Biology subjects

Zunke, F.

Publications and source records attributed to Zunke, F..

6 recordsLinked to original sources

bioRxiv2026

Aberrant FICD-mediated AMPylation drives α-Synuclein pathology and overall protein dyshomeostasis in dopaminergic neurons in Parkinson's disease

BackgroundFilamentation induced by cAMP domain-containing protein (FICD) is an endoplasmic reticulum (ER)-resident adenylyltransferase that catalyzes protein AMPylation, a post-translational modification. Although FICD-mediated AMPylation has been linked to the fine-tuning of proteostasis and neuronal integrity, its role in neurodegenerative diseases characterized by protein dyshomeostasis remains unclear. Parkinsons disease (PD) is defined by dopaminergic neurodegeneration and aggregation of -synuclein (aSyn) as a consequence of impaired protein homeostasis. We therefore investigated whether dysregulated FICD-mediated AMPylation contributes to PD pathogenesis. MethodsWe combined analyses of human post-mortem PD brain tissue with complementary models, including midbrain dopaminergic neurons derived from human induced pluripotent stem cells (hiPSCs) of a PD patient carrying an SNCA gene duplication and its isogenic gene dosage-corrected control line, transgenic mouse models of synucleinopathy, and an aSyn-overexpressing H4 neuroglioma cell model. Genetic and pharmacological modulation of FICD activity was integrated with multi-proteomic approaches, including chemical proteomics-based AMPylation profiling, stable isotope labelling with amino acids in cell culture-based global protein turnover analysis, and whole-proteome profiling to identify AMPylation-associated molecular pathways. ResultsFICD was preferentially expressed in dopaminergic neurons and was upregulated in SNCA duplication PD patient-derived neurons, as well as in the basal ganglia of PD post-mortem brains and synucleinopathy mice. Despite this overall increase, the proportion of FICD-expressing dopaminergic neurons was reduced under PD conditions, suggesting selective vulnerability of dopaminergic neurons to FICD. Mechanistically, FICD selectively AMPylated lysosomal proteins, thereby linking AMPylation to the regulation of degradative pathways. Moreover, hyperactivation of FICD-induced AMPylation triggered ER stress, impaired lysosomal function, reduced protein turnover, and ultimately promoted aSyn aggregation and apoptotic cell death. Importantly, pharmacological inhibition of AMPylation reversed aSyn pathology and neurite degeneration in PD patient-derived neurons. ConclusionsWe identify the pathological relevance of FICD-mediated AMPylation in PD-related neurodegeneration and its contribution to aSyn aggregation through a bidirectional interplay with aSyn pathology. Our findings support FICD-mediated AMPylation as a defining molecular switch regulating intracellular protein homeostasis in PD and highlight the FICD-AMPylation pathway as a potential therapeutic target for restoring aSyn pathology and mitigating disease progression.

neuroscience↗

bioRxiv2025

TNF-α disrupts the malate-aspartate shuttle, driving metabolic rewiring in iPSC-derived enteric neural lineages from Parkinson's Disease patients

Gastrointestinal (GI) dysfunction emerges years before motor symptoms in Parkinsons disease (PD), implicating the enteric nervous system (ENS) in early disease progression. However, the mechanisms linking the PD hallmark protein, -synuclein (-syn), to ENS dysfunction - and whether these mechanisms are influenced by inflammation - remains elusive. Using iPSC-derived enteric neural lineages from patients with -syn triplications, we reveal that TNF- increases mitochondrial--syn interactions, disrupts the malate-aspartate shuttle, and forces a metabolic shift toward glutamine oxidation. These alterations drive mitochondrial dysfunction, characterizing metabolic impairment under cytokine stress. Interestingly, targeting glutamate metabolism with Chicago Sky Blue 6B restores mitochondrial function, reversing TNF--driven metabolic disruption. Our findings position the ENS as a central player in PD pathogenesis, establishing a direct link between cytokines, -syn accumulation, metabolic stress and mitochondrial dysfunction. By uncovering a previously unrecognized metabolic vulnerability in the ENS, we highlight its potential as a therapeutic target for early PD intervention.

neuroscience↗

bioRxiv2024

Tagless LysoIP method for molecular profiling of lysosomal content in clinical samples

Lysosomes are implicated in a wide spectrum of human diseases including monogenic lysosomal storage disorders (LSDs), age-associated neurodegeneration and cancer. Profiling lysosomal content using tag-based lysosomal immunoprecipitation (LysoTagIP) in cell and animal models allowed major discoveries in the field, however studying lysosomal dysfunction in human patients remains challenging. Here, we report the development of the "tagless LysoIP method" to enable rapid enrichment of lysosomes, via immunoprecipitation, using the endogenous integral lysosomal membrane protein TMEM192, directly from clinical samples and human cell lines (e.g. induced Pluripotent Stem Cell (iPSCs) derived neurons). Isolated lysosomes are intact and suitable for subsequent multimodal omics analyses. To validate our approach, we employed the tagless LysoIP to enrich lysosomes from peripheral blood mononuclear cells (PBMCs) derived from fresh blood from patients with CLN3 disease, a neurodegenerative LSD. Metabolic profiling of isolated lysosomes showed massive accumulation of glycerophosphodiesters (GPDs) in patients lysosomes. Interestingly, a patient with a milder phenotype and genotype displayed lower accumulation of lysosomal GPDs, consistent with their potential role as disease biomarkers. Altogether, the tagless LysoIP provides a framework to study native lysosomes from patient samples, identify novel biomarkers and discover human-relevant disease mechanisms.

cell biology↗

bioRxiv2023

Linear ubiquitination at damaged lysosomes induces local NF-κB activation and controls cell survival

Lysosomes are the major cellular organelles responsible for nutrient recycling and degradation of cellular material. Maintenance of lysosomal integrity is essential for cellular homeostasis and lysosomal membrane permeabilization (LMP), induced by lysosomotrophic agents, sensitizes towards cell death. Damaged lysosomes are repaired or degraded via lysophagy, during which glycans, exposed on ruptured lysosomal membranes, are recognized by galectins leading to K48- and K63-linked poly-ubiquitination (poly-Ub) of lysosomal proteins followed by recruitment of the autophagic machinery and degradation. Linear (M1) poly-Ub, catalyzed by the E3 ligase linear ubiquitin chain assembly complex (LUBAC) and removed by the OTU domain-containing deubiquitinase with linear linkage specificity (OTULIN) exerts important functions in immune signaling and cell survival, but the role of M1 poly-Ub in lysosomal homeostasis remains largely unexplored. Here, we demonstrate that damaged lysosomes are decorated with M1 poly-Ub in a LUBAC-, OTULIN- and K63-dependent manner. LMP-induced M1 poly-Ub at damaged lysosomes contributes to lysosome degradation, recruits nuclear factor {kappa}-B (NF-{kappa}B) essential modulator (NEMO) and locally activates inhibitor of NF-B kinase (IKK) to trigger NF-{kappa}B activation in a K63 poly-Ub-dependent manner. Inhibition of lysosomal degradation enhances LMP- and OTULIN-dependent cell death, indicating pro-survival functions of LMP and potentially lysophagy. Finally, we demonstrate that M1 poly-Ub occurs at L-leucyl-leucine methyl ester (LLOMe)-damaged lysosomes in primary mouse neurons and induced pluripotent stem cell (iPSC)-derived primary human dopaminergic neurons. Together, our results reveal novel functions of M1 poly-Ub during lysosomal homeostasis, LMP and degradation of damaged lysosomes, with important implications for NF-{kappa}B signaling, inflammation and cell death.

cell biology↗

bioRxiv2021

Decomposition of the SARS-CoV-2-ACE2 interface reveals a common trend among emerging viral variants

New viral variants of the SARS-CoV-2 virus show enhanced infectivity compared to wild type, resulting in an altered pandemic situation in affected areas. These variants are the B.1.1.7 (United Kingdom), B.1.1.7 with the additional E484K mutation, the B.1.351 variant (South Africa) and the P.1 variant (Brazil). Understanding the binding modalities between these viral variants and the host cell receptor ACE2 allows depicting changes, but also common motifs of virus-host cell interaction. The trimeric spike protein expressed at the viral surface contains the receptor-binding domain (RBD) that forms the molecular interface with ACE2. All the above-mentioned variants carry between one and three amino acid exchanges within the interface-forming region of the RBD, thereby altering the binding interface with ACE2. Using molecular dynamics simulations and decomposition of the interaction energies between the RBD and ACE2, we identified phenylalanine 486, glutamine 498, threonine 500 and tyrosine 505 as important interface-forming residues across viral variants. We also suggest a reduced binding energy between RBD and ACE2 in viral variants with higher infectivity, attributed to residue-specific differences in electrostatic interaction energy. Importantly, individual amino acid exchanges not only influence the affected position, but also alter the conformation of surrounding residues and affect their interaction potential as well. We demonstrate how computational methods can help to identify changed as well as common motifs across viral variants. These identified motifs might play a crucial role, in the strategical development of therapeutic interventions against the fast mutating SARS-CoV-2 virus. Significance StatementThe COVID-19 pandemic caused by the SARS-CoV-2 virus has significantly changed our lives. To date, there is a lack of neutralizing drugs that specifically target SARS-CoV-2. Hope lies in newly developed vaccines that effectively prevent severe cases of acute respiratory syndrome. However, emerging viral variants escape vaccine-induced immune-protection. Therefore, identification of appropriate molecular targets across viral variants is important for the development of second- and third-generation vaccines and inhibitory antibodies. In this study, we identify residues across viral variants that are important for viral binding to the host cell. As such residues cannot be replaced without diminishing infectivity of the virus, these residues represent primary targets for intervention, for example by neutralizing antibodies.

bioinformatics↗

bioRxiv2021

Mutations in the B.1.1.7 SARS-CoV-2 spike protein reduce receptor-binding affinity and induce a flexible link to the fusion peptide

The B.1.1.7 variant of the SARS-CoV-2 virus shows enhanced infectiousness over the wild type virus, leading to increasing patient numbers in affected areas. A number of single amino acid exchanges and deletions within the trimeric viral spike protein characterize this new SARS-CoV-2 variant. Crucial for viral entry into the host cell is the interaction of the spike protein with the cell surface receptor angiotensin-converting enzyme 2 (ACE2) as well as integration of the viral fusion peptide into the host membrane. Respective amino acid exchanges within the SARS-CoV-2 variant B.1.1.7 affect inter-monomeric contact sites within the spike protein (A570D and D614G) as well as the ACE2-receptor interface region (N501Y), which comprises the receptor-binding domain (RBD) of the viral spike protein. However, the molecular consequences of mutations within B.1.1.7 on spike protein dynamics and stability, the fusion peptide, and ACE2 binding are largely unknown. Here, molecular dynamics simulations comparing SARS-CoV-2 wild type with the B.1.1.7 variant revealed inter-trimeric contact rearrangements, altering the structural flexibility within the spike protein trimer. In addition to reduced flexibility in the N-terminal domain of the spike protein, we found increased flexibility in direct spatial proximity of the fusion peptide. This increase in flexibility is due to salt bridge rearrangements induced by the D614G mutation in B.1.1.7 found in pre- and post-cleavage state at the S2 site. Our results also imply a reduced binding affinity for B.1.1.7 with ACE2, as the N501Y mutation restructures the RBD-ACE2 interface, significantly decreasing the linear interaction energy between the RBD and ACE2. Our results demonstrate how mutations found within B.1.1.7 enlarge the flexibility around the fusion peptide and change the RBD-ACE2 interface, which, in combination, might explain the higher infectivity of B.1.1.7. We anticipate our findings to be starting points for in depth biochemical and cell biological analyses of B.1.1.7, but also other highly contagious SARS-CoV-2 variants, as many of them likewise exhibit a combination of the D614G and N501Y mutation.

biophysics↗