Structural Studies of Prion Proteins

Pogrešno nabiranje normalnog, staničnog prionskog proteina (PrPC) u patološki oblik nazvan prion (PrPSc) povezano je s razvojem smrtonosnih neurodegenerativnih poremećaja poznatih kao prionske bolesti ili prijenosne spužvaste encefalopatije. Molekulski mehanizam kojim nastaju prioni još uvijek nije poznat te su stoga strukturna istraživanja oba oblika PrP-a vrlo važna za razjašnjavanje mehanizma pogrešnog nabiranja proteina kod prionskih bolesti. U nekim slučajevima prionske bolesti uzrokovane su mutacijama u genu koji kodira za PrP. Strukturna istraživanja mutanata PrP-a omogućuju bolje razumijevanje početnih stadija konformacijskih promjena prilikom spontanog nastajanja priona kod nasljednih oblika prionskih bolesti. U ovome radu dan je pregled najvažnijih rezultata strukturnih istraživanja PrP-a s naglaskom na istraživanja provedena spektroskopijom NMR na mutantima ljudskog PrP-a.The common industrial process of biodiesel production is a sequence of particular unit operations starting with raw material preparation, followed by transesterification reaction stage and ending with reaction mixture separation and treatment. The main product is biodiesel of prescribed quality while the treatment of glycerol and salt containing phase is carried out so that the by-products obtained maximize profitability of the entire undertaking. The biodiesel production process is often analysed by people of various profiles without proper engineering backgrounds, so that qualitative descriptions are prevailing in the literature. Opposite to that approach, promoted in this paper are the chemical engineering aspects of biodiesel production technology. That approach has not attracted adequate attention so far despite the eminently chemical engineering nature rather than the chemical nature of that process. This will be elaborated in a three-part series. In this, the first one, the necessity of replacing fossil fules with renewable fuels is explained and the biodiesel production put into that context. In particular, the characteristics of industrial biodiesel production are delineated along with the physical and chemical characteristics of homogeneously catalysed transesterification. The systematic approach to the scale-up procedure is described as well

Mapping the prion protein distribution in marsupials: insights from comparing opossum with mouse CNS

The cellular form of the prion protein (PrP(C)) is a sialoglycoprotein widely expressed in the central nervous system (CNS) of mammalian species during neurodevelopment and in adulthood. The location of the protein in the CNS may play a role in the susceptibility of a species to fatal prion diseases, which are also known as the transmissible spongiform encephalopathies (TSEs). To date, little is known about PrP(C) distribution in marsupial mammals, for which no naturally occurring prion diseases have been reported. To extend our understanding of varying PrP(C) expression profiles in different mammals we carried out a detailed expression analysis of PrP(C) distribution along the neurodevelopment of the metatherian South American short-tailed opossum (Monodelphis domestica). We detected lower levels of PrP(C) in white matter fiber bundles of opossum CNS compared to mouse CNS. This result is consistent with a possible role for PrP(C) in the distinct neurodevelopment and neurocircuitry found in marsupials compared to other mammalian species

Synthetic prions with novel strain-specified properties

Prions are infectious proteins that possess multiple self-propagating structures. The information for strains and structural specific barriers appears to be contained exclusively in the folding of the pathological isoform, PrP(Sc). Many recent studies determined that de novo prion strains could be generated in vitro from the structural conversion of recombinant (rec) prion protein (PrP) into amyloidal structures. Our aim was to elucidate the conformational diversity of pathological recPrP amyloids and their biological activities, as well as to gain novel insights in characterizing molecular events involved in mammalian prion conversion and propagation. To this end we generated infectious materials that possess different conformational structures. Our methodology for the prion conversion of recPrP required only purified rec full-length mouse (Mo) PrP and common chemicals. Neither infected brain extracts nor amplified PrP(Sc) were used. Following two different in vitro protocols recMoPrP converted to amyloid fibrils without any seeding factor. Mouse hypothalamic GT1 and neuroblastoma N2a cell lines were infected with these amyloid preparations as fast screening methodology to characterize the infectious materials. Remarkably, a large number of amyloid preparations were able to induce the conformational change of endogenous PrPC to harbor several distinctive proteinase-resistant PrP forms. One such preparation was characterized in vivo habouring a synthetic prion with novel strain specified neuropathological and biochemical properties

Structural Studies of Prion Proteins

Pogrešno nabiranje normalnog, staničnog prionskog proteina (PrPC) u patološki oblik nazvan prion (PrPSc) povezano je s razvojem smrtonosnih neurodegenerativnih poremećaja poznatih kao prionske bolesti ili prijenosne spužvaste encefalopatije. Molekulski mehanizam kojim nastaju prioni još uvijek nije poznat te su stoga strukturna istraživanja oba oblika PrP-a vrlo važna za razjašnjavanje mehanizma pogrešnog nabiranja proteina kod prionskih bolesti. U nekim slučajevima prionske bolesti uzrokovane su mutacijama u genu koji kodira za PrP. Strukturna istraživanja mutanata PrP-a omogućuju bolje razumijevanje početnih stadija konformacijskih promjena prilikom spontanog nastajanja priona kod nasljednih oblika prionskih bolesti. U ovome radu dan je pregled najvažnijih rezultata strukturnih istraživanja PrP-a s naglaskom na istraživanja provedena spektroskopijom NMR na mutantima ljudskog PrP-a.The common industrial process of biodiesel production is a sequence of particular unit operations starting with raw material preparation, followed by transesterification reaction stage and ending with reaction mixture separation and treatment. The main product is biodiesel of prescribed quality while the treatment of glycerol and salt containing phase is carried out so that the by-products obtained maximize profitability of the entire undertaking. The biodiesel production process is often analysed by people of various profiles without proper engineering backgrounds, so that qualitative descriptions are prevailing in the literature. Opposite to that approach, promoted in this paper are the chemical engineering aspects of biodiesel production technology. That approach has not attracted adequate attention so far despite the eminently chemical engineering nature rather than the chemical nature of that process. This will be elaborated in a three-part series. In this, the first one, the necessity of replacing fossil fules with renewable fuels is explained and the biodiesel production put into that context. In particular, the characteristics of industrial biodiesel production are delineated along with the physical and chemical characteristics of homogeneously catalysed transesterification. The systematic approach to the scale-up procedure is described as well

In Search for Universal Models. Dimerization of Nitrosobenzenes and Investigation of Some Basic Chemical Concepts

Jedan od načina istraživanja pojedinih kemijskih koncepata temelji se na izboru odgovarajuaeg konkretnog eksperimentalnog ili teoretskog modela na kojem se ispitivani koncept ili učinak dobro razabiru. U epistemologiji se takvom pristupu obično pridaje termin case study. Sustavna istraživanja kemijskog ponašanja C-nitrozo-spojeva, koja su posljednjih godina provedena u našem laboratoriju, pokazala su da se taj molekulski sustav može iskoristiti kao višestruki kemijski model (case study) za paralelno istraživanje niza temeljnih kemijskih koncepata, selektivnosti, samoorganiziranja, reakcijskih mehanizama u čvrstom stanju, fotokromizma i molekulske logike. Iz niza navedenih primjera, koji se odnose na istraživanja u našem laboratoriju u posljednjih osam godina, razabire se univerzalnost molekulskog modela koji uključuje dimerizacije aromatskih nitrozo-spojeva.One of the most exploited approaches in scientific methodology is studying concepts by using a case study. In chemistry, a case study appears in finding a specific molecular and/or reaction system by which the investigated problem can be observed directly “on substance”. Thus, the studied problem becomes a concrete system, rather than an abstract concept. Most case-study models are designed specifically to deepen one or another chemical problem. However, there exist (although rarely) models, which afford their universality, i.e. could serve for modeling of quite different, and even epistemologically independent concepts. Systematic investigations of the chemistry of C-nitroso compounds performed in our Laboratory in last few years, have resulted in the discovery that such molecular system may be used as a model for studying a series of basic chemical concepts; selectivity, self-assembly, solid-state reaction mechanisms, photochromism and molecular logics. Since it is known that C-nitroso compounds under specific conditions dimerize by forming new N=N bond (Scheme 1), the reaction can be used for studying selectivity in the formation of dimers between different nitroso monomers (Fig. 1 and Fig. 2). The system is very convenient for investigation of the structure-selectivity relations.6 It has already been established, especially in the work of Brian Gowenlock, that nitroso compounds in crystal form appear as dimers. These discoveries inspired us to investigate the kinetics and mechanism of dimerization of nitroso monomers in solid state, because the freshly sublimed nitrosoaromatic compounds appear as crystals of monomers. Consequently, this molecular system is a perfect case-study model for concepts of solid-state reactions, self-organizations in the crystalline forms (Fig. 3), as well as for the interrelation of phase transition and chemical reaction (Fig. 6).8,13 Extending our research of self-organization of nitroso compounds to two-dimensional systems (Fig. 4), we have also demonstrated the ability of such molecules to be models for formation of self-assembly bilayers (Fig. 5), and for direct observations of 2D crystallizations.12 The discovery of photodissociation of crystallized dimers observed by the change in color, also demonstrated that this molecular system could be one of the basic models for molecular logic gates (Fig. 7).16 From the presented examples it follows that this molecular model of dimerizations of aromatic nitroso compounds affords its epistemic universality

Structural rearrangements at physiological pH: Nuclear magnetic resonance insights from the V210I human prion protein mutant

A major focus in prion structural biology studies is unraveling the molecular mechanism leading to the structural conversion of PrP(C) to its pathological form, PrP(Sc). In our recent studies, we attempted to understand the early events of the conformational changes leading to PrP(Sc) using as investigative tools point mutations clustered in the open reading frame of the human PrP gene and linked to genetic forms of human prion diseases. In the work presented here, we investigate the effect of pH on the nuclear magnetic resonance (NMR) structure of recombinant human PrP (HuPrP) carrying the pathological V210I mutation responsible for familial Creutzfeldt-Jakob disease. The NMR structure of HuPrP(V210I) determined at pH 7.2 shows the same overall fold as the previously determined structure of HuPrP(V210I) at pH 5.5. It consists of a disordered N-terminal tail (residues 90-124) and a globular C-terminal domain (residues 125-231) comprising three \u3b1-helices and a short antiparallel \u3b2-sheet. Detailed comparison of three-dimensional structures of HuPrP(V210I) at pH 7.2 and 5.5 revealed significant local structural differences, with the most prominent pH-related structural variations clustered in the \u3b1(2)-\u3b1(3) interhelical region, at the interface of the \u3b2(1)-\u3b1(1) loop, in helices \u3b1(1) and \u3b1(3), and in the \u3b2(2)-\u3b1(2) loop region. The detailed analysis of interactions among secondary structure elements suggests a higher degree of structural ordering of HuPrP(V210I) under neutral-pH conditions, thus implying that spontaneous misfolding of PrP(C) may occur under acidic-pH conditions in endosomal compartments

NMR studies of human prion proteins with pathological mutations

Prion diseases belong to a group of fatal neurodegenerative disorders caused by the conversion of the normal cellular prion protein (PrPC) into its pathogenic form (PrPSc). According to the "protein-only hypothesis" PrPSc is the sole component of the infectious agents. One of the key arguments supporting this hypothesis is the link between inherited prion diseases and mutations in the gene coding for human PrP. Several pathogenic mutations leading to familial prion diseases have been identified in the prion protein gene (PRNP) open reading frame. However, it is still largely unknown how these mutations affect the PrPC\u2192PrPSc conversion. Structural studies on PrP variants carrying familial mutations may provide new clues about the molecular mechanism at early stages of the disease. In the current study we have determined a high-resolution 3D structure of the truncated recombinant HuPrP(90-231) containing the pathological Q212P mutation that is associated with a Gerstmann-Str\ue4ussler-Scheinker (GSS) syndrome. In comparison to the other known PrP structures, structure of Q212P mutant shows some unique structural features. The most remarkable differences involve the C-terminal end of the protein and the \u3b22-\u3b12 loop. Spontaneous generation of PrPSc in inherited prion diseases might be due to the disruptions of the hydrophobic core consisting of \u3b22-\u3b12 loop and \u3b13 helix

A single point mutation, a way to prion disease?

Prion diseases or Transmissible Spongiform Encephalopathies (TSE) are a group of fatal neurodegenerative illnesses affecting humans and animals. They are classified into sporadic, genetic and infectious forms. Genetic prion diseases are caused by mutations in the human prion protein gene and include Gerstmann-Straussler-Scheinker (GSS) syndrome, Fatal Familial Insomnia and genetic Creutzfeldt-Jakob disease (CJD). Approximately 10-15% of all TSE cases in humans are associated with mutations. The development of TSEs is associated with the conversion of the cellular prion protein (PrPC) into a misfolded, pathogenic isoform (PrPSc). Our recent NMR studies were focused on structural characterization of different truncated recombinant human (Hu) PrPs carrying the pathological Q212P (90-231, M129) and V210I(90-231, M129) mutations, and protective E219K (90-231, M129) polymorphism. While Q212P mutation is linked to GSS, the V210I mutation is linked to genetic CJD. The naturally occurring E219K polymorphism in the HuPrP is considered to protect against sCJD. We have demonstrated that the determined structures of variants consist of unstructured N-terminal part (residues 90-124) and well defined C-terminal domain (residues 125-228). Analysis and comparison with the structure of the WT HuPrP revealed that although structures share similar global fold, mutations introduces several local structural differences. The observed differences are mostly clustered at the alpha2-alpha3 inter-helical interface and in the beta2-alpha2 loop region. The determined NMR structures offer new insights on the earliest events of the pathogenic conversion process and could be used for the development of antiprion drugs. More recently we have determined solution state structures of V210I (90-231, M129) pathogenic mutation at two different conditions with pH 5.5 and 7.2. The detailed comparison of three-dimensional structures of HuPrP(V210I) at two different pH values revealed that interactions among secondary structure elements have a higher degree of structural ordering under neutral pH conditions, thus implying that spontaneous misfolding of PrPC may occur under acidic-pH conditions in endosomal compartments

NMR studies of human prion proteins with inherited mutations

Prion diseases are fatal neurodegenerative disorders caused by an aberrant accumulation of the misfolded cellular prion protein (PrPC) conformer, denoted as infectious scrapie isoform or PrPSc. Our understanding of the mechanisms by which mutations cause disease remains limited. In this work results of recent high-resolution NMR structural studies on human prion protein variants carrying pathological mutations are presented