A prion fehérje konformációs átalakulásának vizsgálata = Studies on the conformational transition of the prion protein to protease-resistant forms.

Sajnos a 2006.-évi NF OTKA pályázatok keretét drasztikusan csökkentették. Ennek eredményeképpen az én pályázatom is csak az eredeti költségvetés majdnem 20%-t kapta, (ez egy fiatal OTKA PD ösztöndíj nélküli támogatásnak felel meg), ami természetesen az eredeti kutatási terv töredékes megvalósítására sem volt alkalmas. Sikerként könyvelhető el, hogy ennek ellenére sikerült a kutató csoportot elindítani, kulcs technikákat módszereket beállítani a laborban és kritikus kutatási modellrendszereket létrehozni. Sikerült olyan, az endogén prion fehérjére (PrP) knock-out sejtvonalakat kialakítani, amelyek mutáns PrP-ket, illetve különböző tag-gel megjelölt PrP variánsokat fejeznek ki; illetve nemzetközi együttműködés keretében olyan módszereket sikerült kifejleszteni, ami fehérjék poszt-translációs módosítását képesek detektálni. Másik eljárás, amit kidolgoztunk, nem csak a mutáns prion fehérjék feltekeredését tudja követni, de nagy áteresztő képességű fehérje feltekeredési kísérletekben is hasznosan alkalmazható. Ez utóbbi eljárást a Nature Biotechnology tudományos folyóirat is közlésre érdemesnek találta. | Since the budget of 2006 for NF OTKA proposals was drastically reduced, my NF proposal received support of about just 20% of that was planned (that is close to an F type proposal without PD grant). This support was not sufficient to keep any of the aims of the original proposal. Despite all these, we, I think, managed to use effectively the support we gained. The research group as well as key techniques and critical model systems were successfully established. Particularly, cell lines that are knock-out for the endogenous prion protein were developed expressing various mutant PrP-s. Moreover, in an international collaboration, methods were developed for the detection of post translation modifications of proteins. Most importantly, a technique was developed for monitoring the folding of the mutant PrP variants. This method that is also very useful for High Throughput applications was published in Nature Biotechnology

The prion protein family member Shadoo induces spontaneous ionic currents in cultured cells

Some mutant forms of the cellular prion protein (PrPC) carrying artificial deletions or point mutations associated with familial human prion diseases are capable of inducing spontaneous ionic currents across the cell membrane, conferring hypersensitivity to certain antibiotics to a wide range of cultured cells and primary cerebellar granular neurons (CGNs). These effects are abrogated when the wild type (WT) form is co-expressed, suggesting that they might be related to a physiological activity of PrPC. Interestingly, the prion protein family member Shadoo (Sho) makes cells hypersensitive to the same antibiotics as mutant PrP-s, an effect that is diminished by the co-expression of WT-PrP. Here, we report that Sho engages in another mutant PrP-like activity: it spontaneously induces large ionic currents in cultured SH-SY5Y cells, as detected by whole-cell patch clamping. These currents are also decreased by the co-expression of WT-PrP. Furthermore, deletion of the N-terminal (RXXX)8 motif of Sho, mutation of the eight arginine residues of this motif to glutamines, or replacement of the hydrophobic domain by that of PrP, also diminish Sho-induced ionic currents. Our results suggest that the channel activity that is also characteristic to some pathogenic PrP mutants may be linked to a physiological function of Sho

Retinoid Machinery in Distinct Neural Stem Cell Populations with Different Retinoid Responsiveness

Retinoic acid (RA) is present at sites of neurogenesis in both the embryonic and adult brain. While it is widely accepted that RA signaling is involved in the regulation of neural stem cell differentiation, little is known about vitamin A utilization and biosynthesis of active retinoids in the neurogenic niches, or about the details of retinoid metabolism in neural stem cells and differentiating progenies. Here we provide data on retinoid responsiveness and RA production of distinct neural stem cell/neural progenitor populations. In addition, we demonstrate differentiation-related changes in the expression of genes encoding proteins of the retinoid machinery, including components responsible for uptake (Stra6) and storage (Lrat) of vitamin A, transport of retinoids (Rbp4, CrbpI, CrabpI-II), synthesis (Rdh10, Raldh1-4), degradation of RA (Cyp26a1-c1) and RA signaling (Raralpha,beta,gamma, Rxralpha,beta,gamma). We show that both early embryonic neuroectodermal (NE-4C) stem cells and late embryonic or adult derived radial glia like progenitors (RGl cells) are capable to produce bioactive retinoids but respond differently to retinoid signals. However, while neuronal differentiation of RGl cells can not be induced by RA, neuron formation by NE-4C cells is initiated by both RA and RA-precursors (retinol or retinyl acetate). The data indicate that endogenous RA production, at least in some neural stem cell populations, may result in autocrine regulation of neuronal differentiation

The G127V variant of the prion protein interferes with dimer formation in vitro but not in cellulo

Scrapie prion, PrPSc, formation is the central event of all types of transmissible spongiform encephalopathies (TSEs), while the pathway with possible intermediates and their mechanism of formation from the normal isoform of prion (PrP), remains not fully understood. Recently, the G127V variant of the human PrP is reported to render the protein refractory to transmission of TSEs, via a yet unknown mechanism. Molecular dynamics studies suggested that this mutation interferes with the formation of PrP dimers. Here we analyze the dimerization of 127G and 127VPrP, in both in vitro and a mammalian cell culture system. Our results show that while molecular dynamics may capture the features affecting dimerization in vitro, G127V inhibiting dimer formation of PrP, these are not evidenced in a more complex cellular system. © 2021, The Author(s)

BEAR reveals that increased fidelity variants can successfully reduce the mismatch tolerance of adenine but not cytosine base editors

Base editors allow for precision engineering of the genome. Here, the authors present BEAR, a plasmid-based fluorescence assay for the measurement of CBE and ABE activity, to reveal the mechanism underlying their differences and to increase the yield of edited cells with reduced indel background

Expression of the prion protein family member Shadoo causes drug hypersensitivity that is diminished by the coexpression of the wild type prion protein.

The prion protein (PrP) seems to exert both neuroprotective and neurotoxic activities. The toxic activities are associated with the C-terminal globular parts in the absence of the flexible N-terminus, specifically, the hydrophobic domain (HD) or the central region (CR). The wild type prion protein (PrP-WT), having an intact flexible part, exhibits neuroprotective qualities by virtue of diminishing many of the cytotoxic effects of these mutant prion proteins (PrPDeltaHD, PrPDeltaCR) when coexpressed. The prion protein family member Doppel, which possesses a three dimensional fold similar to the C-terminal part of PrP is also harmful to neuronal and other cells in various models; a phenotype that can also be eliminated by the coexpression of PrP-WT. In contrast, another prion protein family member, Shadoo (Sho), a natively disordered protein possessing structural features similar to the flexible N-terminal tail of PrP, exhibits PrP-WT-like protective properties. Here, we report that contrary to expectations, Sho expression in SH-SY5Y or HEK293 cells induces the same toxic phenotype of drug hypersensitivity as PrPDeltaCR. This effect is exhibited in a dose-dependent manner and is also counteracted by the coexpression of PrP-WT. The opposing effects of Shadoo in different model systems revealed here may be explored to help discern the relationship of the various toxic activities of mutant PrP-s with each other and the neurotoxic effects seen in neurodegenerative diseases, such as transmissible spongiform encephalopathy and Alzheimer's

Position-Dependent Sequence Motif Preferences of SpCas9 are Largely Determined by Scaffold-Complementary Spacer Motifs

Streptococcus pyogenes Cas9 (SpCas9) nuclease exhibits considerable position-dependent sequence preferences. The reason behind these preferences is not well understood and is difficult to rationalise, since the protein establishes interactions with the target-spacer duplex in a sequence-independent manner. We revealed here that intramolecular interactions within the single guide RNA (sgRNA), between the spacer and the scaffold, cause most of these preferences. By using in cellulo and in vitro SpCas9 activity assays with systematically designed spacer and scaffold sequences and by analysing activity data from a large SpCas9 sequence library, we show that some long (>8 nucleotides) spacer motifs, that are complementary to the RAR unit of the scaffold, interfere with sgRNA loading, and that some motifs of more than 4 nucleotides, that are complementary to the SL1 unit, inhibit DNA binding and cleavage. Furthermore, we show that intramolecular interactions are present in the majority of the inactive sgRNA sequences of the library, suggesting that they are the most important intrinsic determinants of the activity of the SpCas9 ribonucleoprotein complex. We also found that in pegRNAs, sequences at the 3′ extension of the sgRNA that are complementary to the SL2 unit are also inhibitory to prime editing, but not to the nuclease activity of SpCas9

Membrane Domain Localization and Interaction of the Prion-Family Proteins, Prion and Shadoo with Calnexin

The cellular prion protein (PrP(C)) is renowned for its infectious conformational isoform PrP(Sc), capable of templating subsequent conversions of healthy PrP(C)s and thus triggering the group of incurable diseases known as transmissible spongiform encephalopathies. Besides this mechanism not being fully uncovered, the protein’s physiological role is also elusive. PrP(C) and its newest, less understood paralog Shadoo are glycosylphosphatidylinositol-anchored proteins highly expressed in the central nervous system. While they share some attributes and neuroprotective actions, opposing roles have also been reported for the two; however, the amount of data about their exact functions is lacking. Protein–protein interactions and membrane microdomain localizations are key determinants of protein function. Accurate identification of these functions for a membrane protein, however, can become biased due to interactions occurring during sample processing. To avoid such artifacts, we apply a non-detergent-based membrane-fractionation approach to study the prion protein and Shadoo. We show that the two proteins occupy similarly raft and non-raft membrane fractions when expressed in N2a cells and that both proteins pull down the chaperone calnexin in both rafts and non-rafts. These indicate their possible binding to calnexin in both types of membrane domains, which might be a necessary requisite to aid the inherently unstable native conformation during their lifetime