Biology subjects

Reiling, N.

Publications and source records attributed to Reiling, N..

7 recordsLinked to original sources

bioRxiv2025

M. tuberculosis meets European Lead Factory - identification and structural characterization of novel Rv0183 inhibitors using X-ray crystallography

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of mortality worldwide. Proteins involved in lipid metabolism, such as the monoacylglycerol lipase Rv0183, play critical roles during both the active and dormant phases of Mtb and present novel targets for therapeutic intervention. Through high-throughput screening at the European Lead Factory, we identified a novel chemotype characterized by a hydroxypyrrolidine ring, which demonstrated potent inhibition of Rv0183 and promising results in whole cell bacterial studies. Subsequent co-crystallization studies of this chemotype with Rv0183 revealed non-covalent interactions within the lipases binding pocket, elucidating the inhibitory mechanism. Comparative analysis, augmented by AI-driven 3D-point-cloud approaches, distinguished Rv0183s ligand-binding cavity from that of human monoacylglycerol lipase, implying the possibility for species-selective inhibition. This selectivity was further supported by molecular docking simulations which validated the experimental binding affinities and predicted strong, specific binding modes. Our study presents not only the structural basis for the inhibition of Rv0183 by these novel hydroxypyrrolidine-based inhibitors but also demonstrates the utility of integrating computational and empirical methods to achieve species-specific targeting. This approach could minimize off-target effects in humans, marking a significant step toward developing more effective antitubercular therapies. The potential to selectively inhibit Mtb in its dormant state could lead to treatments that prevent the persistence and resurgence of the disease, addressing a crucial gap in the fight against tuberculosis.

biochemistry↗

bioRxiv2025

Revealing the unexpected interplay between the Proteasome Activator PA200 and the immunoproteasome

The proteasome activator PA200 binds to the catalytic core of the proteasome, the 20S, and activates its proteolytic activities. The cellular function of PA200 is poorly understood and appears to be cell type and differentiation specific. Recent evidence suggests that PA200 not only binds to the standard 20S (s20S) proteasome but also to the specialized immunoproteasome (i20S) which plays a key role in anti-viral and anti-tumor immunity. We here investigated the interaction of PA200 and the immunoproteasome in detail. We show the very first cryo-EM structures of the singly- and doubly-capped i20S-PA200 complexes that revealed no major difference regarding the first binding event of PA200 to the i20S vs. the s20S. However, first PA200 binding triggered a subtle and long range allosteric bending of the i20S barrel which was not seen in the s20S-PA200 complexes. This resulted in major structural rearrangements in the opposite unbound ring - the displacement of atoms up to 5.4 [A] and the increase in its outer diameter - thereby increasing the occupancy of the second PA200 binding site. Mass photometry confirmed higher occupancy of PA200 to the i20S versus the s20S. Binding of PA200 to the i20S enhanced proteasomal activation compared to the s20S. Co-expression of PA200 and the i20S in cells and tissues, however, is restricted but their interaction is favored upon co-expression. The expression of PA200 and the catalytic subunits of the i20S is differentially regulated depending on the cellular context. Our data also suggest that PA200 has the potential to regulate i20S gene expression whereas the i20S has no effects on PA200 expression. Overall, this work sheds new light on the interaction of PA200 with the i20S from a structural, mechanistic and cellular point of view. Importantly, we identify PA200 as a key regulator of the i20S whenever PA200 and the catalytic subunits of the i20S are co-expressed in the same cell.

biochemistry↗

bioRxiv2024

The proteasome activator PA200 regulates tumor cell invasion

Proteasome activator 200 (PA200) is upregulated in non-small cell lung cancer (NSCLC) and linked to poor prognosis. We previously demonstrated that the overexpression of PA200 in NSCLC is associated with immune evasion and reduced responsiveness to immune checkpoint inhibitors. The cell autonomous function of PA200 in cancer growth, however, is not solved. We here demonstrate that deletion of PA200 in two distinct lung cancer cell lines induced cell-specific alterations in proteasome composition and activities with a minor direct impact on overall proteasome activity. Deficiency of PA200 in lung cancer cells did not consistently alter tumor cell growth in vitro and in vivo. However, we observed concerted inhibition of tumor cell migration and invasion with conserved downregulation of the integrin ITGB3 and transcriptional dysregulation of multiple cell adhesion and ECM regulators. Our transcriptome profiling revealed a striking disparity in the transcriptional response to PA200 deletion in the two lung cancer cell lines. Together with our PA200 interactome analysis that uncovered an unexpected cell-dependent profile of PA200-interacting proteins, our data indicate that the function of PA200 is cell-specific and depends on the cellular context. In conclusion, we here demonstrate that PA200 cell-autonomously regulates the invasive capacities of tumor cells thereby potentially promoting lung cancer spread and metastasis formation. This mechanism might add to PA200-related immune evasion and may contribute to the observed poor prognosis of PA200-overexpressing lung cancer patients.

cancer biology↗

bioRxiv2023

GlnA3Mt is able to glutamylate spermine but it is not essential for the detoxification of spermine in Mycobacterium tuberculosis

Mycobacterium tuberculosis is well adapted to survive and persist in the infected host, escaping the host immune response. Since polyamines, which are synthesized by infected macrophages are able to inhibit the growth of M. tuberculosis, the pathogen needs strategies to cope with toxic spermine. The actinomycete Streptomyces coelicolor, closely related to M. tuberculosis makes use of a gamma-glutamylation pathway to functionally neutralize spermine. We therefore considered whether a similar pathway would be functional in M. tuberculosis. In the current study we demonstrated that M. tuberculosis growth was inhibited by the polyamine spermine. Using a glutamine synthetase-based in vitro enzymatic activity assay we determined that GlnA3Mt (Rv1878) is a gamma-glutamylspermine synthetase. In an in vitro phosphate release assay we showed that purified His-Strep-GlnA3Mt as well as native GlnA3Mt prefer spermine as a substrate to putrescine, cadaverine, spermidine or other monoamines and amino acids, suggesting that GlnA3Mt may play a specific role in the detoxification of the polyamine spermine. However, the deletion of the glnA3 gene in M. tuberculosis did not result in growth inhibition or enhanced sensitivity of M. tuberculosis in the presence of high spermine concentrations. Subsequent RNAsequencing of M. tuberculosis bacteria revealed that the gene cluster consisting of the efflux pump-encoding rv3065-rv3066-rv3067 genes is upregulated upon spermine treatment, suggesting its involvement in bacterial survival under elevated spermine concentrations. IMPORTANCEAntibiotics for the treatment of Mycobacterium tuberculosis infections attack classical bacterial targets, such as the cell envelope or the ribosome. Upon M. tuberculosis infection macrophages synthesize the polyamine spermine which - at elevated concentrations - is toxic for M. tuberculosis. Based on our investigations of spermine resistance in the closely related actinomycete Streptomyces coelicolor, we hypothesized that the glutamyl-sperminesynthetase GlnA3 may be responsible for resistance against toxic spermine. Here we show that the mycobacterial glutamyl-sperminesynthetase indeed can inactivate spermine by glutamylation. However, GlnA3 is probably not the only resistance mechanism since a glnA3 mutant of M. tuberculosis can survive under spermine stress. Gene expression studies suggest that an efflux pump may participate in resistance. The functional role of GlnA3Mt as well as of the spermine transporter in the pathogenicity of M. tuberculosis is of special interest for their validation as new targets of novel anti-tubercular drugs.

microbiology↗

bioRxiv2022

Lipobiotin-capture magnetic bead assay for isolation, enrichment and detection of Mycobacterium tuberculosis from saliva

BackgroundPulmonary Tuberculosis (TB) is diagnosed through sputum samples. As sputum sampling is challenging in children and cachexic patients, the development of diagnostic tests using saliva appears promising but has been discouraged due to low bacterial load and the poor sensitivity. We present a novel and rapid method to enrich Mycobacterium tuberculosis (Mtb) from saliva, which may serve as a basis for a diagnostic saliva test. MethodsLipobiotin-functionalized magnetic beads (LMBs) were incubated with Mtb-spiked PBS and saliva from healthy donors as well as with saliva from TB patients. Flow cytometry was used to evaluate the capacity of the beads to bind Mtb, while real-time quantitative polymerase chain reaction (qPCR) was utilized to detect Mtb and estimate the amount of mycobacterial DNA in different sample types. ResultsWe found that LMBs bind Mtb efficiently when compared to non-functionalized beads. The development of an qPCR assay based on the use of LMBs (LMB assay) allowed us to enrich mycobacterial DNA in spiked sample types, including PBS and saliva from healthy donors (enrichment of up to 8.7 fold). In Mtb-spiked saliva samples, we found that the LMB assay improved the detection rate of 102 bacteria in a volume of 5 ml from 0 out of 15 (0%) to 6 out of 15 (40%). Consistent with that, the LMB assay increased the rate of correctly identified saliva samples from TB patients in two independent cohorts. ConclusionsImplementation of the principle of the LMB-based assay may improve the sensitivity of existing diagnostic techniques, e.g. by functionalizing materials that facilitate Mtb sampling from the oral cavity.

microbiology↗

bioRxiv2021

Tuberculostearic acid (TSA)-containing phosphatidylinositols as reliable marker to determine Mycobacterium tuberculosis bacterial burden

It is estimated that approximately one-fourth of the world's population is infected with strains of the Mycobacterium tuberculosis complex (MTBC), the causative agents of tuberculosis (TB). In this study, we present rationally developed molecular markers for bacterial burden, which are derived from mycobacterial phospholipids. Using lipidomic approaches, we show that tuberculostearic acid (TSA)-containing phosphatidylinositols (PI) are present in all clinically relevant MTBC lineages investigated. For the major abundant lipid PI 16:0_19:0 (TSA), a detection limit equivalent to 102 colony forming units (CFU) was determined for bacterial cultures and approximately 103 for cell culture systems. We further developed a mass spectrometry based targeted lipid assay, which - in contrast to bacterial quantification on solid medium - can be performed within several hours including sample preparation. Translation of this indirect and culture-free detection approach allowed the determination of pathogen loads in infected murine macrophages, human neutrophils and murine lung tissue. We show that marker lipids inferred from the mycobacterial PIs are increased in peripheral blood mononuclear cells (PBMCs) of TB patients beyond the lipid metabolic background in comparison to healthy controls. In a small cohort of drug-susceptible TB patients elevated levels of these marker molecules were detected at therapy start and declined following successful anti-tuberculosis treatment. The concentration of TSA-containing PIs can be used as correlate for reliable and rapid quantification of Mycobacterium tuberculosis (Mtb) burden in experimental in vitro model systems and may also provide a clinically relevant tool for monitoring TB therapy. One Sentence SummaryTuberculostearic acid containing phosphatidylinositols represent a novel, fast to measure, reliable correlate of Mycobacterium tuberculosis bacterial burden in experimental model systems, which makes a future clinical application conceivable.

biochemistry↗

bioRxiv2020

WNT6-ACC2-induced accumulation of triacylglycerol rich lipid droplets is exploited by Mycobacterium tuberculosis

In view of emerging drug-resistant tuberculosis, host directed therapies are urgently needed to improve treatment outcomes with currently available anti-tuberculosis therapies. One option is to interfere with the formation of lipid-laden “foamy” macrophages in the infected host. Here, we provide evidence that WNT6, a member of the evolutionary conserved WNT signaling pathway, promotes foam cell formation by regulating key lipid metabolic genes including acetyl-CoA carboxylase-2 (ACC2) during pulmonary TB. In addition, we demonstrate that Mycobacterium tuberculosis (Mtb) facilitates its intracellular growth and dissemination in the host by exploiting the WNT6-ACC2 pathway. Using genetic and pharmacological approaches, we show that lack of functional WNT6 or ACC2 significantly reduces intracellular TAG levels, Mtb growth and necrotic cell death of macrophages. In combination with the anti-TB drug isoniazid, pharmacological inhibition of ACC2 improved anti-mycobacterial treatment in vitro and in vivo. Therefore, we propose the WNT6-ACC2 signaling pathway as a promising target for a host-directed therapy to reduce intracellular replication of Mtb by modulating neutral lipid metabolism.Competing Interest StatementDrs. N. Reiling and J. Brandenburg (Research Center Borstel, Leibniz Lung Center, 23845 Borstel, Germany) have filed a patent application entitled ACC inhibitors as means and methods for treating mycobacterial diseases(WO2018007430A1, patent pending).View Full Text

immunology↗