MACC1 regulates clathrin-mediated endocytosis and receptor recycling of transferrin receptor and EGFR in colorectal cancer

Metastasis Associated in Colon Cancer 1 (MACC1) is a novel prognostic, predictive and causal biomarker for tumor progression and metastasis in many cancer types, including colorectal cancer. Besides its clinical value, little is known about its molecular function. Its similarity to SH3BP4, involved in regulating uptake and recycling of transmembrane receptors, suggests a role of MACC1 in endocytosis. By exploring the MACC1 interactome, we identified the clathrin-mediated endocytosis (CME)-associated proteins CLTC, DNM2 and AP-2 as MACC1 binding partners. We unveiled a MACC1-dependent routing of internalized transferrin receptor towards recycling. Elevated MACC1 expression caused also the activation and internalization of EGFR, a higher rate of receptor recycling, as well as earlier and stronger receptor activation and downstream signaling. These effects are limited by deletion of CME-related protein interaction sites in MACC1. Thus, MACC1 regulates CME and receptor recycling, causing increased growth factor-mediated downstream signaling and cell proliferation. This novel mechanism unveils potential therapeutic intervention points restricting MACC1-driven metastasis

Validation of a Saliva-Based Test for the Molecular Diagnosis of SARS-CoV-2 Infection

Background. Since the beginning of the pandemic, clinicians and researchers have been searching for alternative tests to improve the screening and diagnosis of the SARS-CoV-2 infection. Currently, the gold standard for virus identification is the nasopharyngeal (NP) swab. Saliva samples, however, offer clear, practical, and logistical advantages but due to a lack of collection, transport, and storage solutions, high-throughput saliva-based laboratory tests are difficult to scale up as a screening or diagnostic tool. With this study, we aimed to validate an intralaboratory molecular detection method for SARS-CoV-2 on saliva samples collected in a new storage saline solution, comparing the results to NP swabs to determine the difference in sensitivity between the two tests. Methods. In this study, 156 patients (cases) and 1005 asymptomatic subjects (controls) were enrolled and tested simultaneously for the detection of the SARS-CoV-2 viral genome by RT-PCR on both NP swab and saliva samples. Saliva samples were collected in a preservative and inhibiting saline solution (Biofarma Srl). Internal method validation was performed to standardize the entire workflow for saliva samples. Results. The identification of SARS-CoV-2 conducted on saliva samples showed a clinical sensitivity of 95.1% and specificity of 97.8% compared to NP swabs. The positive predictive value (PPV) was 81% while the negative predictive value (NPV) was 99.5%. Test concordance was 97.6% (Cohen's Kappa=0.86; 95% CI 0.81-0.91). The LoD of the test was 5 viral copies for both samples. Conclusions. RT-PCR assays conducted on a stored saliva sample achieved similar performance to those on NP swabs, and this may provide a very effective tool for population screening and diagnosis. Collection of saliva in a stabilizing solution makes the test more convenient and widely available; furthermore, the denaturing properties of the solution reduce the infective risks belonging to sample manipulation

PA28αβ reduces size and increases hydrophilicity of 20S immunoproteasome peptide products

The specific roles that immunoproteasome variants play in MHC class I antigen presentation are unknown at present. To investigate the biochemical properties of different immunoproteasome forms and unveil the molecular mechanisms of PA28 activity, we performed in vitro degradation of full-length proteins by 20S, 26S, and PA28{alpha}{beta}-20S immunoproteasomes and analyzed the spectrum of peptides released. Notably, PA28{alpha}{beta}-20S immunoproteasomes hydrolyze proteins at the same low rates as 20S alone, which is in line with PA28, neither stimulating nor preventing entry of unfolded polypeptides into the core particle. Most importantly, binding of PA28{alpha}{beta} to 20S greatly reduces the size of proteasomal products and favors the release of specific, more hydrophilic, longer peptides. Hence, PA28{alpha}{beta} may either allosterically modify proteasome active sites or act as a selective "smart" sieve that controls the efflux of products from the 20S proteolytic chamber

DCAF8, a novel MuRF1 interaction partner, promotes muscle atrophy

The muscle-specific RING-finger protein MuRF1 constitutes a bona fide ubiquitin ligase that routes proteins like Myosin heavy chain (MyHC) to proteasomal degradation during muscle atrophy. In two unbiased screens we identified DCAF8 as a new MuRF1 binding partner. MuRF1 physically interacts with DCAF8 and both proteins localize to overlapping structures in muscle cells. Noteworthy, similar to MuRF1, DCAF8 levels increase during atrophy and the down-regulation of either protein substantially impedes muscle wasting and MyHC degradation in C2C12 myotubes, a model system for muscle differentiation and atrophy. DCAF proteins typically serve as substrate receptors in Cullin 4-type (Cul4) ubiquitin ligases (CRL) and we demonstrate that DCAF8 and MuRF1 associate with the subunits of such a protein complex. Because genetic downregulation of DCAF8 and inhibition of Cullin activity also impair myotube atrophy in C2C12 cells, our data imply that the DCAF8 promotes muscle wasting by targeting proteins like MyHC as an integral substrate receptor of a CRL4A ubiquitin ligase

The newly identified MEK1 tyrosine phosphorylation target MACC1 is druggable by approved MEK1 inhibitors to restrict colorectal cancer metastasis

Cancer metastasis causes >90% of cancer deaths and remains a major treatment challenge. Here we deciphered the impact of tyrosine phosphorylation of MACC1, a causative driver for cancer metastasis, for cancer cell signaling and novel interventions to restrict cancer metastasis. We identified MACC1 as new MEK1 substrate. MEK1 directly phosphorylates MACC1, leading to accelerated and increased ERK1 activation. Mutating in silico predicted hierarchical MACC1 tyrosine phosphorylation sites abrogates MACC1-induced migration, invasion, and MET expression, a transcriptional MACC1 target. Targeting MEK1 by RNAi or clinically applicable MEK1 inhibitors AZD6244 and GSK1120212 reduces MACC1 tyrosine phosphorylation and restricts MACC1-induced metastasis formation in mice. Although MEK1 levels, contrary to MACC1, are not of prognostic relevance for CRC patients, MEK1 expression was found indispensable for MACC1-induced metastasis. This study identifies MACC1 as new MEK1 substrate for tyrosine phosphorylation decisively impacting cell motility, tumor growth, and metastasis. Thus, MAP kinase signaling is not linear leading to ERK activation, but branches at the level of MEK1. This fundamental finding opens new therapeutic options for targeting the MEK1/MACC1 axis as novel vulnerability in patients at high risk for metastasis. This might be extended from CRC to further solid tumor entities

Schopenhauer on Christ, Suffering and the Negation of the Will

This paper seeks to illuminate Schopenhauer’s notion of the negation or denial of the will by investigating the figure of the saint within his philosophy. We argue that various discussions in Schopenhauer’s works of the possible role of Christ as exemplar reveals an underlying Christological understanding of self-denial as a model for the saint. The connection between Schopenhauer’s approach to Christ and Kant’s Christology in Religion within the Bounds of Mere Reason is also explored. We note how Schopenhauer builds upon on the Kantian demythologization of the Incarnated Christ in order to uncover the moral and existential spirit that underlies Christianity. In addition, we outline how a recognition of the symbolic importance of Christ for Schopenhauer suggests features of the denial of the will that have been overlooked in the literature, involving the intellectual transcendence of suffering through the cultivation of a state of emotional unresponsiveness. It is argued that this reflects a deeper connection between Schopenhauer’s philosophy and Christian thought than is often recognised