Identifizierung von physiologischen und artifiziellen Liganden von GABARAP und Charakterisierung der resultierenden Interaktionen

$\gamma$-aminobutyric acid (GABA) receptors (type A) (GABA$_{A}$ receptors) mediate fast synaptic inhibition in the central nervous system. They are of particular pharmacological importance and are targets for drugs used to treat mental disorders or to modulate sleep and mood. Sorting, transport and degradation of neurotransmitter receptors are important for the construction and maintenance of functional synapses and are fundamental processes to modulate synaptic plasticity. The GABA$_{A}$ receptor-associated protein (GABARAP) belongs to the MAP-LC3 protein family which is involved in vesicular transport processes, like autophagocytosis and intra-golgi transport. GABARAP binds to a GABA$_{A}$ receptor subunit and participates in its transport events. The exact mechanisms and the function of this interaction are not yet known. The objective of the present work was the i dentificati on of artificial peptide and physiological protein ligands of GABARAP and the characterisation of the resulting interactions. This enhances on the one hand the understanding of the binding mechanisms of any ligand to GABARAP and on the other hand, can facilitate the design of new drugs capable of modulating the function of GABARAP or the GABA$_{A}$ receptor. The knowledge of new GABARAP interaction partners additionally contributes to a better understanding of the cellular functions of GABARAP and the procedures at the postsynaptic membrane. By employing a phage display selection procedure, artificial high-affinity peptide ligands of GABARAP could successfully be identified. The resulting interactions could be characterised via enzyme-linked immunosorbent assay, fluorescence titration, surface-plasmon resonance and nuclear magnetic resonance spectroscopy. A consensus motif was derived from the GABARAP-binding peptide sequences. It was used to search protein databases to identify putative GABARAP-binding proteins. For the first time, the human cellular proteins calreticulin (CRT) and clathrin heavy chain (CHC) were identified to bind GABARAP. The corresponding interactions could be characterised via surface plasmon resonance, pulldown analysis and nuclear magnetic resonance spectroscopy. The results of the present work make a substantial contribution to an enhanced understanding of the GABARAP binding specificity and its cellular function

Inhibition of Polyglutamine Misfolding with D-Enantiomeric Peptides Identified by Mirror Image Phage Display Selection

Nine heritable diseases are known that are caused by unphysiologically elongated polyglutamine tracts in human proteins leading to misfolding, aggregation and neurodegeneration. Current therapeutic strategies include efforts to inhibit the expression of the respective gene coding for the polyglutamine-containing proteins. There are, however, concerns that this may interfere with the physiological function of the respective protein. We aim to stabilize the protein’s native conformation by D-enantiomeric peptide ligands to prevent misfolding and aggregation, shift the equilibrium between aggregates and monomers towards monomers and dissolve already existing aggregates into non-toxic and functional monomers. Here, we performed a mirror image phage display selection on the polyglutamine containing a fragment of the androgen receptor. An elongated polyglutamine tract in the androgen receptor causes spinal and bulbar muscular atrophy (SBMA). The selected D-enantiomeric peptides were tested for their ability to inhibit polyglutamine-induced androgen receptor aggregation. We identified D-enantiomeric peptide QF2D-2 (sqsqwstpqGkwshwprrr) as the most promising candidate. It binds to an androgen receptor fragment with 46 consecutive glutamine residues and decelerates its aggregation, even in seeded experiments. Therefore, QF2D-2 may be a promising drug candidate for SBMA treatment or even for all nine heritable polyglutamine diseases, since its aggregation-inhibiting property was shown also for a more general polyglutamine target

In Vitro Reconstitution of the Highly Active and Natively Folded Recombinant Human Superoxide Dismutase 1 Holoenzyme

SOD1 is an antioxidant enzyme that exists as a highly stable dimer in healthy humans. Each subunit contains an intramolecular disulfide bond and coordinates one zinc and one copper ion. The dimer is destabilized in the absence of the ions and disruption of the disulfide bond, which leads to the formation of small oligomers and subsequently larger insoluble aggregates. An acquired toxic function of destabilized SOD1 is postulated to be associated with amyotrophic lateral sclerosis (ALS), which is a neurodegenerative disease characterized by peripheral and central paralysis and by 3‐ to 5‐year median survival after diagnosis. In this study, we present a protocol for heterologous expression of human SOD1 in E. coli and total reconstitution of the holoenzyme, which exhibits the highest reported specific activity (four‐fold higher) of recombinant hSOD1. Biophysical characterization confirms the native state of this protein. The presented protocol provides highly active hSOD1 that will benefit in vitro investigations of this protein

A So-Far Overlooked Secondary Conformation State in the Binding Mode of SARS-CoV-2 Spike Protein to Human ACE2 and Its Conversion Rate Are Crucial for Estimating Infectivity Efficacy of the Underlying Virus Variant

Since its outbreak in 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread with high transmission efficiency across the world, putting health care as well as economic systems under pressure. During the course of the pandemic, the originally identified SARS-CoV-2 variant has been multiple times replaced by various mutant versions, which showed enhanced fitness due to increased infection and transmission rates. In order to find an explanation for why SARS-CoV-2 and its emerging mutated versions showed enhanced transmission efficiency compared with SARS-CoV (2002), an enhanced binding affinity of the spike protein to human angiotensin converting enzyme 2 (hACE2) has been proposed by crystal structure analysis and was identified in cell culture models. Kinetic analysis of the interaction of various spike protein constructs with hACE2 was considered to be best described by a Langmuir-based 1:1 stoichiometric interaction. However, we demonstrate in this report that the SARS-CoV-2 spike protein interaction with hACE2 is best described by a two-step interaction, which is defined by an initial binding event followed by a slower secondary rate transition that enhances the stability of the complex by a factor of ~190 (primary versus secondary state) with an overall equilibrium dissociation constant (KD) of 0.20 nM. In addition, we show that the secondary rate transition is not only present in SARS-CoV-2 wild type (“wt”; Wuhan strain) but also found in the B.1.1.7 variant, where its transition rate is 5-fold increased.IMPORTANCE The current SARS-CoV-2 pandemic is characterized by the high infectivity of SARS-CoV-2 and its derived variants of concern (VOCs). It has been widely assumed that the reason for its increased cell entry compared with SARS-CoV (2002) is due to alterations in the viral spike protein, where single amino acid residue substitutions can increase affinity for hACE2. So far, the interaction of a single unit of the CoV-2 spike protein has been described using the 1:1 Langmuir interaction kinetic. However, we demonstrate here that there is a secondary state binding step that may be essential for novel VOCs in order to further increase their infectivity. These findings are important for quantitatively understanding the infection process of SARS-CoV-2 and characterization of emerging SARS-CoV-2 variants of spike proteins. Thus, they provide a tool for predicting the potential infectivity of the respective viral variants based on secondary rate transition and secondary complex stability

Development of an α-synuclein fibril and oligomer specific tracer for diagnosis of Parkinson's disease, dementia with Lewy bodies and multiple system atrophy

The development of specific disease-associated PET tracers is one of the major challenges, the realization of which in neurodegenerative diseases would enable not only the efficiency of diagnosis but also support the development of disease-modifying therapeutics. Parkinson's disease (PD) is the most common neurodegenerative movement disorder and is characterized by neuronal fibrillary inclusions composed of aggregated α-synuclein (α-syn). However, these deposits are not only found in PD, but also in other related diseases such as multiple system atrophy (MSA) and dementia with Lewy bodies (DLB), which are grouped under the term synucleinopathies. In this study, we used NGS-guided phage display selection to identify short peptides that bind aggregated α-syn. By surface plasmon resonance (SPR)-based affinity screening, we identified the peptide SVLfib-5 that recognizes aggregated α-syn with high complex stability and sequence specificity. Further analysis SPR showed that SVLfib-5 is not only specific for aggregated α-syn, but in particular recognizes fibrillary and oligomeric structures. Moreover, fluorescence microscopy of human brain tissue sections from PD, MSA, and DLB patients with SVLfib-5 allowed specific recognition of α-syn and a clear discrimination between diseased and non-diseased samples. These findings provide the basis for the further development of an α-syn PET tracer for early diagnosis and monitoring of disease progression and therapy progress

In Vitro Reconstitution of the Highly Active and Natively Folded Recombinant Human Superoxide Dismutase 1 Holoenzyme

SOD1 is an antioxidant enzyme that exists as a highly stable dimer in healthy humans. Each subunit contains an intramolecular disulfide bond and coordinates one zinc and one copper ion. The dimer is destabilized in the absence of the ions and disruption of the disulfide bond, which leads to the formation of small oligomers and subsequently larger insoluble aggregates. An acquired toxic function of destabilized SOD1 is postulated to be associated with amyotrophic lateral sclerosis (ALS), which is a neurodegenerative disease characterized by peripheral and central paralysis and by 3‐ to 5‐year median survival after diagnosis. In this study, we present a protocol for heterologous expression of human SOD1 in E. coli and total reconstitution of the holoenzyme, which exhibits the highest reported specific activity (four‐fold higher) of recombinant hSOD1. Biophysical characterization confirms the native state of this protein. The presented protocol provides highly active hSOD1 that will benefit in vitro investigations of this protein

Sequence-specific 1^{1}H, 15^{15}N, and 13^{13}C resonance assignments of the autophagy-related protein LC3_{3}C

Autophagy is a versatile catabolic pathway for lysosomal degradation of cytoplasmic material. While the phenomenological and molecular characteristics of autophagic non-selective (bulk) decomposition have been investigated for decades, the focus of interest is increasingly shifting towards the selective mechanisms of autophagy. Both, selective as well as bulk autophagy critically depend on ubiquitin-like modifiers belonging to the Atg8 (autophagy-related 8) protein family. During evolution, Atg8 has diversified into eight different human genes. While all human homologues participate in the formation of autophagosomal membrane compartments, microtubule-associated protein light chain 3C (LC3C) additionally plays a unique role in selective autophagic clearance of intracellular pathogens (xenophagy), which relies on specific protein–protein recognition events mediated by conserved motifs. The sequence-specific 1H, 15N, and 13C resonance assignments presented here form the stepping stone to investigate the high-resolution structure and dynamics of LC3C and to delineate LC3C’s complex network of molecular interactions with the autophagic machinery by NMR spectroscopy