pathologies.

Protection of tissue physicochemical properties using polyfunctional crosslinkers.

Understanding complex biological systems requires the system-wide characterization of both molecular and cellular features. Existing methods for spatial mapping of biomolecules in intact tissues suffer from information loss caused by degradation and tissue damage. We report a tissue transformation strategy named stabilization under harsh conditions via intramolecular epoxide linkages to prevent degradation (SHIELD), which uses a flexible polyepoxide to form controlled intra- and intermolecular cross-link with biomolecules. SHIELD preserves protein fluorescence and antigenicity, transcripts and tissue architecture under a wide range of harsh conditions. We applied SHIELD to interrogate system-level wiring, synaptic architecture, and molecular features of virally labeled neurons and their targets in mouse at single-cell resolution. We also demonstrated rapid three-dimensional phenotyping of core needle biopsies and human brain cells. SHIELD enables rapid, multiscale, integrated molecular phenotyping of both animal and clinical tissues.

Chaperone AMPylation modulates aggregation and toxicity of neurodegenerative disease-associated polypeptides.

Proteostasis is critical to maintain organismal viability, a process counteracted by aging-dependent protein aggregation. Chaperones of the heat shock protein (HSP) family help control proteostasis by reducing the burden of unfolded proteins. They also oversee the formation of protein aggregates. Here, we explore how AMPylation, a posttranslational protein modification that has emerged as a powerful modulator of HSP70 activity, influences the dynamics of protein aggregation. We find that adjustments of cellular AMPylation levels in Caenorhabditis elegans directly affect aggregation properties and associated toxicity of amyloid-ß (Aß), of a polyglutamine (polyQ)-extended polypeptide, and of α-synuclein (α-syn). Expression of a constitutively active C. elegans AMPylase FIC-1(E274G) under its own promoter expedites aggregation of Aß and α-syn, and drastically reduces their toxicity. A deficiency in AMPylation decreases the cellular tolerance for aggregation-prone polyQ proteins and alters their aggregation behavior. Overexpression of FIC-1(E274G) interferes with cell survival and larval development, underscoring the need for tight control of AMPylase activity in vivo. We thus define a link between HSP70 AMPylation and the dynamics of protein aggregation in neurodegenerative disease models. Our results are consistent with a cytoprotective, rather than a cytotoxic, role for such protein aggregates.

In vitro AMPylation Assays Using Purified, Recombinant Proteins.

Post-translational protein modifications (PTMs) orchestrate the activity of individual proteins and ensure their proper function. While modifications such as phosphorylation or glycosylation are well understood, more unusual modifications, including nitrosylation or AMPylation remain comparatively poorly characterized. Research on protein AMPylation-which refers to the covalent addition of an AMP moiety to the side chains of serine, threonine or tyrosine-has undergone a renaissance (Yarbrough et al., 2009; Engel et al., 2012; Ham et al., 2014; Woolery et al., 2014; Preissler et al., 2015; Sanyal et al., 2015; Truttmann et al., 2016; Truttmann et al., 2017). The identification and characterization of filamentation (fic) domain-containing AMPylases sparked new interest in this PTM (Kinch et al., 2009; Yarbrough et al., 2009). Based on recent in vivo and in vitro studies, we now know that secreted bacterial AMPylases covalently attach AMP to members of the Rho family of GTPases, while metazoan AMPylases modify HSP70 family proteins in the cytoplasm and the endoplasmic reticulum (ER) (Itzen et al., 2011; Hedberg and Itzen, 2015; Truttmann and Ploegh, 2017). AMPylation is thought to trap HSP70 in a primed yet transiently disabled state that cannot participate in protein refolding reactions (Preissler et al., 2015). In vitro AMPylation experiments are key to assess the activity, kinetics and specificity of protein AMPylation catalyzed by pro- and eukaryotic enzymes. These simple assays require recombinant AMPylases, target proteins (Rho GTPases, HSP70s), as well as ATP as a nucleotide source. Here, we describe strategies to qualitatively and quantitatively study protein AMPylation in vitro.

Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation.

Strategies for site-specific protein modification are highly desirable for the construction of conjugates containing non-genetically-encoded functional groups. Ideally, these strategies should proceed under mild conditions, and be compatible with a wide range of protein targets and non-natural moieties. The transpeptidation reaction catalyzed by bacterial sortases is a prominent strategy for protein derivatization that possesses these features. Naturally occurring or engineered variants of sortase A from Staphylococcus aureus catalyze a ligation reaction between a five-amino-acid substrate motif (LPXTG) and oligoglycine nucleophiles. By pairing proteins and synthetic peptides that possess these ligation handles, it is possible to install modifications onto the protein N- or C-terminus in site-specific fashion. As described in this unit, the successful implementation of sortase-mediated labeling involves straightforward solid-phase synthesis and molecular biology techniques, and this method is compatible with proteins in solution or on the surface of live cells. © 2017 by John Wiley & Sons, Inc.

rAMPing Up Stress Signaling: Protein AMPylation in Metazoans.

Protein AMPylation - the covalent attachment of an AMP residue to amino acid side chains using ATP as the donor - is a post-translational modification (PTM) increasingly appreciated as relevant for both normal and pathological cell signaling. In metazoans single copies of filamentation induced by cAMP (fic)-domain-containing AMPylases - the enzymes responsible for AMPylation - preferentially modify a set of dedicated targets and contribute to the perception of cellular stress and its regulation. Pathogenic bacteria can exploit AMPylation of eukaryotic target proteins to rewire host cell signaling machinery in support of their propagation and survival. We review endogenous as well as parasitic protein AMPylation in metazoans and summarize current views of how fic-domain-containing AMPylases contribute to cellular proteostasis.

Hepta-Mutant Staphylococcus aureus Sortase A (SrtA7m) as a Tool for in Vivo Protein Labeling in Caenorhabditis elegans.

In vivo protein ligation is of emerging interest as a means of endowing proteins with new properties in a controlled fashion. Tools to site-specifically and covalently modify proteins with small molecules, peptides, or other proteins in living cells are few and far between. Here, we describe the development of a Staphylococcus aureus sortase (SrtA)-based protein ligation approach for site-specific conjugation of fluorescent dyes and ubiquitin (Ub) to modify proteins in Caenorhabditis elegans. Hepta-mutant SrtA (SrtA7m) expressed in C. elegans is functional and supports in vitro sortase reactions in a low-Ca2+ environment. Feeding SrtA7m-expressing C. elegans with small peptide-based probes such as (Gly)3- biotin or (Gly)3-fluorophores enables in vivo target protein modification. SrtA7m also catalyzes the circularization of suitably modified linear target proteins in vivo and allows the installation of F-box domains on targets to induce their degradation in a ubiquitin-dependent manner. This is a noninvasive method to achieve in vivo protein labeling, protein circularization, and targeted degradation in C. elegans. This technique should improve our ability to monitor and alter the function of intracellular proteins in vivo.

Unrestrained AMPylation targets cytosolic chaperones and activates the heat shock response.

Protein AMPylation is a conserved posttranslational modification with emerging roles in endoplasmic reticulum homeostasis. However, the range of substrates and cell biological consequences of AMPylation remain poorly defined. We expressed human and Caenorhabditis elegans AMPylation enzymes-huntingtin yeast-interacting protein E (HYPE) and filamentation-induced by cyclic AMP (FIC)-1, respectively-in Saccharomyces cerevisiae, a eukaryote that lacks endogenous protein AMPylation. Expression of HYPE and FIC-1 in yeast induced a strong cytoplasmic Hsf1-mediated heat shock response, accompanied by attenuation of protein translation, massive protein aggregation, growth arrest, and lethality. Overexpression of Ssa2, a cytosolic heat shock protein (Hsp)70, was sufficient to partially rescue growth. In human cell lines, overexpression of active HYPE similarly induced protein aggregation and the HSF1-dependent heat shock response. Excessive AMPylation also abolished HSP70-dependent influenza virus replication. Our findings suggest a mode of Hsp70 inactivation by AMPylation and point toward a role for protein AMPylation in the regulation of cellular protein homeostasis beyond the endoplasmic reticulum.

Recent advances in sortase-catalyzed ligation methodology.

The transpeptidation reaction catalyzed by bacterial sortases continues to see increasing use in the construction of novel protein derivatives. In addition to growth in the number of applications that rely on sortase, this field has also seen methodology improvements that enhance reaction performance and scope. In this opinion, we present an overview of key developments in the practice and implementation of sortase-based strategies, including applications relevant to structural biology. Topics include the use of engineered sortases to increase reaction rates, the use of redesigned acyl donors and acceptors to mitigate reaction reversibility, and strategies for expanding the range of substrates that are compatible with a sortase-based approach.

The Caenorhabditis elegans Protein FIC-1 Is an AMPylase That Covalently Modifies Heat-Shock 70 Family Proteins, Translation Elongation Factors and Histones.

Protein AMPylation by Fic domain-containing proteins (Fic proteins) is an ancient and conserved post-translational modification of mostly unexplored significance. Here we characterize the Caenorhabditis elegans Fic protein FIC-1 in vitro and in vivo. FIC-1 is an AMPylase that localizes to the nuclear surface and modifies core histones H2 and H3 as well as heat shock protein 70 family members and translation elongation factors. The three-dimensional structure of FIC-1 is similar to that of its human ortholog, HYPE, with 38% sequence identity. We identify a link between FIC-1-mediated AMPylation and susceptibility to the pathogen Pseudomonas aeruginosa, establishing a connection between AMPylation and innate immunity in C. elegans.