Biology subjects

Geibel, S.

Publications and source records attributed to Geibel, S..

7 recordsLinked to original sources

bioRxiv2026

Cryo-EM structures of mycobacterial MCC reveal carrier-domain translocation between catalytic sites

3-Methylcrotonyl-CoA carboxylase (MCC) is a biotin-dependent enzyme complex that catalyzes an essential step in leucine degradation. In mycobacteria, MCC is formed by AccA1 and AccD1, but its structural organization has remained poorly defined. Here, we report two high-resolution cryo-EM structures of the endogenous, biotin-bound 6{beta}6 complex from Mycobacterium smegmatis. The MCC architecture comprises a central hexameric carboxyltransferase ({beta}/CT) core flanked by trimeric biotin carboxylase (/BC) modules. The structures capture BC- and CT-engaged conformations, in which the biotin carboxyl carrier protein (BCCP) engages either the BC or CT active site, providing structural evidence for long-range carrier-domain translocation. Conformational rearrangements within the BC module suggest a role in regulating BCCP engagement. Together, our findings provide insights into carrier-domain dynamics in mycobacterial MCC.

biochemistry↗

bioRxiv2026

Structural Basis of Substrate Selectivity and Catalysis in the Mycobacterial Long-Chain Acyl-CoA Carboxylase

Long-chain acyl-CoA carboxylase (LCC) is an essential enzyme complex in mycobacteria that generates acyl-CoA precursors for mycolic acid and complex lipid biosynthesis, yet its architecture and mechanism of substrate selection have remained unclear. Here we determine pre- and post-reaction states of the endogenous 868-kDa LCC complex from Mycobacterium smegmatis by cryo-electron microscopy at 2.1-3.7 [A] resolution. These structures visualize ATP-dependent redistribution of the biotin carboxyl carrier protein. LCC assembles into an asymmetric 8:2:4:2 organization of AccA3, AccD4, AccD5, and AccE5, with two biotin carboxylase modules flexibly tethered to a heterohexameric carboxyltransferase core. We define the structural basis of substrate selectivity within the CT core: AccD5 selectively binds the short-chain substrate C3-CoA, whereas AccD4 accommodates the long-chain substrate C16-CoA. In addition, we resolve AccD5-centered assemblies that associate with biotin carboxylase modules yet lack AccD4, providing structural evidence that distinct carboxyltransferase cores can engage shared modules to generate alternative holoenzyme architectures. Together, these findings define LCC and AccD5-centered assemblies as elements of a combinatorial acyl-CoA carboxylase platform and establish the structural principles governing assembly-specific function in mycobacteria.

biochemistry↗

bioRxiv2024

Superior target genes and pathways for RNAi mediated pest control revealed by genome wide analysis in the red flour beetle Tribolium castaneum

An increasing human population, the emergence of resistances against pesticides and their potential impact on the environment call for the development of new eco-friendly pest control strategies. RNA interference (RNAi) based pesticides have emerged as new option with the first products entering the market. Essentially, double stranded RNAs targeting essential genes of pests are either expressed in the plants or sprayed on their surface. Upon feeding, pests mount an RNAi response and die. However, it has remained unclear, whether RNAi based insecticides should target the same pathways as classic pesticides or whether the different mode of action would favor other processes. Moreover, there is no consensus on the best genes to be targeted. We performed a genome-wide screen in the red flour beetle to identify 905 RNAi target genes. Based on a validation screen and clustering, we identified the 192 most effective target genes in that species. The transfer to oral application in other beetle pests revealed a list of 34 superior target genes, which are an excellent starting point for application in other pests. GO and KEGG analyses of our genome wide dataset revealed that genes with high efficacy belonged mainly to basic cellular processes such as gene expression and protein homeostasis - processes not targeted by classic insecticides. In summary, our work revealed the best target genes and target processes for RNAi based pest control and we propose a procedure to transfer our short list of superior target genes to other pests.

genomics↗

bioRxiv2022

Functional characterization of putative ecdysone transporters in lepidopteran pests

The insect steroid hormone ecdysone plays a critical role in insect development. Several recent studies have shown that ecdysone is transported through Organic Anion Transporting Polypeptides (OATPs) in insects such as flies and mosquitoes. However, the conservation of this mechanism across other arthropods and the role of this transporter in canonical ecdysone pathways are less well studied. Herein we functionally characterized the putative ecdysone transporter OATP74D from two major agricultural moth pests: Helicoverpa armigera (cotton bollworm) and Spodoptera frugiperda (fall armyworm). Phylogenetic analysis of OATP transporters across the superphylum Ecdysozoa revealed that Oatp74D is well represented among arthropod species and appeared only at the root of the arthropod lineage. Partial disruption of Oatp74D in S. frugiperda decreased embryo hatching rate and larval survival, suggesting that this gene is essential for development in vivo. Depletion and re-expression of OatP74D in the lepidoptera cell line RP-HzGUT-AW1(MG) confirmed the genes role in ecdysone import and demonstrated that OATP74D is essential for the transcriptional activation of ecdysone responsive genes including caspase-3, implicating this transporter in cell death pathways. Establishment of a simple and robust luciferase assay using the RP-HzGUT- AW1(MG) cell line demonstrated that both HaOATP74D and SfOATP74D are inhibited by rifampicin, a well-known organic anion transporter inhibitor. Overall, this work sheds more light on ecdysone uptake mechanisms across insect species and broadens our knowledge of the physiological roles of OATPs in the transportation of endogenous substrates. Author SummaryThe insect steroid hormone ecdysone is critical in regulating many aspects of insects life, including development and reproduction. A passive diffusion model was never functionally resolved, but was strongly supported until an organic anion transporting polypeptide was identified to mediate the transport of the hormone. The OATP74D, belonging to the Solute carrier superfamily, has been identified and functionally characterized for the first time in Drosophila melanogaster. Although phylogenetic analysis suggests that the Drosophila Oatp74D is probably conserved among several insect species, the theory for transporter mediated ecdysone uptake cannot be generalized to all insects without concrete proof. In here we provide functional evidence that the Oatp74D of two lepidopteran pest species: Helicoverpa armigera and Spodoptera frugiperda, is highly required for insect survival and development. Furthermore, we reveal that the OATP74D is necessary to regulate the expression of several ecdysone response genes, including caspase-3 which is involved in programmed cell death. In addition, we have developed a cell-based platform for screening chemical compounds against the lepidopteran orthologs of Oat74D and rifampicin was functionally shown to inhibit ecdysone uptake. Taken all together, our study reveals that Oatp74D is conserved among several arthropod species in the ecdysone pathway and given the high necessity for an effective control of these two lepidopteran species, we hypothesized that OATP74D could serve as a possible drug target in those two species.

genetics↗

bioRxiv2022

Genetic characterization of ABC pesticide transporters establishes Drosophila as an in vivo model for toxicogenetics

Pesticides remain one of the most effective ways of controlling agricultural and public health insects, but much is still unknown regarding how these compounds reach their targets. Specifically, the role of ABC transporters in pesticide absorption and excretion is poorly understood, especially compared to the detailed knowledge in mammalian systems. Here, we present a comprehensive characterization of pesticide transporters in the model insect D. melanogaster. An RNAi screen was performed, which knocked down individual ABCs in targeted epithelial tissue, examining the subsequent changes in sensitivity to the pesticides spinosad and fipronil. This simultaneously implicated a novel ABC drug transporter CG4562 but also highlighted a predominant role for the P-glycoprotein orthologue Mdr65. Further characterization of the P-glycoprotein family was performed via transgenic overexpression and immunolocalization, finding that Mdr49 and Mdr50 play enigmatic roles in pesticide toxicology perhaps determined by their different subcellular localizations within the midgut. Lastly, heterologous expression of the Mdr65 orthologue from the major malaria vector Anopheles gambiae was used to establish an in vivo characterization system for the characterization of P-glycoproteins from non-model insects in D. melanogaster. This study provides the basis for establishing Drosophila as a model for toxicology research regarding drug transporters.

genetics↗

bioRxiv2020

Environmental RNA interference in two-spotted spider mite, Tetranychus urticae, reveals dsRNA processing requirements for efficient RNAi response

Comprehensive understanding of pleiotropic roles of RNAi machinery highlighted the conserved chromosomal functions of RNA interference. The consequences of the evolutionary variation in the core RNAi pathway genes are mostly unknown, but may lead to the species-specific functions associated with gene silencing. The two-spotted spider mite, Tetranychus urticae, is a major polyphagous chelicerate pest capable of feeding on over 1,100 plant species and developing resistance to pesticides used for its control. A well annotated genome, susceptibility to RNAi and economic importance, make T. urticae an excellent candidate for development of an RNAi protocol that enables high-throughput genetic screens and RNAi-based pest control. Here, we show that the length of the exogenous dsRNA critically determines its processivity and ability to induce RNAi in vivo. A combination of the long dsRNAs and the use of dye to trace the ingestion of dsRNA enabled the identification of genes involved in membrane transport and 26S proteasome degradation as sensitive RNAi targets. Our data demonstrate that environmental RNAi can be an efficient reverse genetics and pest control tool in T. urticae. In addition, the species-specific properties together with the variation in the components of the RNAi machinery make T. urticae a potent experimental system to study the evolution of RNAi pathways.

molecular biology↗

bioRxiv2020

The helix bundle domain of primase RepB' is required for dinucleotide formation and extension

During DNA replication, primases synthesize oligonucleotide primers on single-stranded template DNA, which are then extended by DNA polymerases to synthesize a complementary DNA strand. Primase RepB of plasmid RSF1010 initiates DNA replication on two 40 nucleotide long inverted repeats, termed ssiA and ssiB, within the oriV of RSF1010. RepB consists of a catalytic domain and a helix bundle domain which are connected by long -helix 6 and an unstructured linker. Previous work has demonstrated that RepB requires both domains for initiation of dsDNA synthesis in DNA replication assays. However, the precise functions of these two domains in primer synthesis have been unknown. Here we report that both domains of RepB are required to synthesizes a 10-12 nucleotide long DNA primer whereas the isolated domains are inactive. Mutational analysis of the catalytic domain indicates that the solvent-exposed W50 plays a critical role in resolving a hairpin structures formed by ssiA and ssiB. Three structurally conserved aspartates (D77, D78 and D134) of RepB catalyse the nucleotidyl transfer reaction. Mutations on the helix bundle domain are identified that either reduce the primer length to a dinucleotide (R285A) or abolish primer synthesis (D238A) indicating that the helix bundle domain is required to form and extend the initial dinucleotide synthesized by the catalytic domain.

biochemistry↗