40 research outputs found
The Caenorhabditis elegans Protein FIC-1 Is an AMPylase That Covalently Modifies Heat-Shock 70 Family Proteins, Translation Elongation Factors and Histones
No full textProtein 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.National Institutes of Health (U.S.) (P41 GM103403)Swiss National Science Foundation (Advanced Postdoc Mobility Fellowship)National Institutes of Health (U.S.) (NIH Pioneer Award (DP01)
BID-F1 and BID-F2 Domains of Bartonella henselae Effector Protein BepF Trigger Together with BepC the Formation of Invasome Structures
No full textThe gram-negative, zoonotic pathogen Bartonella henselae (Bhe) translocates seven distinct Bartonella effector proteins (Beps) via the VirB/VirD4 type IV secretion system (T4SS) into human cells, thereby interfering with host cell signaling [1], [2]. In particular, the effector protein BepG alone or the combination of effector proteins BepC and BepF trigger massive F-actin rearrangements that lead to the establishment of invasome structures eventually resulting in the internalization of entire Bhe aggregates [2], [3]. In this report, we investigate the molecular function of the effector protein BepF in the eukaryotic host cell. We show that the N-terminal [E/T]PLYAT tyrosine phosphorylation motifs of BepF get phosphorylated upon translocation but do not contribute to invasome-mediated Bhe uptake. In contrast, we found that two of the three BID domains of BepF are capable to trigger invasome formation together with BepC, while a mutation of the WxxxE motif of the BID-F1 domain inhibited its ability to contribute to the formation of invasome structures. Next, we show that BepF function during invasome formation can be replaced by the over-expression of constitutive-active Rho GTPases Rac1 or Cdc42. Finally we demonstrate that BID-F1 and BID-F2 domains promote the formation of filopodia-like extensions in NIH 3T3 and HeLa cells as well as membrane protrusions in HeLa cells, suggesting a role for BepF in Rac1 and Cdc42 activation during the process of invasome formation.Swiss National Science Foundation (grant 31003A-132979)Howard Hughes Medical Institute (grant 5500550)Swiss Initiative for Systems Biology (grant 51RT-0_126008 (InfectX)
The Role of Flavin-Containing Monooxygenase in Intestinal Barrier Formation
No full textThe maintenance of a healthy intestine is critical for preserving overall health and preventing disease development. The intestinal barrier consists of specialized epithelial cells covered by a protective mucus bilayer. In the colon, this bilayer of mucus functions to prevent bacteria and harmful metabolites from invading the epithelium and increasing permeability. Intestinal barrier permeability is associated with various pathologies, including inflammatory bowel disease. However, the physiological and molecular stimuli driving intestinal barrier dysfunction and its relationship to gastrointestinal disease onset and progression remain poorly understood. This dissertation investigates mechanisms underlying the formation and maintenance of an intestinal barrier capable of resisting systemic and environmental stress.
Here, I demonstrate that an intestinal enzyme responsive to environmental stress in C. elegans contributes to maintaining intestinal barrier integrity in mice. Specifically, I identify the stress resistance and longevity gene, flavin-containing monooxygenase (FMO)-2, as a critical regulator of intestinal barrier integrity in C. elegans. I show that fmo-2 is upregulated following exposure to the barrier-damaging chemical, dextran sodium sulfate. Furthermore, I find that fmo-2 is required for the expression of a key actin protein essential for maintaining microvilli integrity, establishing its role in the structural maintenance of the C. elegans intestine.
FMOs are a highly conserved family of enzymes involved in xenobiotic and endogenous metabolism. To investigate whether mammalian FMOs play a similar role to fmo-2, I generated an intestinal epithelium-specific, conditional knockout model of the mammalian homolog, Fmo5. Extensive characterization of this mouse model revealed that acute loss of FMO5 in mice leads to rapid deterioration of crypt architecture in the colon. I further observed that FMO5 loss disrupts the localization of colonic goblet cells, which synthesize and secrete mucus, coinciding with a depletion of the mucosal barrier in mice. Interestingly, these effects are strongly sex-dependent, with dramatic phenotypes observed in female animals and only moderate changes in males. This finding suggests that FMO5 may interact with sex-dependent differences observed in human gastrointestinal diseases.
To elucidate the mechanism underlying the FMO5-mediated phenotype, I ruled out microbiota dysregulation as a primary cause and instead identified a role for FMO5 in maintaining endoplasmic reticulum (ER) homeostasis. Specifically, I observed a significant increase in mucin-related ER stress in female Fmo5IntKO mice. Supporting this mechanism, treatment of female Fmo5IntKO mice with an ER stress-resolving bile acid fully rescued defects in crypt architecture, goblet cell localization, and mucus barrier thickness. Together, these findings implicate FMO5 as a regulator of the intestinal epithelium and highlight its potential relevance to diseases affecting intestinal barrier integrity.
To conclude this project, I returned to the C. elegans model to investigate the role of fmo-2 in detecting microbial stimuli during exposure to the intestine-targeting pathogen Enterococcus faecalis. I identified neural signaling pathways to the intestine involved in this process and demonstrated that fmo-2 overexpression is sufficient to enhance survival following E. faecalis exposure.
The data presented here provide novel insights into goblet cell regulation and mucus barrier formation and maintenance. This work establishes a foundation for utilizing these models to explore gastrointestinal disease etiology and identify therapeutic strategies targeting FMOs to improve human health.PhDMolecular and Integrative PhysiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/197160/1/meschall_1.pd
Defining the Role of C. elegans fmo-4 in Longevity and Stress Resistance
No full textAging is the leading risk factor for chronic diseases, with nearly 95% of adults over the age of 60 affected with at least one chronic condition. As the global population trends older, understanding the mechanisms underlying age-related decline has become increasingly important for public health. Chronic conditions such as heart disease, cancer, and diabetes not only impact individual quality of life, but also place a significant burden on healthcare resources. Therefore, elucidating the biological processes that drive aging is crucial for developing interventions that promote healthier aging and reduce the prevalence of age-related diseases.
Because of its fundamental role in cellular function and energy production, metabolism has emerged as a major area of interest in aging research. Broadly, my work centers on understanding how metabolic pathways influence aging, with the ultimate goal of uncovering potential treatments to extend lifespan and enhance healthspan. This dual approach contributes significantly both to our fundamental understanding of the mechanisms of aging and to the development of practical therapeutic interventions.
Specifically, my research investigates the role of fmo-4, a gene that promotes longevity, healthspan, and stress resistance in Caenorhabditis elegans. I discovered that fmo-4 functions downstream of multiple nutrient-sensing longevity pathways, including dietary restriction and the inhibition of mTOR signaling, implicating fmo-4 as a major regulator of aging. I also found that fmo-4 is sufficient to extend lifespan when overexpressed either ubiquitously or specifically in the hypodermis. Upon investigation of downstream mechanisms, I established that fmo-4 extends lifespan and promotes resistance to paraquat stress, which increases the formation of free radicals, by interacting with key genes in the endoplasmic reticulum and the mitochondria that regulate calcium signaling between these organelles. These findings highlight the importance of intracellular calcium homeostasis in the aging process as well as the importance of fmo-4 in calcium metabolism.
Building on this foundational work, I next explored how fmo-4 influences mitochondrial physiology. Given that fmo-4 plays a critical role in regulating calcium signaling between the endoplasmic reticulum and mitochondria – a process essential for maintaining mitochondrial health – I hypothesized that fmo-4 expression would significantly affect key aspects of mitochondria metabolism. My findings indicate that fmo-4 modulates mitochondrial metabolism to promote longevity and stress resistance by influencing the tricarboxylic acid (TCA) cycle and its metabolites, including malate and fumarate, as well as regulating mitochondrial dynamics, such as fission and fusion. These results reveal an intricate relationship between cellular organelles and metabolic pathways in lifespan extension.
In addition to studying fmo-4’s impact on metabolism and longevity, my work also explores its translational potential for human health. I found that fmo-4 expression in C. elegans can serve as a valuable readout for identifying pro-longevity compounds, such as deguelin. Importantly, I confirmed that deguelin requires fmo-4 for its longevity- and healthspan-promoting effects. These data demonstrate the potential for using FMOs as biomarkers to screen for therapeutics that can promote longevity and healthspan in humans. By bridging the gap between basic research and applied science, my research aims to accelerate the development of interventions that mitigate age-related decline and improve quality of life for aging populations.
Together, the findings presented in this thesis enhance our understanding of the metabolic mechanisms that regulate aging while supporting the long-term goal of developing therapeutics that promote human health and longevity.PhDCellular & Molecular BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/199441/1/atuckow_1.pd
Regulation of Longevity by Flavin-Containing Monooxygenases
No full textAs the average age of the world’s population skews older, improving human healthspan and lifespan has become a priority. To that end, biogerontology focuses on identifying the genes and pathways that influence aging using model animals, such as mice and the nematode, Caenorhabditis elegans, with the ultimate goal of identifying therapeutic targets to extend lifespan and healthspan. Our previous work established C. elegans flavin-containing monooxygenase-2 (fmo-2) as 1) necessary and sufficient to increase lifespan downstream of dietary restriction and hypoxia-mediated longevity and 2) sufficient to increase resistance to many toxic stresses. fmo-2 encodes for a protein that is most likely an ER transmembrane protein, like mammalian FMOs, and shares ~88% of its catalytic residues with mammalian FMO5 and C. elegans FMO-1 and FMO- 4. Consequently, our central hypothesis is that the regulation of stress resistance and longevity is a conserved property of C. elegans and mammalian FMOs.
To that end, we initially hypothesized that overexpression of mouse FMOs in mammalian cell lines would also convey stress resistance. We find that mouse FMO overexpression conveys broad stress resistance against heavy metal, oxidative, ER, and UV stresses. We further find that FMO overexpression results in a shift from carbohydrate metabolism to mitochondrial metabolism and that FMOs regulate multiple endogenous metabolic pathways, including central carbon metabolism, essential amino acid metabolism, and energy metabolism. These data establish a conserved stress resistance role for FMOs.
Since mammalian FMO overexpression regulates cellular metabolism, we hypothesized that nematode fmo-2 overexpression would regulate endogenous metabolism and that one or more of these metabolic pathways would be required for fmo-2-mediated longevity. We find that fmo-2 interacts with one carbon metabolism to regulate longevity through modulations of the transmethylation pathway. We also find that fmo-2 interacts with tryptophan metabolism and the kynurenine pathway to regulate longevity as well. Last, due to one carbon metabolism and the kynurenine pathway regulating stress and longevity differently, we conclude that fmo-2 likely regulates longevity and stress resistance through separable pathways. Ultimately, we determine that fmo-2 regulates endogenous metabolism to affect stress resistance, like mammalian FMOs, and that these metabolic effects are also required for longevity.
C. elegans have two other fmos that encode for ER transmembrane proteins, fmo-1 and fmo-4. Since these are induced similarly to fmo-2 in multiple longevity pathways (e.g dietary restriction and hypoxia), we hypothesized that these fmos would also be necessary and sufficient to increase longevity downstream of these pathways. Interestingly, we find that both fmo-1 and fmo-4 are required multiple longevity pathways, including sDR, IF, and fmo-2 overexpression-mediated longevity. When overexpressed, fmo-1 and fmo-4 are sufficient to increase longevity, and fmo-4 is sufficient to increase oxidative stress resistance. Nematode fmos regulate multiple overlapping and distinct metabolic pathways with mammalian Fmos, such as glutathione metabolism, cysteine and methionine metabolism, and energy metabolism. We conclude that fmo-1 and fmo-4 function downstream of fmo-2 in regulation longevity and stress resistance, but the exact genetic and metabolic mechanisms of these pathways remain under investigation.
Collectively, these findings enhance our understanding of conserved FMO roles, including stress resistance and regulation of endogenous metabolic pathways. Further, these findings establish FMOs as regulators of C. elegans longevity with future work aiming to determine if this role also extends to mammalian FMOs with the long-term goal of identifying therapeutic targets that increase human healthspan and lifespan.PhDCellular & Molecular BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/174385/1/mhowingt_1.pd
Cell Nonautonomous Regulation of Longevity and Behavior
No full textAging is a complex biological process influenced by environmental, genetic, and physiological factors. Recent progress in aging research indicates that diverse environmental stressors, including changes in nutrient supply, oxygen levels, and temperature, can significantly improve health and extend lifespan across species. The beneficial effects of these environmental stressors are frequently mediated by cell nonautonomous signaling, where sensory cells relay critical information to peripheral tissues to initiate a protective stress response. Intriguingly, activating these neural pathways can enhance health and longevity even when external stressors are not present. Understanding the pathways that regulate aging, particularly those involving cell nonautonomous signaling, is crucial for developing effective treatments that promote healthspan and lifespan. This dissertation explores how sensory perception and neural circuits influence longevity, health, and behavior using Caenorhabditis elegans as a model organism.
This work first examines the interaction between sensory perception and dietary restriction (DR), a well-established longevity intervention. I demonstrate that tactile cues resembling bacterial food disrupt the lifespan extension provided by DR. This effect relies on primary mechanoreceptors and downstream neurotransmitters, including dopamine and tyramine, as well as neuropeptides like insulin and GnRH. These findings reveal a novel sensory pathway linking touch to systemic longevity regulation, emphasizing the role of neural sensory perception in aging. My collaborators and I then investigate the response to low oxygen, or hypoxia, another environmental longevity intervention. Hypoxia-induced longevity requires serotonin signaling through ADF serotonergic neurons and SER-7 receptor activity in RIS interneurons. This pathway also engages additional neuromodulators, such as tyramine, GABA, and NLP-17. By dissecting these molecular mechanisms, we identify potential therapeutic targets for neural aging interventions, such as modulating hypoxic signaling components in specific neuronal subsets to minimize adverse effects.
In addition to signaling from the nervous system to peripheral tissues in longevity pathways, I also explore cell nonautonomous signals originating from peripheral tissues and modifying the nervous system. This retrograde signaling is explored in the context of fmo-2, a gene involved in longevity and metabolism. My collaborators and I find that altering fmo-2 expression affects exploratory behavior through modifying tryptophan metabolism, linking broad metabolic changes to altered neural signaling. Normal exploratory behavior can be restored in fmo-2 mutants through modifying serotonin and quinolinic acid synthesis, highlighting the interconnectedness of metabolism, behavior, and longevity, while also illustrating the challenges posed by pleiotropic effects in longevity interventions.
Finally, this dissertation addresses methodological challenges in C. elegans research. By developing a metabolically inactive bacterial food source and automated fluorescent image quantification techniques, I improve our capacity to study neural signaling and metabolic pathways with greater throughput and precision. Collectively, this work explores the intricate interplay between sensory perception, neural signaling, and peripheral longevity pathways. Ultimately, these findings could contribute to the broader goal of identifying conserved mechanisms that promote health and longevity in humans.PhDMolecular and Integrative PhysiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/199097/1/esdean_1.pd
Fic and non-Fic AMPylases: protein AMPylation in metazoans
No full textProtein 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
Chaperone AMPylation modulates aggregation and toxicity of neurodegenerative disease-associated polypeptides
No full textAbstractProteostasis 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 post-translational 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 C.elegans directly affect aggregation properties and associated toxicity of amyloid-β (Aβ), 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 Aβ 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. Over-expression 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 cyto-protective, rather than a cytotoxic role for such protein aggregates.</jats:p
Combined action of the type IV secretion effector proteins BepC and BepF promotes invasome formation of Bartonella henselae on endothelial and epithelial cells
No full textBartonella 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 DeltabepA-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 DeltabepA-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
