Showing papers by "Lutz Schmitt published in 2021"

The ABC transporter G subfamily in Arabidopsis thaliana.

Abstract: ABC transporters are ubiquitously present in all kingdoms and mediate the transport of a large spectrum of structurally different compounds. Plants possess high numbers of ABC transporters in relation to other eukaryotes; the ABCG subfamily in particular is extensive. Earlier studies demonstrated that ABCG transporters are involved in important processes influencing plant fitness. This review summarizes the functions of ABCG transporters present in the model plant Arabidopsis thaliana. These transporters take part in diverse processes such as pathogen response, diffusion barrier formation, or phytohormone transport. Studies involving knockout mutations reported pleiotropic phenotypes of the mutants. In some cases, different physiological roles were assigned to the same protein. The actual transported substrate(s), however, still remain to be determined for the majority of ABCG transporters. Additionally, the proposed substrate spectrum of different ABCG proteins is not always reflected by sequence identities between ABCG members. Applying only reverse genetics is thereby insufficient to clearly identify the substrate(s). We therefore stress the importance of in vitro studies in addition to in vivo studies in order to (i) clarify the substrate identity; (ii) determine the transport characteristics including directionality; and (iii) identify dimerization partners of the half-size proteins, which might in turn affect substrate specificity.

Structure and Function of Hepatobiliary ATP Binding Cassette Transporters.

Abstract: The liver is beyond any doubt the most important metabolic organ of the human body. This function requires an intensive crosstalk within liver cellular structures, but also with other organs. Membrane transport proteins are therefore of upmost importance as they represent the sensors and mediators that shuttle signals from outside to the inside of liver cells and/or vice versa. In this review, we summarize the known literature of liver transport proteins with a clear emphasis on functional and structural information on ATP binding cassette (ABC) transporters, which are expressed in the human liver. These primary active membrane transporters form one of the largest families of membrane proteins. In the liver, they play an essential role in for example bile formation or xenobiotic export. Our review provides a state of the art and comprehensive summary of the current knowledge of hepatobiliary ABC transporters. Clearly, our knowledge has improved with a breath-taking speed over the last few years and will expand further. Thus, this review will provide the status quo and will lay the foundation for new and exciting avenues in liver membrane transporter research.

Structure and efflux mechanism of the yeast pleiotropic drug resistance transporter Pdr5.

Abstract: Pdr5, a member of the extensive ABC transporter superfamily, is representative of a clinically relevant subgroup involved in pleiotropic drug resistance. Pdr5 and its homologues drive drug efflux through uncoupled hydrolysis of nucleotides, enabling organisms such as baker’s yeast and pathogenic fungi to survive in the presence of chemically diverse antifungal agents. Here, we present the molecular structure of Pdr5 solved with single particle cryo-EM, revealing details of an ATP-driven conformational cycle, which mechanically drives drug translocation through an amphipathic channel, and a clamping switch within a conserved linker loop that acts as a nucleotide sensor. One half of the transporter remains nearly invariant throughout the cycle, while its partner undergoes changes that are transmitted across inter-domain interfaces to support a peristaltic motion of the pumped molecule. The efflux model proposed here rationalises the pleiotropic impact of Pdr5 and opens new avenues for the development of effective antifungal compounds. Pdr5 is an ABC transporter conferring multidrug resistance to pathogenic fungi. Here, structural analysis of Pdr5 provides insights into the transport mechanism featuring asymmetric movements of Pdr5 domain and enabling efflux of a broad spectrum of compounds.

Numaswitch: an efficient high-titer expression platform to produce peptides and small proteins

Abstract: The production of peptides as active pharmaceutical ingredients (APIs) by recombinant technologies is of emerging interest. A reliable production platform, however, is still missing due the inherent characteristics of peptides such as proteolytic sensitivity, aggregation and cytotoxicity. We have developed a new technology named Numaswitch solving present limitations. Numaswitch was successfully employed for the production of diverse peptides and small proteins varying in length, physicochemical and functional characteristics, including Teriparatide, Linaclotide, human β-amyloid and Serum amyloid A3. Additionally, the potential of Numaswitch for a cost-efficient commercial production is demonstrated yielding > 2 g Teriparatide per liter fermentation broth in a quality meeting API standard.

Structure and efflux mechanism of the yeast pleiotropic drug resistance transporter Pdr5

Abstract: Pdr5, a member of the extensive ABC transporter superfamily, is representative of a clinically relevant subgroup involved in pleiotropic drug resistance. Through the coupling of nucleotide hydrolysis with drug efflux, Pdr5 homologues enable pathogenic species to survive in the presence of chemically diverse antifungal agents. Our structural and functional results reveal details of an ATP-driven conformational cycle, which mechanically drives drug translocation through an amphipathic channel, and a clamping switch within a conserved linker loop that acts as a nucleotide sensor. One half of the transporter remains nearly invariant throughout the cycle, while its partner undergoes changes that are transmitted across interdomain interfaces to support a peristaltic motion of the pumped molecule. The efflux model proposed here rationalises the pleiotropic impact of Pdr5 and opens avenues for the development of effective antifungal compounds.

Importance of the leader peptide sequence on the lanthipeptide secretion level.

Abstract: Lanthipeptides are ribosomally synthesized and posttranslationally modified peptides. Their precursor peptide comprises of an N-terminal leader peptide and a C-terminal core peptide. Here, the leader peptide is crucial for enzyme recognition especially for the modification enzymes and acts furthermore as a secretion signal for the lanthipeptide exporter. The core peptide is the target site for the posttranslational modifications and contains dehydrated amino acids and lanthionine rings. Nisin produced by the Gram-positive bacterium Lactococcus lactis is one of the best-studied lanthipeptides and used as a model system to study their modification and secretion processes. Nisin is secreted as a precursor peptide. Here, we present an in vivo secretion analysis of NisT in the absence of the modification machinery allowing the secretion of leader peptide mutants and their impact solely on the secretion activity of NisT. Additionally, we created leader peptide hybrids to provide new insights, how the secretion is effected by unnatural leader peptides. The focus on the secretion activity of the transporter alone enabled us to determine the recognition site of NisT within the leader peptide of nisin.

Novel intracellular phospholipase B from Pseudomonas aeruginosa with activity towards endogenous phospholipids affects biofilm assembly

Abstract: Pseudomonas aeruginosa is a severe threat to immunocompromised patients due to its numerous virulence factors and multiresistance against antibiotics. This bacterium produces and secretes various toxins with hydrolytic activities including phospholipases A, C and D. However, the function of intracellular phospholipases for bacterial virulence has still not been established. Here we demonstrate that the hypothetical gene pa2927 of P. aeruginosa encodes a novel phospholipase B named PaPlaB. PaPlaB isolated from detergent-solubilized membranes of E. coli rapidly degraded various GPLs including endogenous GPLs isolated from P. aeruginosa cells. Cellular localization studies suggest that PaPlaB is peripherally bound to the inner and outer membrane of E. coli, yet the active form was predominantly associated with the cytoplasmic membrane. In vitro activity of purified and detergent-stabilized PaPlaB increases at lower protein concentrations. The size distribution profile of PaPlaB oligomers revealed that decreasing protein concentration triggers oligomer dissociation. These results indicate that homooligomerisation regulates PaPlaB activity by a yet unknown mechanism, which might be required for preventing bacteria from self-disrupting the membrane. We demonstrated that PaPlaB is an important determinant of the biofilm lifestyle of P. aeruginosa, as shown by biofilm quantification assay and confocal laser scanning microscopic analysis of biofilm architecture. This novel intracellular phospholipase B with a putative virulence role contributes to our understanding of membrane GPL degrading enzymes and may provide a target for new therapeutics against P. aeruginosa biofilms.

The many facets of bile acids in the physiology and pathophysiology of the human liver.

Abstract: Bile acids perform vital functions in the human liver and are the essential component of bile. It is therefore not surprising that the biology of bile acids is extremely complex, regulated on different levels, and involves soluble and membrane receptors as well as transporters. Hereditary disorders of these proteins manifest in different pathophysiological processes that result in liver diseases of varying severity. In this review, we summarize our current knowledge of the physiology and pathophysiology of bile acids with an emphasis on recently established analytical approaches as well as the molecular mechanisms that underlie signaling and transport of bile acids. In this review, we will focus on ABC transporters of the canalicular membrane and their associated diseases. As the G protein-coupled receptor, TGR5, receives increasing attention, we have included aspects of this receptor and its interaction with bile acids.

A new twist in ABC transporter mediated multidrug resistance − Pdr5 is a drug/proton co-transporter

Abstract: The two major efflux pump systems that are involved in multidrug resistance (MDR) are (i) ATP binding cassette (ABC) transporters and (ii) secondary transporters. While the former use binding and hydrolysis of ATP to facilitate export of cytotoxic compounds, the latter utilize electrochemical gradients to expel their substrates. Pdr5 from Saccharomyces cerevisiae is a prominent member of eukaryotic ATP binding cassette (ABC) transporters that are involved in multidrug resistance (MDR) and used as a frequently studied model system. Although investigated for decades, the underlying molecular mechanisms of drug transport and substrate specificity remain elusive. Here, we provide electrophysiological data on the reconstituted Pdr5 demonstrating that this MDR efflux pump does not only actively translocate its substrates across the lipid bilayer, but at the same time generates a proton motif force in the presence of Mg2+-ATP and substrates by acting as a proton/drug co-transporter. Similar observations have not yet been reported for any other MDR efflux pump. We conclude from these results that the mechanism of MDR conferred by Pdr5 and likely other transporters is more complex than the sole extrusion of cytotoxic compounds and involves secondary coupled processes suitable to increase the effectiveness.

A MademoiseLLE domain binding platform links the key RNA transporter to endosomes

Abstract: Spatiotemporal expression is mostly achieved by transport and translation of mRNAs at defined subcellular sites. An emerging mechanism mediating mRNA trafficking is microtubule-dependent co-transport of mRNAs on shuttling endosomes. Although progress has been made in identifying various components of the endosomal mRNA transport machinery, a mechanistic understanding of how these RNA-binding proteins are connected to endosomes is still lacking. Here, we demonstrate that a flexible MademoiseLLE (MLLE) domain platform within Rrm4 of Ustilago maydis is crucial for endosomal attachment. Our structure/function analysis uncovered three MLLE domains at the C-terminus of Rrm4 with a functionally defined hierarchy. MLLE3 recognizes two PAM2-like sequences of the adaptor protein Upa1 and is essential for endosomal shuttling of Rrm4. MLLE1 and MLLE2 are most likely accessory domains that exhibit a variant binding mode for interaction with currently unknown partners. Thus, endosomal attachment of the mRNA transporter is orchestrated by a sophisticated MLLE domain binding platform.

Eine rekombinante Produktionsplattform für Peptide

Abstract: The production of peptides as active pharmaceutical ingredients (APIs) by recombinant technologies is of emerging interest. A reliable production platform, however, is still missing due to inherent peptide characteristics such as proteolytic sensitivity, aggregation, and cytotoxicity. We developed a new approach — Numaswitch™. It solves present limitations and provides a cost-efficient production platform for diverse peptides and hard-to-be-expressed proteins.

Quantification and surface localization of the hemolysin A type 1 secretion system at the endogenous level and under conditions of overexpression

Abstract: Secretion systems are essential for Gram-negative bacteria as these nanomachineries allow a communication with the outside world by exporting proteins into the extracellular space or directly into the cytosol of a host cell. For example, type one secretion systems (T1SS) secrete a broad range of substrates across both membranes into the extracellular space. One well-known example is the hemolysin A (HlyA) T1SS from Escherichia coli (E. coli), which consists of an ABC transporter (HlyB), a membrane fusion protein (HlyD), the outer membrane protein TolC and the substrate HlyA, a member of the family of RTX (repeats in toxins) toxins. Here, we determined the amount of TolC at the endogenous level (parental strain, UTI89) and under conditions of overexpression (T7 expression system, BL21(DE3)-BD). The overall amount of TolC was not influenced by the overexpression of the HlyBD complex. Moving one step further, we determined the localization of the HlyA T1SS by super-resolution microscopy. In contrast to other bacterial secretion systems, no polarization was observed with respect to endogenous or overexpression levels. Additionally, the cell growth and division cycle did not influence the polarization. Most importantly, the size of the observed T1SS clusters did not correlate with the recently proposed outer membrane islands. These data indicate that T1SS cluster at the outer membrane generating domains of so far not described identity. Importance Uropathogenic Escherichia coli (UPEC) strains cause about 110 million urinary tract infections each year worldwide representing a global burden to the healthcare system. UPEC secrete many virulence factors among these the TX toxin hemolysin A via a cognate T1SS into the extracellular space. In this study, we determined the endogenous copy number of the HlyA T1SS in UTI89 and analyzed the surface localization in BL21(DE3)-BD and UTI89, respectively. With approximately 800 copies of the T1SS in UTI89, this is one of the highest expressed bacterial secretion systems. Furthermore and in clear contrast to other secretion systems, no polarized surface localization was detected. Finally, quantitative analysis of the super-resolution data revealed that clusters of the HlyA T1SS are not related to the recently identified outer membrane protein islands. These data provide insights into the quantitative molecular architecture of the HlyA T1SS.

Lantibiotika — hoffnungsvolle Alternative gegen Antibiotikaresistenz?

Abstract: Nisin is one of the most studied lantibiotics which are antimicrobial peptides. Nowadays the knowledge about the Nisin-modification system is profound and can be explored to express and modify lantibiotics with new or specific antimicrobial features. Here we highlight recent advances that include a strategy on bypassing natural occurring resistances against antimicrobial peptides.