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Membrane proteins frequently assemble into higher order homo- or hetero-oligomers within their natural lipid environment. This complex formation can modulate their folding, activity as well as substrate selectivity. Non-disruptive methods avoiding critical steps, such as membrane disintegration, transfer into artificial environments or chemical modifications are therefore essential to analyze molecular mechanisms of native membrane protein assemblies. The combination of cell-free synthetic biology, nanodisc-technology and non-covalent mass spectrometry provides excellent synergies for the analysis of membrane protein oligomerization within defined membranes. We exemplify our strategy by oligomeric state characterization of various membrane proteins including ion channels, transporters and membrane-integrated enzymes assembling up to hexameric complexes. We further indicate a lipid-dependent dimer formation of MraY translocase correlating with the enzymatic activity. The detergent-free synthesis of membrane protein/nanodisc samples and the analysis by LILBID mass spectrometry provide a versatile platform for the analysis of membrane proteins in a native environment.
Cell-free-synthesized voltage-gated proton channels: Approaches to the study of protein dynamics
(2018)
We often only realize how important health is when diseases manifest themselves through their symptoms and, ultimately, in a diagnosis. Over time, we suffer from many diseases starting with the first childhood disease to colds to gastrointestinal infections. Most diseases pass harmlessly and symptoms fade away. However, not all diseases are so harmless. Alzheimer’s disease, breast cancer, Parkinson’s disease, and colorectal cancer usually cause severe illness with high mortality rates. In pharmaceutical research, efforts are therefore being made to determine the molecular basis of them in order to provide patients with potential relief and, at best, healing. A special group of regulators, involved in the previously mentioned diseases, are voltage-gated proton channels. Thus, the understanding of their structure, function, and potential drug interaction is of great importance for humanity.
Voltage-gated proton channels are localized in the cell membrane. As their name indicates, they are controlled by voltage changes. Depolarization of the cell membrane induces conformational changes that open these channels allowing protons to pass through. Here, the transfer is based on a passive process driven by a concentration gradient between two individual compartments separated by the cell membrane. Voltage-gated proton channels are highly selective for protons and show a temperature- and pH-dependent gating behavior. However, little is known about their channeling mechanism. Previous experimental results are insufficient for understanding the key features of proton channeling.
In this thesis, for the first time, the cell-free production of voltage-sensing domains (VSD) of human voltage-gated proton channels (hHV1) and zebrafish voltage-sensing phosphatases (DrVSP) is described. Utilizing the cell free approach, parameters concerning protein stability, folding and labeling can be easily addressed. Furthermore, the provision of a membrane mimetic in form of detergent micelles, nanodiscs, or liposomes for co-translational incorporations of these membrane proteins is simple and efficient. Both VSDs were successfully produced up to 3 mg/ml. Furthermore, the cell-free synthesis enabled for the first time studies of lipid-dependent co-translational VSD insertions into nanodiscs and liposomes. Cell-free produced VSDs were shown to be active, and to exist mainly as dimers. In addition, also their activation was stated to be lipid-dependent, which has not been described so far. Solution-state NMR experiments were performed with fully and selectively labeled cell-free produced VSDs. With respect to the development of potential drug candidates, I could demonstrate the inhibition of the VSDs by 2-guanidinobenzimidazole (2GBI). Determined KD values were comparable to literature data for the human construct. For the first time, a low affinity for 2GBI of the zebrafish VSD could be described.
In future, the combination of a fast, easy and cheap cell-free production of fully or selectively labeled VSDs and their analysis by solution state NMR will enable structure determinations as well as inhibitor binding studies and protein dynamic investigations of those proteins. The results of these investigations will serve as a basis for example for the development of new drugs. In addition, a detailed description of the lipid-dependent activity might be helpful in controlling the function of voltage-gated proton channels in cancer cells and thereby reducing their growth or disturbing their cell homeostasis in general.
Attention-deficit/hyperactivity disorder (ADHD) is a common, highly heritable neurodevelopmental disorder. Genetic loci have not yet been identified by genome-wide association studies. Rare copy number variations (CNVs), such as chromosomal deletions or duplications, have been implicated in ADHD and other neurodevelopmental disorders. To identify rare (frequency ≤1%) CNVs that increase the risk of ADHD, we performed a whole-genome CNV analysis based on 489 young ADHD patients and 1285 adult population-based controls and identified one significantly associated CNV region. In tests for a global burden of large (>500 kb) rare CNVs, we observed a nonsignificant (P=0.271) 1.126-fold enriched rate of subjects carrying at least one such CNV in the group of ADHD cases. Locus-specific tests of association were used to assess if there were more rare CNVs in cases compared with controls. Detected CNVs, which were significantly enriched in the ADHD group, were validated by quantitative (q)PCR. Findings were replicated in an independent sample of 386 young patients with ADHD and 781 young population-based healthy controls. We identified rare CNVs within the parkinson protein 2 gene (PARK2) with a significantly higher prevalence in ADHD patients than in controls (P=2.8 × 10(-4) after empirical correction for genome-wide testing). In total, the PARK2 locus (chr 6: 162 659 756-162 767 019) harboured three deletions and nine duplications in the ADHD patients and two deletions and two duplications in the controls. By qPCR analysis, we validated 11 of the 12 CNVs in ADHD patients (P=1.2 × 10(-3) after empirical correction for genome-wide testing). In the replication sample, CNVs at the PARK2 locus were found in four additional ADHD patients and one additional control (P=4.3 × 10(-2)). Our results suggest that copy number variants at the PARK2 locus contribute to the genetic susceptibility of ADHD. Mutations and CNVs in PARK2 are known to be associated with Parkinson disease.
The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.