Biology subjects

Matic, I.

Publications and source records attributed to Matic, I..

9 recordsLinked to original sources

bioRxiv2025

Phosphoribosyl ubiquitination of SNARE proteins regulate autophagy in Legionella infection

Legionella pneumophila is an intracellular pathogen that causes Legionnaires disease. The bacteria releases effector proteins some of which remodel the host autophagic-lysosomal pathways. One effector is RavZ, which delipidates ATG8 proteins, making Legionella-infected cells deficient in autophagy. Here we show that SidE effectors mediate the phosphoribosyl ubiquitination (PR-Ub) of autophagic SNARE proteins STX17 and SNAP29. STX17 modification induces recruitment of STX17+ membranes from the endoplasmic reticulum to Legionella-containing phagosomes to form replicative vacuoles. Using proximity labeling, biochemistry and Legionella infection studies, we have discovered a mechanism by which autophagy is hijacked by bacteria to recruit ER membranes to the bacterial vacuole which has autophagy markers but do not fuse with lysosomes. Mass spectrometric identification of PR-Ub sites and mutational studies show that PR-Ub modification of STX17 alters its interaction with ATG14L, which drives the recruitment of ER membranes to the bacterial vacuole in a PI3K dependent manner. On the other hand, PR-Ub of SNAP29 inhibits the formation of the autophagosomal SNARE complex (STX17-SNAP29-VAMP8) by steric hindrance, thereby preventing the fusion of bacterial vacuoles with lysosomes.

cell biology↗

bioRxiv2025

Versatile Modular Antibodies for Sensitive and Specific Detection of Poly-ADP-Ribose

ADP-ribosylation is a chemically versatile modification of proteins, RNA and DNA that regulates various important signaling pathways, many of which are implicated in human diseases. Despite being discovered 60 years ago in the form of poly-ADP-ribosylation - the most studied form of this modification - investigating ADP-ribosylation at the molecular level has historically been challenging. By applying serine ADP-ribosylation-based antibody engineering technology, we have developed the first site-specific, as well as sensitive, mono-ADP-ribosylation modular antibodies. Here, we extend the scope of this technology to poly-ADP-ribosylation. By combining serine poly-ADP-ribosylated peptides as antigens, PARP1 serine mono- and poly-ADP-ribosylation for validation, phage display and the SpyTag protein ligation systems, we developed modular antibodies that are highly specific for poly-ADP-ribosylation. SpyTag-based coupling of horseradish peroxidase at specific positions, distant from the antigen-binding region of the antibody, yields a format that simplifies immunoblotting while dramatically enhancing poly-ADP-ribosylation detection sensitivity. Additionally, the creation of synthetic immunoglobulin formats - mouse, rabbit and human - enables straightforward co-detection of mono- and poly-ADP-ribosylation in cells by immunofluorescence. Our new tools improve and simplify the detection of poly-ADP-ribosylation, particularly in immunoblotting, enabling specific investigations of this key cellular signal in the context of other distinct forms of ADP-ribosylation.

biochemistry↗

bioRxiv2024

Amyloid Beta Precursor Protein contributes to brain aging and learning decline in short-lived turquoise killifish (Nothobranchius furzeri)

Brain aging is a key risk factor for many neurodegenerative diseases, yet its molecular and cellular mechanisms remain elusive. Amyloid-beta precursor protein (APP) is among the most studied proteins linked to brain pathology; however, its role in non-pathological brain aging remains poorly characterized. Here, we investigate the natural impact of APP on normal brain aging using the short-lived turquoise killifish (Nothobranchius furzeri), which exhibits rapid and spontaneous age-related decline. We found that a pyroglutamated APP derivative (APPpE11) accumulates intra-neuronally in an age-dependent manner, co-localizing with a marker of cell death. We found that intraneuronal APPpE11 is also present in brains from healthy elderly humans, suggesting deep evolutionary conservation. To determine APPs role in spontaneous brain aging, we knock-out "amyloid precursor protein a" (appa) in killifish via CRISPR/Cas9. The lack of appa mitigated brain aging from a proteome-wide perspective, reduced age-related cell death and inflammation, and improved neuronal activity and learning capacity in aged individuals. Our findings show an ancestral and previously unrecognized role of amyloid-beta precursor protein in non-pathological brain aging, making it an ideal target for anti-aging interventions.

neuroscience↗

bioRxiv2024

Single-nucleosome imaging reveals principles of transient multiscale chromatin unfolding triggered by histone ADP-ribosylation at DNA lesions

Timely access to DNA lesions is crucial for genome integrity. This process requires profound remodeling of densely packed chromatin to establish a repair-competent architecture. However, limited resolution has made it impossible to fully understand these remodeling events. Here, combining microirradiation with live-cell multiscale imaging, we report that DNA damage-induced changes in genome packing rely on the conformational behaviour of the chromatin fiber. Immediately after damage, a transient increase in nucleosome mobility switches chromatin from a densely-packed state to a looser conformation, making it accessible to repair. While histone poly-ADP-ribosylation is required to trigger this switch, mono-ADP-ribosylation is sufficient to maintain the open-chromatin state. The removal of these histone marks by the ARH3 hydrolase then leads to chromatin recondensation. Together, our multiscale study of chromatin dynamics establishes a global model: distinct waves of histone ADP-ribosylation control nucleosome mobility, triggering a transient breathing of chromatin, crucial for initiating the DNA damage response.

cell biology↗

bioRxiv2023

Untargeted proteomics identifies plant substrates of the bacterial-derived ADP-ribosyltransferase AvrRpm1

One class of enzymes that plant pathogens employ to manipulate innate immunity and physiology of the infected cells are host-targeted ADP-ribosyltransferases. The bacterial pathogen Pseudomonas syringae uses its type III secretion system to inject several effector proteins with ADP-ribosyltransferase activity into plant cells. One of them, AvrRpm1, ADP-ribosylates the plasma membrane-associated RPM1-INTERACTING PROTEIN 4 (RIN4) in Glycine max and Arabidopsis thaliana to attenuate targeted secretion of defense-promoting compounds. Substrate identification of host-targeted ADP-ribosyltransferases is complicated by the biochemical lability of the protein modification during plant protein extraction and in several cases required prior knowledge on plant immune signaling pathways that are impaired by the ADP-ribosylating type III effector. Using the AvrRpm1-RIN4 pair as a proof-of-concept, we present an untargeted proteomics workflow for enrichment and detection of ADP-ribosylated proteins and peptides from plant cell extracts that in several cases provides site-resolution for the modification.

plant biology↗

bioRxiv2023

Serine ubiquitination of p62 regulates Nrf2 dependent redox homeostasis

The KEAP1-Nrf2 axis is essential for the cellular response against metabolic and oxidative stress. KEAP1 is an adaptor protein of Cullin-3 ubiquitin ligase that controls the cellular levels of Nrf2, a critical transcription factor of several cytoprotective genes. Oxidative stress, defective autophagy and pathogenic infections activate Nrf2 signaling through phosphorylation of the adaptor protein p62, which competes with Nrf2 for binding to KEAP1. Here we show that phosphoribosyl-linked serine ubiquitination of p62 catalyzed by SidE effectors of Legionella pneumophila controls Nrf2 signaling and cell metabolism upon Legionella infection. Serine ubiquitination of p62 sterically blocks its binding to KEAP1, resulting in Nrf2 ubiquitination and degradation. This reduces Nrf2-dependent antioxidant synthesis in the early phase of infection. Levels of serine ubiquitinated p62 diminish in the later stage of infection allowing the expression of Nrf2-target genes; resulting in a differential regulation of the host metabolome and proteome in a Nrf2 dependent manner.

cell biology↗

bioRxiv2022

Hypoxia promotes osteogenesis via regulation of the mito-nuclear communication

Bone marrow mesenchymal stem cells (MSCs) reside in a hypoxic niche that maintains their differentiation potential. Several studies have highlighted the critical role of hypoxia (low oxygen concentration) in the regulation of stem cell function, reporting differentiation defects following a switch to normoxia (high oxygen concentration). However, the molecular events triggering changes in stem cell fate decisions in response to high oxygen remain elusive. Here, we study the impact of normoxia in the mito-nuclear communication, with regards to stem cell differentiation. We show that normoxia-cultured MSCs undergo profound transcriptional alterations which cause irreversible osteogenesis defects. Mechanistically, high oxygen promotes chromatin compaction and histone hypo-acetylation, particularly on promoters and enhancers of osteogenic genes. Although normoxia induces rewiring of metabolism, resulting in high acetyl-CoA levels, histone hypo-acetylation occurs due to trapping of acetyl-CoA inside mitochondria, likely due to lower CiC activity. Strikingly, restoring the cytosolic acetyl-CoA pool via acetate supplementation remodels the chromatin landscape and rescues the osteogenic defects. Collectively, our results demonstrate that the metabolism-chromatin-osteogenesis axis is heavily perturbed in response to high oxygen and identify CiC as a novel, oxygen-sensitive regulator of MSC function.

cell biology↗

bioRxiv2021

Structural basis for protein glutamylation by the Legionella pseudokinase SidJ

Legionella pneumophila (LP) avoids phagocytosis by secreting nearly 300 effector proteins into the host cytosol. SidE family of effectors (SdeA, SdeB, SdeC and SidE) employ phosphoribosyl serine ubiquitination to target an array of host Rab GTPases and innate immune factors. To suppress the deleterious toxicity of SidE enzymes in a timely manner, LP employs a metaeffector named SidJ. Upon activation by host Calmodulin (CaM), SidJ executes an ATP-dependent glutamylation to modify the catalytic residue Glu860 in the mono-ADP-ribosyl transferase (mART) domain of SdeA. SidJ is a unique glutamylase that adopts a kinase-like fold but contains two nucleotide-binding pockets. There is a lack of consensus about the substrate recognition and catalytic mechanism of SidJ. Here, we determined the cryo-EM structure of SidJ in complex with its substrate SdeA in two different states of catalysis. Our structures reveal that both phosphodiesterase (PDE) and mART domains of SdeA make extensive contacts with SidJ. In the pre-glutamylation state structure of the SidJ-SdeA complex, adenylylated E860 of SdeA is inserted into the non-canonical (migrated) nucleotide-binding pocket of SidJ. Structure-based mutational analysis indicates that SidJ employs its migrated pocket for the glutamylation of SdeA. Finally, using mass spectrometry, we identified several transient autoAMPylation sites close to both the catalytic pockets of SidJ. Our data provide unique insights into the substrate recognition and the mechanism of protein glutamylation by the pseudokinase SidJ.

biochemistry↗

bioRxiv2020

Serine-ubiquitination regulates Golgi morphology and the secretory pathway upon Legionella infection

SidE family of Legionella effectors catalyze non-canonical phosphoribosyl-linked ubiquitination (PR-ubiquitination) of host proteins during bacterial infection. SdeA localizes predominantly to ER and partially to the Golgi apparatus, and mediates serine ubiquitination of multiple ER and Golgi proteins. Here we show that SdeA causes disruption of Golgi integrity due to its ubiquitin ligase activity. The Golgi linking proteins GRASP55 and GRASP65 are PR-ubiquitinated on multiple serine residues, thus preventing their ability to cluster and form oligomeric structures. In addition, we found that the functional consequence of Golgi disruption is not linked to the recruitment of Golgi membranes to the growing Legionella-containing vacuoles. Instead, it affects the secretory pathway, including cytokine release in cells. Taken together, our study sheds light on the Golgi manipulation strategy by which Legionella hijacks the secretory pathway and promotes bacterial infection.

cell biology↗