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.

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.

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.

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.

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.

HypE-specific nanobodies as tools to modulate HypE-mediated target AMPylation.

The covalent addition of mono-AMP to target proteins (AMPylation) by Fic domain-containing proteins is a poorly understood, yet highly conserved post-translational modification. Here, we describe the generation, evaluation, and application of four HypE-specific nanobodies: three that inhibit HypE-mediated target AMPylation in vitro and one that acts as an activator. All heavy chain-only antibody variable domains bind HypE when expressed as GFP fusions in intact cells. We observed localization of HypE at the nuclear envelope and further identified histones H2-H4, but not H1, as novel in vitro targets of the human Fic protein. Its role in histone modification provides a possible link between AMPylation and regulation of gene expression.

Bartonella henselae trimeric autotransporter adhesin BadA expression interferes with effector translocation by the VirB/D4 type IV secretion system.

The Gram-negative, zoonotic pathogen Bartonella henselae is the aetiological agent of cat scratch disease, bacillary angiomatosis and peliosis hepatis in humans. Two pathogenicity factors of B. henselae - each displaying multiple functions in host cell interaction - have been characterized in greater detail: the trimeric autotransporter Bartonella adhesin A (BadA) and the type IV secretion system VirB/D4 (VirB/D4 T4SS). BadA mediates, e.g. binding to fibronectin (Fn), adherence to endothelial cells (ECs) and secretion of vascular endothelial growth factor (VEGF). VirB/D4 translocates several Bartonella effector proteins (Beps) into the cytoplasm of infected ECs, resulting, e.g. in uptake of bacterial aggregates via the invasome structure, inhibition of apoptosis and activation of a proangiogenic phenotype. Despite this knowledge of the individual activities of BadA or VirB/D4 it is unknown whether these major virulence factors affect each other in their specific activities. In this study, expression and function of BadA and VirB/D4 were analysed in a variety of clinical B. henselae isolates. Data revealed that most isolates have lost expression of either BadA or VirB/D4 during in vitro passages. However, the phenotypic effects of coexpression of both virulence factors was studied in one clinical isolate that was found to stably coexpress BadA and VirB/D4, as well as by ectopic expression of BadA in a strain expressing VirB/D4 but not BadA. BadA, which forms a dense layer on the bacterial surface, negatively affected VirB/D4-dependent Bep translocation and invasome formation by likely preventing close contact between the bacterial cell envelope and the host cell membrane. In contrast, BadA-dependent Fn binding, adhesion to ECs and VEGF secretion were not affected by a functional VirB/D4 T4SS. The obtained data imply that the essential virulence factors BadA and VirB/D4 are likely differentially expressed during different stages of the infection cycle of Bartonella.

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.

FICD deficiency protects mice from hypertrophy-induced heart failure via BiP-mediated activation of the UPR ER and ER-phagy.

Cardiomyocytes require the HSP70 chaperone BiP to maintain proteostasis in the endoplasmic reticulum (ER) following cardiac stress. The adenylyl transferase (AMPylase) FICD is increasingly recognized to regulate BiP activity through the post-translational addition of an adenosine monophosphate moiety to BiP surface residues. However, the physiological impact of FICD-mediated BiP regulation in the context of cardiovascular health is unknown. Here, we find that FICD deficiency prevents pressure overload-associated heart failure, hypertrophy, and fibrosis, and that FICD knockout mice maintain normal cardiac function after cardiac pressure overload. At a cellular level, we observe that FICD-mediated BiP AMPylation blunts the induction of the unfolded protein response (UPR ER ) and impairs BiP interaction with FAM134B, an ER-phagy receptor, thus limiting ER-phagy induction under stress. In contrast, FICD loss significantly increases BiP-dependent UPR ER induction and ER-phagy in stressed cardiomyocytes. We also uncover cell type-specific consequences of FICD activity in response to ER stress, positioning FICD as a critical proteostasis regulator in cardiac tissue. Our results highlight a novel regulatory paradigm controlling stress resilience in cardiomyocytes and offer a rationale to consider FICD as a therapeutic target to treat cardiac hypertrophy.

Bartonella henselae engages inside-out and outside-in signaling by integrin ß1 and talin1 during invasome-mediated bacterial uptake.

The VirB/D4 type IV secretion system (T4SS) of the bacterial pathogen Bartonella henselae (Bhe) translocates seven effector proteins (BepA-BepG) into human cells that subvert host cellular functions. Two redundant pathways dependent on BepG or the combination of BepC and BepF trigger the formation of a bacterial uptake structure termed the invasome. Invasome formation is a multi-step process consisting of bacterial adherence, effector translocation, aggregation of bacteria on the cell surface and engulfment, and eventually, complete internalization of the bacterial aggregate occurs in an F-actin-dependent manner. In the present study, we show that Bhe-triggered invasome formation depends on integrin-ß1-mediated signaling cascades that enable assembly of the F-actin invasome structure. We demonstrate that Bhe interacts with integrin ß1 in a fibronectin- and VirB/D4 T4SS-independent manner and that activated integrin ß1 is essential for both effector translocation and the actin rearrangements leading to invasome formation. Furthermore, we show that talin1, but not talin2, is required for inside-out activation of integrin ß1 during invasome formation. Finally, integrin-ß1-mediated outside-in signaling by FAK, Src, paxillin and vinculin is necessary for invasome formation. This is the first example of a bacterial entry process that fully exploits the bi-directional signaling capacity of integrin receptors in a talin1-specific manner.

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.

Combined action of the type IV secretion effector proteins BepC and BepF promotes invasome formation of Bartonella henselae on endothelial and epithelial cells.

Bartonella henselae (Bhe) can invade human endothelial cells (ECs) by two distinguishable entry routes: either individually by endocytosis or as large bacterial aggregates by invasome-mediated internalization. Only the latter process is dependent on a functional VirB/VirD4 type IV secretion system (T4SS) and the thereby translocated Bep effector proteins. Here, we introduce HeLa cells as a new cell system suitable to study invasome formation. We describe a novel route to trigger invasome formation by the combined action of the effectors BepC and BepF. Co-infections of either HUVEC or HeLa cells with the Bep-deficient ΔbepA-G mutant expressing either BepC or BepF restores invasome formation. Likewise, ectopic expression of a combination of BepC and BepF in HeLa cells enables invasome-mediated uptake of the Bhe ΔbepA-G mutant strain. Further, eGFP-BepC and eGFP-BepF fusion proteins localize to the cell membrane and, upon invasome formation, to the invasome. Furthermore, the combined action of BepC and BepF inhibits endocytic uptake of inert microspheres. Finally, we show that BepC and BepF-triggered invasome formation differs from BepG-triggered invasome formation in its requirement for cofilin1, while the Rac1/Scar1/WAVE/Arp2/3 and Cdc42/WASP/Arp2/3 signalling pathways are required in both cases.

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.

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.

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.

A translocated protein of Bartonella henselae interferes with endocytic uptake of individual bacteria and triggers uptake of large bacterial aggregates via the invasome.

Bartonella henselae enters human endothelial cells (ECs) by two alternative routes: either by endocytosis, giving rise to Bartonella-containing vacuoles or by invasome-mediated internalization. Only the latter process depends on the type IV secretion system VirB/VirD4 and involves the formation of cell surface-associated bacterial aggregates, which get engulfed by EC membranes in an F-actin-dependent manner, eventually resulting in their complete internalization. Here, we report that among the VirB/VirD4-translocated effector proteins BepA-BepG only BepG is required for triggering invasome-mediated internalization. Expression of BepG in the Bep-deficient DeltabepA-G mutant restored invasome-mediated internalization. Likewise, ectopic expression of BepG in ECs also restored invasome-mediated internalization of the DeltabepA-G mutant, while no discernable cytoskeletal rearrangements were triggered in uninfected cells. Rather, BepG inhibited endocytic uptake of B. henselae into Bartonella-containing vacuoles and other endocytic processes, that is, invasin-mediated uptake of Yersinia enterocolitica and uptake of inert microspheres. BepG thus triggers invasome-mediated internalization primarily by inhibiting bacterial endocytosis. Bacteria accumulating on the cell surface then induce locally the F-actin rearrangements characteristic for the invasome. These cytoskeletal changes encompass both the rearrangement of pre-existing F-actin fibres and the de novo polymerization of cortical F-actin in the periphery of the invasome by Rac1/Scar1/WAVE- and Cdc42/WASP-dependent pathways that involve the recruitment of the Arp2/3 complex.

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.