Cleavage site-directed antibodies reveal the prion protein in humans is shed by ADAM10 at Y226 and associates with misfolded protein deposits in neurodegenerative diseases.

Proteolytic cell surface release ('shedding') of the prion protein (PrP), a broadly expressed GPI-anchored glycoprotein, by the metalloprotease ADAM10 impacts on neurodegenerative and other diseases in animal and in vitro models. Recent studies employing the latter also suggest shed PrP (sPrP) to be a ligand in intercellular communication and critically involved in PrP-associated physiological tasks. Although expectedly an evolutionary conserved event, and while soluble forms of PrP are present in human tissues and body fluids, for the human body neither proteolytic PrP shedding and its cleavage site nor involvement of ADAM10 or the biological relevance of this process have been demonstrated thus far. In this study, cleavage site prediction and generation (plus detailed characterization) of sPrP-specific antibodies enabled us to identify PrP cleaved at tyrosin 226 as the physiological and apparently strictly ADAM10-dependent shed form in humans. Using cell lines, neural stem cells and brain organoids, we show that shedding of human PrP can be stimulated by PrP-binding ligands without targeting the protease, which may open novel therapeutic perspectives. Site-specific antibodies directed against human sPrP also detect the shed form in brains of cattle, sheep and deer, hence in all most relevant species naturally affected by fatal and transmissible prion diseases. In human and animal prion diseases, but also in patients with Alzheimer`s disease, sPrP relocalizes from a physiological diffuse tissue pattern to intimately associate with extracellular aggregated deposits of misfolded proteins characteristic for the respective pathological condition. Findings and research tools presented here will accelerate novel insight into the roles of PrP shedding (as a process) and sPrP (as a released factor) in neurodegeneration and beyond.

Hypoxia-Induced Adaptations of N-Glycomes and Proteomes in Breast Cancer Cells and Their Secreted Extracellular Vesicles.

The hypoxic tumor microenvironment significantly impacts cellular behavior and intercellular communication, with extracellular vesicles (EVs) playing a crucial role in promoting angiogenesis, metastasis, and host immunosuppression, and presumed cancer progression and metastasis are closely associated with the aberrant surface N-glycan expression in EVs. We hypothesize that hypoxic tumors synthesize specific hypoxia-induced N-glycans in response to or as a consequence of hypoxia. This study utilized nano-LC-MS/MS to integrate quantitative proteomic and N-glycomic analyses of both cells and EVs derived from the MDA-MB-231 breast cancer cell line cultured under normoxic and hypoxic conditions. Whole N-glycome and proteome profiling revealed that hypoxia has an impact on the asparagine N-linked glycosylation patterns and on the glycolysis/gluconeogenesis proteins in cells in terms of altered N-glycosylation for their adaptation to low-oxygen conditions. Distinct N-glycan types, high-mannose glycans like Man3 and Man9, were highly abundant in the hypoxic cells. On the other hand, alterations in the sialylation and fucosylation patterns were observed in the hypoxic cells. Furthermore, hypoxia-induced EVs exhibit a signature consisting of mono-antennary structures and specific N-glycans (H4N3F1S2, H3N3F1S0, and H7N4F3S2; H8N4F1S0 and H8N6F1S2), which are significantly associated with poor prognoses for breast tumors, presumably altering the interactions within the tumor microenvironment to promote tumorigenesis and metastasis. Our findings provide an overview of the N-glycan profiles, particularly under hypoxic conditions, and offer insights into the potential biomarkers for tracking tumor microenvironment dynamics and for developing precision medicine approaches in oncology.

Anchorless risk or released benefit? An updated view on the ADAM10-mediated shedding of the prion protein.

The prion protein (PrP) is a broadly expressed glycoprotein linked with a multitude of (suggested) biological and pathological implications. Some of these roles seem to be due to constitutively generated proteolytic fragments of the protein. Among them is a soluble PrP form, which is released from the surface of neurons and other cell types by action of the metalloprotease ADAM10 in a process termed 'shedding'. The latter aspect is the focus of this review, which aims to provide a comprehensive overview on (i) the relevance of proteolytic processing in regulating cellular PrP functions, (ii) currently described involvement of shed PrP in neurodegenerative diseases (including prion diseases and Alzheimer's disease), (iii) shed PrP's expected roles in intercellular communication in many more (patho)physiological conditions (such as stroke, cancer or immune responses), (iv) and the need for improved research tools in respective (future) studies. Deeper mechanistic insight into roles played by PrP shedding and its resulting fragment may pave the way for improved diagnostics and future therapeutic approaches in diseases of the brain and beyond.

Autosomal dominantly inherited myopathy likely caused by the TNNT1 variant p.(Asp65Ala).

Nemaline myopathies (NEMs) are genetically and clinically heterogenous. Biallelic or monoallelic variants in TNNT1, encoding slow skeletal troponin T1 (TnT1), cause NEM. We report a 2-year-old patient and his mother carrying the heterozygous TNNT1 variant c.194A>C/p.(Asp65Ala) that occurred de novo in the mother. Both had muscle hypotrophy and muscle weakness. Muscle pathology in the proband's mother revealed slow twitch type 1 fiber hypotrophy and fast twitch type 2 fiber hypertrophy that was confirmed by a reduced ratio of slow skeletal myosin to fast skeletal myosin type 2a. Reverse transcription polymerase chain reaction and immunoblotting data demonstrated increased levels of high-molecular-weight TnT1 isoforms in skeletal muscle of the proband's mother that were also observed in some controls. In an overexpression system, complex formation of TnT1-D65A with tropomyosin 3 (TPM3) was enhanced. The previously reported TnT1-E104V and TnT1-L96P mutants showed reduced or no co-immunoprecipitation with TPM3. Our studies support pathogenicity of the TNNT1 p.(Asp65Ala) variant.

Inducing prion protein shedding as a neuroprotective and regenerative approach in pathological conditions of the brain: from theory to facts.

In the last decades, the role of the prion protein (PrP) in neurodegenerative diseases has been intensively investigated, initially in prion diseases of humans (e.g., Creutzfeldt-Jakob disease) and animals (e.g., scrapie in sheep, chronic wasting disease in deer and elk, or "mad cow disease" in cattle). Templated misfolding of physiological cellular prion protein (PrPC) into an aggregation-prone isoform (termed PrP "Scrapie" (PrPSc)), self-replication and spreading of the latter inside the brain and to peripheral tissues, and the associated formation of infectious proteopathic seeds (termed "prions") are among the essential pathogenic mechanisms underlying this group of fatal and transmissible spongiform encephalopathies. Later, key roles of the correctly folded PrPC were identified in more common human brain diseases (such as Alzheimer's disease or Parkinson's disease) associated with the misfolding and/or accumulation of other proteins (such as amyloid-ß, tau or α-synuclein, respectively). PrPC has also been linked with neuroprotective and regenerative functions, for instance in hypoxic/ischemic conditions such as stroke. However, despite a mixed "bouquet" of suggested functions, our understanding of pathological and, especially, physiological roles played by PrPC in the brain and beyond is certainly incomplete. Interactions with various other proteins at the cell surface or within intracellular compartments may account for the functional diversity linked with PrPC. Moreover, conserved endogenous proteolytic processing of PrPC generates several defined PrPC fragments, possibly holding intrinsic functions in physiological and pathological conditions, thus making the "true and complete biology" of this protein more complicated to be elucidated. Here, we focus on one of those released PrPC fragments, namely shed PrP (sPrP), generated by a membrane-proximate ADAM10-mediated cleavage event at the cell surface. Similar to other soluble PrPC fragments (such as the N1 fragment representing PrP's released N-terminal tail upon the major α-cleavage event) or experimentally employed recombinant PrP, sPrP is being suggested to act neuroprotective in Alzheimer's disease and other protein misfolding diseases. Several lines of evidence on extracellular PrPC (fragments) suggest that induction of PrPC release could be a future therapeutic option in various brain disorders. Our recent identification of a substrate-specific approach to stimulate the shedding by ADAM10, based on ligands binding to cell surface PrPC, may further set the stage for research into this direction.

Ligands binding to the prion protein induce its proteolytic release with therapeutic potential in neurodegenerative proteinopathies.

The prion protein (PrPC) is a central player in neurodegenerative diseases, such as prion diseases or Alzheimer's disease. In contrast to disease-promoting cell surface PrPC, extracellular fragments act neuroprotective by blocking neurotoxic disease-associated protein conformers. Fittingly, PrPC release by the metalloprotease ADAM10 represents a protective mechanism. We used biochemical, cell biological, morphological, and structural methods to investigate mechanisms stimulating this proteolytic shedding. Shed PrP negatively correlates with prion conversion and is markedly redistributed in murine brain in the presence of prion deposits or amyloid plaques, indicating a sequestrating activity. PrP-directed ligands cause structural changes in PrPC and increased shedding in cells and organotypic brain slice cultures. As an exception, some PrP-directed antibodies targeting repetitive epitopes do not cause shedding but surface clustering, endocytosis, and degradation of PrPC. Both mechanisms may contribute to beneficial actions described for PrP-directed ligands and pave the way for new therapeutic strategies against currently incurable neurodegenerative diseases.