Receptor Proteins in Selective Autophagy

Autophagy has long been thought to be an essential but unselective bulk degradation pathway. However, increasing evidence suggests selective autophagosomal turnover of a broad range of substrates. Bifunctional autophagy receptors play a key role in selective autophagy by tethering cargo to the site of autophagosomal engulfment. While the identity of molecular components involved in selective autophagy has been revealed at least to some extent, we are only beginning to understand how selectivity is achieved in this process. Here, we summarize the mechanistic and structural basis of receptor-mediated selective autophagy

Autophagosome content profiling using proximity biotinylation proteomics coupled to protease digestion in mammalian cells

The ascorbate peroxidase APEX2 is commonly used to study the neighborhood of a protein of interest by proximity-dependent biotinylation. Here, we describe a protocol for sample processing compatible with immunoblotting and mass spectrometry, suitable to specifically map the content of autophagosomes and potentially other short-lived endomembrane transport vesicles without the need of subcellular fractionation. By combining live-cell biotinylation with pro-teinase K digestion of cell homogenates, proteins enriched in membrane -pro-tected compartments can be readily enriched and identified.For complete details on the use and execution of this protocol, please refer to Zellner et al. (2021)

Proteotoxicity of polyglutamine expansion proteins: Cellular mechanisms and their modulation by molecular chaperones

Proteins are central to all biological processes. To become functionally active, newly synthesized protein chains must fold into unique three-dimensional conformations. A group of proteins, known as molecular chaperones, are essential for protein folding to occur with high efficiency in cells. Their main role is to prevent off-pathway reactions during folding that lead to misfolding and aggregation. A number of human diseases are known to result from aberrant folding reactions. The formation of insoluble protein aggregates in neurons is a hallmark of neurodegenerative diseases including Huntington’s disease (HD). These disorders are though to result from the acquisition of dominant, toxic functions of misfolded proteins. HD is caused by a CAG trinucleotide expansion that results in the expansion of a polyglutamine (polyQ) tract in the protein Huntingtin (Htt). The disorder is characterized by a progressive loss of specific neurons and the formation of inclusions containing aggregated Htt. Aggregate formation is causally linked to the progressive HD neuropathology, though it is not clear whether large insoluble, fibrillar structures or smaller assemblies of Htt are the toxic agents. Toxicity could arise from the recruitment of other polyQ-containing proteins, i.e. transcription factors, into the inclusions resulting in a loss of their normal cellular functions. Here, soluble Htt oligomers have been found to accumulate in the nucleus and to inhibit the function of the transcription factors TBP and CBP in cells. Aberrant interaction of toxic Htt with the benign polyQ repeat of TBP structurally destabilized the transcription factor, independent of the formation of insoluble coaggregates and caused transcriptional dysregulation. Chaperones of the Hsp70 family protect against this deactivation by modulating the conformation of Htt. This protective effect of Hsp70 was found to be based on a cooperation between Hsp70 and the chaperonin TRiC. Both chaperone systems cooperate in eliminating toxic polyQ oligomers, which may resemble the potentially pathogenic, prefibrillar states of other amyloidogenic disease proteins, and in stabilizing mutant Htt in a soluble, oligomeric state that is not associated with toxicity. TRiC and Hsp70 appear to be part of an effective chaperone network preventing the formation of harmful, amyloidogenic proteins species. They act synergistically on Htt, reminiscent of their sequential action in assisting the folding of newly-synthesized proteins

ALS-linked loss of Cyclin-F function affects HSP90

The founding member of the F-box protein family, Cyclin-F, serves as a substrate adaptor for the E3 ligase Skp1-Cul1-F-box (SCF)Cyclin-F which is responsible for ubiquitination of proteins involved in cell cycle progression, DNA damage and mitotic fidelity. Missense mutations in CCNF encoding for Cyclin-F are associated with amyotrophic lateral sclerosis (ALS). However, it remains elusive whether CCNF mutations affect the substrate adaptor function of Cyclin-F and whether altered SCFCyclin-F–mediated ubiquitination contributes to pathogenesis in CCNF mutation carriers. To address these questions, we set out to identify new SCFCyclin-F targets in neuronal and ALS patient–derived cells. Mass spectrometry–based ubiquitinome profiling of CCNF knockout and mutant cell lines as well as Cyclin-F proximity and interaction proteomics converged on the HSP90 chaperone machinery as new substrate candidate. Biochemical analyses provided evidence for a Cyclin-F–dependent association and ubiquitination of HSP90AB1 and implied a regulatory role that could affect the binding of a number of HSP90 clients and co-factors. Together, our results point to a possible Cyclin-F loss-of-function–mediated chaperone dysregulation that might be relevant for ALS

Characterization of pixelated silicon detectors for daily quality assurance measurements in proton therapy

The advanced imaging and delivery techniques in proton therapy allow conformal high-dose irradiation of the target volume with high accuracy using pencil beam scanning or beam shaping apertures. These irradiation methods increasingly include small radiation fields with large dose gradients, which require detector systems with high spatial resolution for quality assurance. In addition the measurement of all success parameters for daily quality assurance with only one proton field and one simple detector system would save a lot of time in clinical usage. Based on their good spatial resolution and high rate compatibility, pixelated silicon detectors could meet the new requirements. To assess their applicability in proton therapy, ATLAS pixelated silicon detectors are used to measure the lateral beam profile with high spatial resolution. Furthermore, a dose dependent detector calibration is presented to allow the measurement of the requested output constancy. A strategy to verify the proton energy during the daily quality assurance is under study. Promising results from proof-of-principle measurements at the West German Proton Therapy Centre in Essen, Germany, have been obtained.Comment: 5 pages, 6 figures, accepted for publication in the proceedings of TIPP 2021 to be published in Journal of Physics: Conference Serie

Direct Detection of Photoinduced Charge Transfer Complexes in Polymer:Fullerene Blends

We report transient electron paramagnetic resonance (trEPR) measurements with sub-microsecond time resolution performed on a P3HT:PCBM blend at low temperature. The trEPR spectrum immediately following photoexcitation reveals signatures of spin-correlated polaron pairs. The pair partners (positive polarons in P3HT and negative polarons in PCBM) can be identified by their characteristic g-values. The fact that the polaron pair states exhibit strong non-Boltzmann population unambiguously shows that the constituents of each pair are geminate, i.e. originate from one exciton. We demonstrate that coupled polaron pairs are present even several microseconds after charge transfer and suggest that they embody the intermediate charge transfer complexes which form at the donor/acceptor interface and mediate the conversion from excitons into free charge carriers

Lipid and protein content profiling of isolated native autophagic vesicles

Autophagy is responsible for clearance of an extensive portfolio of cargoes, which are sequestered into vesicles, called autophagosomes, and are delivered to lysosomes for degradation. The pathway is highly dynamic and responsive to several stress conditions. However, the phospholipid composition and protein contents of human autophagosomes under changing autophagy rates are elusive so far. Here, we introduce an antibody-based FACS-mediated approach for the isolation of native autophagic vesicles and ensured the quality of the preparations. Employing quantitative lipidomics, we analyze phospholipids present within human autophagic vesicles purified upon basal autophagy, starvation, and proteasome inhibition. Importantly, besides phosphoglycerides, we identify sphingomyelin within autophagic vesicles and show that the phospholipid composition is unaffected by the different conditions. Employing quantitative proteomics, we obtain cargo profiles of autophagic vesicles isolated upon the different treatment paradigms. Interestingly, starvation shows only subtle effects, while proteasome inhibition results in the enhanced presence of ubiquitin–proteasome pathway factors within autophagic vesicles. Thus, here we present a powerful method for the isolation of native autophagic vesicles, which enabled profound phospholipid and cargo analyses

NMR evaluation of total statin content and HMG-CoA reductase inhibition in red yeast rice (Monascus spp.) food supplements

Background Red yeast rice (i.e., rice fermented with Monascus spp.), as a food supplement, is claimed to be blood cholesterol-lowering. The red yeast rice constituent monacolin K, also known as lovastatin, is an inhibitor of the hydroxymethylglutaryl-CoA (HMG-CoA) reductase. This article aims to develop a sensitive nuclear magnetic resonance (NMR) method to determine the total statin content of red yeast rice products. Methods The total statin content was determined by a 400 MHz 1H NMR spectroscopic method, based on the integration of the multiplet at δ 5.37-5.32 ppm of a hydrogen at the hexahydronaphthalene moiety in comparison to an external calibration with lovastatin. The activity of HMG-CoA reductase was measured by a commercial spectrophotometric assay kit. Results The NMR detection limit for total statins was 6 mg/L (equivalent to 0.3 mg/capsule, if two capsules are dissolved in 50 mL ethanol). The relative standard deviations were consistently lower than 11%. The total statin concentrations of five red yeast rice supplements were between 1.5 and 25.2 mg per specified daily dose. A dose-dependent inhibition of the HMG-CoA reductase enzyme activity by the red yeast rice products was demonstrated. Conclusion A simple and direct NMR assay was developed to determine the total statin content in red yeast rice. The assay can be applied for the determination of statin content for the regulatory control of red yeast rice products

Comprehensive investigation of lateral dose profile and output factor measurements in small proton fields from different delivery techniques

Background and purpose: As a part of the commissioning and quality assurance in proton beam therapy, lateral dose profiles and output factors have to be acquired. Such measurements can be performed with point detectors and are especially challenging in small fields or steep lateral penumbra regions as the detector's volume effect may lead to perturbations. To address this issue, this work aims to quantify and correct for such perturbations of six point detectors in small proton fields created via three different delivery techniques. Methods: Lateral dose profile and output measurements of three proton beam delivery techniques (pencil beam scanning, pencil beam scanning combined with collimators, passive scattering with collimators) were performed using high-resolution EBT3 films, a PinPoint 3D 31022 ionization chamber, a microSilicon diode 60023 and a microDiamond detector 60019 (all PTW Freiburg, Germany). Detector specific lateral dose response functions K(x,y) acting as the convolution kernel transforming the undisturbed dose distribution D(x,y) into the measured signal profiles M(x,y) were applied to quantify perturbations of the six investigated detectors in the proton fields and correct the measurements. A signal theoretical analysis in Fourier space of the dose distributions and detector's K(x,y) was performed to aid the understanding of the measurement process with regard to the combination of detector choice and delivery technique. Results: Quantification of the lateral penumbra broadening and signal reduction at the fields center revealed that measurements in the pencil beam scanning fields are only compromised slightly even by large volume ionization chambers with maximum differences in the lateral penumbra of 0.25 mm and 4% signal reduction at the field center. In contrast, radiation techniques with collimation are not accurately represented by the investigated detectors as indicated by a penumbra broadening up to 1.6 mm for passive scattering with collimators and 2.2 mm for pencil beam scanning with collimators. For a 3 mm diameter collimator field, a signal reduction at field center between 7.6% and 60.7% was asserted. Lateral dose profile measurements have been corrected via deconvolution with the corresponding K(x,y) to obtain the undisturbed D(x,y). Corrected output ratios of the passively scattered collimated fields obtained for the microDiamond, microSilicon and PinPoint 3D show agreement better than 0.9% (one standard deviation) for the smallest field size of 3 mm. Conclusion: Point detector perturbations in small proton fields created with three delivery techniques were quantified and found to be especially pronounced for collimated small proton fields with steep dose gradients. Among all investigated detectors, the microSilicon diode showed the smallest perturbations. The correction strategies based on detector's K(x,y) were found suitable for obtaining unperturbed lateral dose profiles and output factors. Approximation of K(x,y) by considering only the geometrical averaging effect has been shown to provide reasonable prediction of the detector's volume effect. The findings of this work may be used to guide the choice of point detectors in various proton fields and to contribute toward the development of a code of practice for small field proton dosimetry.</p