Biology subjects

Sass, P.

Publications and source records attributed to Sass, P..

4 recordsLinked to original sources

bioRxiv2024

Development of a small-molecule epigenetic regenerative therapy. Subcutaneous administration of alginate formulations with high loads of zebularine and retinoic acid promotes tissue growth, vascularization and innervation and induces extensive epigenetic repatterning

Recently, zebularine, a small-molecule epigenetic inhibitor and retinoic acid, acting as a transcriptional activator, have been found to induce tissue regeneration. In this study, the pro-regenerative properties of zebularine and retinoic acid were combined with the potential of the alginate carrier to expand its therapeutic possibilities. Alginate formulations of zebularine and retinoic acid were developed for subcutaneous administration to mice. Hydrophilic zebularine formed a homogenous formulation with extreme drug loadings reaching 240 mg of zebularine per 1 ml of 2% sodium alginate, while hydrophobic retinoic acid, 0.8 mg/ml, dispersed as fine crystals. Cell culture tests exhibited no significant cytotoxicity of the alginate formulations. Subcutaneous administration of zebularine and retinoic acid in 2% sodium alginate promoted regenerative responses in a mouse model of ear pinna punch wound mice involving the restoration of tissue architecture, nerve and vessel growth, and extensive epigenetic and transcriptional repatterning with no adverse effects observed in the animals. Significant trancriptomic responses to the epigenetic treatment included the induction of epithelium development genes contrasted with the downregulation of muscle development genes on day 7 post-injury. Among the remarkable changes in global gene methylation are those in neurodevelopmental genes. In vitro studies showed rapid zebularine but no retinoic acid discharge from the alginate formulations. Live ultrasound imaging demonstrated gradual absorption of the subcutaneously injected alginate formulations, which may explain the in vivo activity of retinoic acid following subcutaneous administration. Effective induction of tissue regeneration together with a high safety profile and of the subcutaneously administered pro-regenerative alginate formulations opens the way to testing further regenerative therapies for hard-to-reach lesions.

bioengineering↗

bioRxiv2022

In vivo and In vitro Characterization of the ClpC AAA+ ATPase of Chlamydia trachomatis

Bacterial AAA+ unfoldases are crucial for bacterial physiology by recognizing specific substrates and, typically, unfolding them for degradation by a proteolytic component. The caseinolytic protease (Clp) system is one example where a hexameric unfoldase (e.g., ClpC) interacts with the tetradecameric proteolytic core ClpP. Unfoldases can have both ClpP-dependent and ClpP-independent roles in protein homeostasis, development, virulence, and cell differentiation. ClpC is an unfoldase predominantly found in Gram-positive bacteria and mycobacteria. Intriguingly, the obligate intracellular Gram-negative pathogen Chlamydia, an organism with a highly reduced genome, also encodes a ClpC ortholog, implying an important function for ClpC in chlamydial physiology. Here, we used a combination of in vitro and in vivo approaches to gain insight into the function of chlamydial ClpC. ClpC exhibits intrinsic ATPase and chaperone activities, with a primary role for the Walker B motif in the first nucleotide binding domain (NBD1). Furthermore, ClpC binds ClpP1P2 complexes via ClpP2 to form the functional protease ClpCP2P1 in vitro, which degraded arginine-phosphorylated {beta}-casein. In vivo experiments confirmed that higher order complexes of ClpC are present in chlamydial cells. Importantly, the in vivo data further revealed severe negative effects of both overexpression and depletion of ClpC in Chlamydia as revealed by a significant reduction in chlamydial growth. Here again, NBD1 was critical for ClpC function. Hence, we provide the first mechanistic insight into the molecular and cellular function of chlamydial ClpC, which supports its essentiality in Chlamydia. ClpC is, therefore, a potential novel target for the development of anti-chlamydial agents. SignificanceChlamydia trachomatis is an obligate intracellular pathogen and the worlds leading cause of preventable infectious blindness and bacterial sexually transmitted infections. Due to the high prevalence of chlamydial infections along with negative effects of current broad-spectrum treatment strategies, new anti-chlamydial agents with novel targets are desperately needed. In this context, bacterial Clp proteases have emerged as promising new antibiotic targets, since they often play central roles in bacterial physiology and, for some bacterial species, are even essential for survival. Here, we report on the chlamydial AAA+ unfoldase ClpC, its functional reconstitution and characterization, individually and as part of the ClpCP2P1 protease, and establish an essential role for ClpC in chlamydial growth and intracellular development, thereby identifying ClpC as a potential target for anti-chlamydial compounds.

microbiology↗

bioRxiv2022

Antibiotic acyldepsipeptides stimulate the Streptomyces Clp-ATPase/ClpP complex for accelerated proteolysis

Clp proteases consist of a proteolytic, tetradecameric core ClpP and AAA+ Clp-ATPases. Streptomycetes, producers of a plethora of secondary metabolites, encode up to five different ClpP homologs and the composition of their unusually complex Clp protease machinery has remained unsolved. Here, we report on the composition of the house-keeping Clp protease in Streptomyces, consisting of a hetero-tetradecameric core built of ClpP1, ClpP2 and the cognate Clp-ATPases ClpX, ClpC1 or ClpC2, all interacting with ClpP2 only. ADEP antibiotics dysregulate the Clp protease for unregulated proteolysis. We observed that ADEP binds Streptomyces ClpP1, but not ClpP2, thereby not only triggering the degradation of non-native protein substrates but also accelerating Clp-ATPase-dependent proteolysis. The explanation is the concomitant binding of ADEP and Clp-ATPases to opposite sides of the ClpP1P2 barrel, hence revealing a third, so far unknown mechanism of ADEP action, i.e., the accelerated proteolysis of native protein substrates by the Clp protease. SignificanceClp proteases are antibiotic and anti-cancer drug targets. Composed of the proteolytic core ClpP and a regulatory Clp-ATPase, the protease machinery is important for protein homeostasis and regulatory proteolysis. The acyldepsipeptide antibiotic ADEP targets ClpP and has shown promise for treating multi-resistant and persistent bacterial infections. The molecular mechanism of ADEP is multi-layered. Here, we present a new way how ADEP can deregulate the Clp protease system. Clp-ATPases and ADEP bind to opposite sides of Streptomyces ClpP, accelerating the degradation of natural Clp protease substrates. We also demonstrate the composition of the major Streptomyces Clp protease complex, a heteromeric ClpP1P2 core with the Clp-ATPases ClpX, ClpC1 or ClpC2 exclusively bound to ClpP2, and the killing mechanism of ADEP in Streptomyces.

microbiology↗

bioRxiv2020

Antibiotic-induced degradation of FtsZ reveals distinct stages of Bacillus subtilis FtsZ ring assembly and constriction

Cell division is a central and essential process in most bacteria, and also due to its complexity and highly coordinated nature, it has emerged as a promising new antibiotic target pathway in recent years. We have previously shown that ADEP antibiotics preferably induce the degradation of the major cell division protein FtsZ, thereby primarily leading to a depletion of the cytoplasmic FtsZ pool that is needed for treadmilling FtsZ rings. To further investigate the physiological consequences of ADEP treatment, we here studied the effect of ADEP on the different stages of the FtsZ ring in rod-shaped bacteria. Our data reveal the disintegration of early FtsZ rings during ADEP treatment in Bacillus subtilis, indicating an essential role of the cytoplasmic FtsZ pool and thus FtsZ ring dynamics during initiation and maturation of the divisome. However, progressed FtsZ rings finalized cytokinesis once the septal peptidoglycan synthase PBP2b, a late stage cell division protein, colocalized at the division site, thus implying that the concentration of the cytoplasmic FtsZ pool and FtsZ ring dynamics are less critical during the late stages of divisome assembly and progression.

microbiology↗