Absence Of Nucleic Acids Research Articles

Structural basis of archaeal RNA polymerase transcription elongation and Spt4/5 recruitment.

Archaeal transcription is carried out by a multi-subunit RNA polymerase (RNAP) that is highly homologous in structure and function to eukaryotic RNAP II. Among the set of basal transcription factors, only Spt5 is found in all domains of life, but Spt5 has been shaped during evolution, which is also reflected in the heterodimerization of Spt5 with Spt4 in Archaea and Eukaryotes. To unravel the mechanistic basis of Spt4/5 function in Archaea, we performed structure-function analyses using the archaeal transcriptional machinery of Pyrococcus furiosus (Pfu). We report single-particle cryo-electron microscopy reconstructions of apo RNAP and the archaeal elongation complex (EC) in the absence and presence of Spt4/5. Surprisingly, Pfu Spt4/5 also binds the RNAP in the absence of nucleic acids in a distinct super-contracted conformation. We show that the RNAP clamp/stalk module exhibits conformational flexibility in the apo state of RNAP and that the enzyme contracts upon EC formation or Spt4/5 engagement. We furthermore identified a contact of the Spt5-NGN domain with the DNA duplex that stabilizes the upstream boundary of the transcription bubble and impacts Spt4/5 activity in vitro. This study, therefore, provides the structural basis for Spt4/5 function in archaeal transcription and reveals a potential role beyond the well-described support of elongation.

Development of a methodology for large-scale production of prions for biological and structural studies.

Prion diseases are a group of infectious neurodegenerative diseases produced by the conversion of the normal prion protein (PrPC) into the disease-associated form (PrPSc). Extensive evidence indicate that the main or sole component of the infectious agent is PrPSc, which can replicate in affected individuals in the absence of nucleic acids. However, the mechanism of PrPC-to-PrPSc conversion remains elusive, which has been attributed to the lack of sufficient structural information of infectious PrPSc and a reliable system to study prion replication in vitro. In this article we adapted the Protein Misfolding Cyclic Amplification (PMCA) technology for rapid and efficient generation of highly infectious prions in large-scale. Murine prions of the RML strain were efficiently propagated in volumes up to 1,000-fold larger than conventional PMCA. The large-scale PMCA (LS-PMCA) procedure enabled to produce highly infectious prions, which maintain the strain properties of the seed used to begin the reaction. LS-PMCA was shown to work with various species and strains of prions, including mouse RML and 301C strains, hamster Hyper prion, cervid CWD prions, including a rare Norwegian CWD prion, and human CJD prions. We further improved the LS-PMCA into a bioreactor format that can operate under industry-mimicking conditions for continuous and unlimited production of PrPSc without the need to keep adding brain-derived prions. In our estimation, this bioreactor can produce in 1d an amount of prions equivalent to that present in 25 infected animals at the terminal stage of the disease. Our LS-PMCA technology may provide a valuable tool to produce large quantities of well-defined and homogeneous infectious prions for biological and structural studies.

A Mechanistic Overview of the Cellular Pathology and Prion-Like Propagation of α-Synuclein in Parkinson's Disease: A Narrative Review

Introduction: Prions are quaternary protein complexes made up of misfolded protein isoforms (PrPSc) that are able to aggregate and self-replicate in the absence of nucleic acids. They do this by incorporating and inducing the misfolding of normally-folded protein isoforms (PrPC) in a template-directed fashion, before fragmenting and continuing to propagate at increasingly greater concentrations in cells. Parkinson’s disease (PD) is a disease affecting the central nervous system (CNS), primarily characterised by a marked loss of dopaminergic neurons from the pars compacta of the substantia nigra in the midbrain (snPC). Lewy bodies (LBs) and Lewy neurites (LNs) are protein inclusions made primarily of the protein α-synuclein (α-Syn) and are implicated in defective neural signalling and neuronal cell death in PD. LBs/LNs have been hypothesised to cause symptoms of PD via propagation throughout the CNS in a prion-like mechanism. Methods: A narrative literature review was conducted to synthesise current and past research surrounding the prion-like propagation of α-Syn in PD, and models connecting cellular pathology to pathophysiology. Results were critically analysed and implications were determined. Results: α-Syn is a conformationally flexible protein normally involved in presynaptic regulation and dopamine homeostasis. In PD, α-Syn takes on a pathogenic, β-sheet rich conformation resulting from random cellular events or inoculation, that acts in a prion-like manner, inducing the misfolding of normal protein isoforms. Pathogenic α-Syn is propagated between neurons via exosomal secretion and leads to neurotoxicity by loss-of-function causing disruption of dopamine homeostasis, and proteasomal saturation and inhibition. Due to prion-like propagation, and differential susceptibility of neurons to pathogenic α-Syn-mediated neurotoxicity, models of PD progression and symptomatology have been suggested with differing degrees of success. Discussion: This narrative review aims to build on previous knowledge by clearly describing and evaluating the mechanisms of prion-like propagation and neurotoxicity of pathogenic α-Syn in PD, comparing them to traditional prion mechanisms observed for PrPSc, and models of PD which connect cellular pathology with pathophysiology. Conclusion: This review provides insight into the cellular mechanisms behind PD and can be used to propel research in this areas via identification of future areas of inquiry and pharmacological targets, among others.

Synthesis, Self‐Assembly, and Nucleic Acid Recognition of an Acylhydrazone‐Conjugated Cationic Tetraphenylethene Ligand

AbstractSupramolecular polymers are of interest in the pursuit of multivalent nucleic acids recognition. However, their formation often relies on non‐covalent forces that are also at play in the interaction with nucleic acids. In this work, we designed a novel compound (TPE‐Gir) combining a tetraphenylethene aromatic core tethered to four quaternary ammoniums through acylhydrazone spacers, and we investigated in detail its self‐assembly and interaction with different types of nucleic acids. The spectroscopic analyses indicate the self‐assembly of regular fluorescent nanoparticles (observed by DLS and TEM) in the absence of nucleic acids, the strong propensity to intercalate into single‐stranded DNA, the ability to bind into the minor groove of double‐stranded DNA, and the selective binding to G‐quadruplex (G4) structures by fitting within a wide G4‐groove. Those recognition events are quantified by isothermal titration calorimetry and the proposed binding models are supported by docking simulations.

Constant pH molecular dynamics of porcine circovirus 2 capsid protein reveals a mechanism for capsid assembly.

Spatiotemporal regulation of viral capsid assembly ensures the selection of the viral genome for encapsidation. The porcine circovirus 2 is the smallest autonomously replicating pathogenic virus, yet how PCV2 capsid assembly is regulated to occur within the nucleus remains unknown. We report that pure PCV2 capsid proteins, in the absence of nucleic acids, require acidic conditions to assemble into empty capsids in vitro. By employing constant pH replica exchange molecular dynamics, we unveil the atomistic mechanism of pH-dependency for capsid assembly. The results show that an appropriate protonation configuration for a cluster of acidic amino acids is necessary to appropriately position the GH-loop for driving the capsid assembly. We demonstrate that assembly is prohibited at neutral pH because deprotonation of these residues results in their electrostatic repulsion, shifting the GH-loop to a position incompatible with capsid assembly. We propose that encapsulation of nucleic acids overcomes this repulsion to suitably position the GH-loop. Our findings provide the first atomic resolution mechanism of capsid assembly regulation. These findings are useful for the development of therapeutics that inhibit PCV2 self-assembly.

Assembly of Capsids from Hepatitis B Virus Core Protein Progresses through Highly Populated Intermediates in the Presence and Absence of RNA.

The genetic material of viruses is protected by protein shells that are assembled from a large number of subunits in a process that is efficient and robust. Many of the mechanistic details underpinning efficient assembly of virus capsids are still unknown. The assembly mechanism of hepatitis B capsids has been intensively researched using a truncated core protein lacking the C-terminal domain responsible for binding genomic RNA. To resolve the assembly intermediates of hepatitis B virus (HBV), we studied the formation of nucleocapsids and empty capsids from full-length hepatitis B core proteins, using time-resolved small-angle X-ray scattering. We developed a detailed structural model of the HBV capsid assembly process using a combination of analysis with multivariate curve resolution, structural modeling, and Bayesian ensemble inference. The detailed structural analysis supports an assembly pathway that proceeds through the formation of two highly populated intermediates, a trimer of dimers and a partially closed shell consisting of around 40 dimers. These intermediates are on-path, transient and efficiently convert into fully formed capsids. In the presence of an RNA oligo that binds specifically to the C-terminal domain the assembly proceeds via a similar mechanism to that in the absence of nucleic acids. Comparisons between truncated and full-length HBV capsid proteins reveal that the unstructured C-terminal domain has a significant impact on the assembly process and is required to obtain a more complete mechanistic understanding of HBV capsid formation. These results also illustrate how combining scattering information from different time-points during time-resolved experiments can be utilized to derive a structural model of protein self-assembly pathways.

Full atomistic model of prion structure and conversion.

Prions are unusual protein assemblies that propagate their conformationally-encoded information in absence of nucleic acids. The first prion identified, the scrapie isoform (PrPSc) of the cellular prion protein (PrPC), caused epidemic and epizootic episodes [1]. Most aggregates of other misfolding-prone proteins are amyloids, often arranged in a Parallel-In-Register-β-Sheet (PIRIBS) [2] or β-solenoid conformations [3]. Similar folding models have also been proposed for PrPSc, although none of these have been confirmed experimentally. Recent cryo-electron microscopy (cryo-EM) and X-ray fiber-diffraction studies provided evidence that PrPSc is structured as a 4-rung β-solenoid (4RβS) [4, 5]. Here, we combined different experimental data and computational techniques to build the first physically-plausible, atomic resolution model of mouse PrPSc, based on the 4RβS architecture. The stability of this new PrPSc model, as assessed by Molecular Dynamics (MD) simulations, was found to be comparable to that of the prion forming domain of Het-s, a naturally-occurring β-solenoid. Importantly, the 4RβS arrangement allowed the first simulation of the sequence of events underlying PrPC conversion into PrPSc. This study provides the most updated, experimentally-driven and physically-coherent model of PrPSc, together with an unprecedented reconstruction of the mechanism underlying the self-catalytic propagation of prions.

Loop-Mediated Isothermal Amplification (LAMP): A Rapid and Sensitive Tool for Quality Assessment of Meat Products.

Loop-mediated isothermal amplification (LAMP) is a novel method that amplifies target nucleic acids under isothermal conditions. It is a rapid, specific, and sensitive method, which does not require costly thermal cyclers for the detection of nucleic acids. Thus, it is suitable for on-site detection assays under low-resource settings. It can also be integrated on compact lab-on-a-chip devices for the development of micro-total analysis systems. This review discusses LAMP-based methods, as well as LAMP-based centrifugal, microfluidic, and other fluid-handling devices, which have been developed for the assessment of meat quality parameters that are related to the presence or absence of nucleic acids, for example, animal species identification and microbiological quality. Advances in improving the rapidity, specificity, and sensitivity of LAMP techniques for the assessment of these meat quality parameters are also discussed in this review.

Charge neutralization as the major factor for the assembly of nucleocapsid-like particles from C-terminal truncated hepatitis C virus core protein.

BackgroundHepatitis C virus (HCV) core protein, in addition to its structural role to form the nucleocapsid assembly, plays a critical role in HCV pathogenesis by interfering in several cellular processes, including microRNA and mRNA homeostasis. The C-terminal truncated HCV core protein (C124) is intrinsically unstructured in solution and is able to interact with unspecific nucleic acids, in the micromolar range, and to assemble into nucleocapsid-like particles (NLPs) in vitro. The specificity and propensity of C124 to the assembly and its implications on HCV pathogenesis are not well understood.MethodsSpectroscopic techniques, transmission electron microscopy and calorimetry were used to better understand the propensity of C124 to fold or to multimerize into NLPs when subjected to different conditions or in the presence of unspecific nucleic acids of equivalent size to cellular microRNAs.ResultsThe structural analysis indicated that C124 has low propensity to self-folding. On the other hand, for the first time, we show that C124, in the absence of nucleic acids, multimerizes into empty NLPs when subjected to a pH close to its isoelectric point (pH ≈ 12), indicating that assembly is mainly driven by charge neutralization. Isothermal calorimetry data showed that the assembly of NLPs promoted by nucleic acids is enthalpy driven. Additionally, data obtained from fluorescence correlation spectroscopy show that C124, in nanomolar range, was able to interact and to sequester a large number of short unspecific nucleic acids into NLPs.DiscussionTogether, our data showed that the charge neutralization is the major factor for the nucleocapsid-like particles assembly from C-terminal truncated HCV core protein. This finding suggests that HCV core protein may physically interact with unspecific cellular polyanions, which may correspond to microRNAs and mRNAs in a host cell infected by HCV, triggering their confinement into infectious particles.

Prions and prion-like pathogens in neurodegenerative disorders.

Prions are unique elements in biology, being able to transmit biological information from one organism to another in the absence of nucleic acids. They have been identified as self-replicating proteinaceous agents responsible for the onset of rare and fatal neurodegenerative disorders—known as transmissible spongiform encephalopathies, or prion diseases—which affect humans and other animal species. More recently, it has been proposed that other proteins associated with common neurodegenerative disorders, such as Alzheimer’s and Parkinson’s disease, can self-replicate like prions, thus sustaining the spread of neurotoxic entities throughout the nervous system. Here, we review findings that have contributed to expand the prion concept, and discuss if the involved toxic species can be considered bona fide prions, including the capacity to infect other organisms, or whether these pathogenic aggregates share with prions only the capability to self-replicate.

Investigation of the structure and dynamics of the capsid-spacer peptide 1-nucleocapsid fragment of the HIV-1 gag polyprotein by solution NMR spectroscopy.

Structural studies of HIV-1 Gag, the primary structural polyprotein involved in retroviral assembly, have been challenging, owing to its flexibility and conformational heterogeneity. Using residual dipolar couplings, we show that the four structural units of the capsid (CA)-spacer peptide 1 (SP1)-nucleocapsid (NC) fragment of HIV-1 Gag (namely, the N- and C-terminal domains of capsid, and the N- and C-terminal Zn knuckles of nucleocapsid) have the same structures as their individually isolated counterparts, and tumble semi-independently of one another in the absence of nucleic acids. Nucleic acids bind exclusively to the nucleocapsid domain and fix the orientation of the two Zn knuckles relative to one another so that the nucleocapsid domain/nucleic acid complex behaves as a single structural unit. The low (15) N-{(1) H} heteronuclear NOE values (≤0.4), the close to zero values for the residual dipolar couplings of the backbone amides, and minimal deviations from random-coil chemical shifts for the C-terminal tail of capsid and SP1, both in the absence and presence of nucleic acids, indicate that these regions are intrinsically disordered in the context of CA-SP1-NC.

Investigation of the Structure and Dynamics of the Capsid-Spacer Peptide 1-Nucleocapsid Fragment of the HIV-1 Gag Polyprotein by Solution NMR Spectroscopy

Structural studies of HIV-1 Gag, the primary structural polyprotein involved in retroviral assembly, have been challenging, owing to its flexibility and conformational heterogeneity. Using residual dipolar couplings, we show that the four structural units of the capsid (CA)–spacer peptide 1 (SP1)–nucleocapsid (NC) fragment of HIV-1 Gag (namely, the N- and C-terminal domains of capsid, and the N- and C-terminal Zn knuckles of nucleocapsid) have the same structures as their individually isolated counterparts, and tumble semi-independently of one another in the absence of nucleic acids. Nucleic acids bind exclusively to the nucleocapsid domain and fix the orientation of the two Zn knuckles relative to one another so that the nucleocapsid domain/nucleic acid complex behaves as a single structural unit. The low 15N–{1H} heteronuclear NOE values (≤0.4), the close to zero values for the residual dipolar couplings of the backbone amides, and minimal deviations from random-coil chemical shifts for the C-terminal tail of capsid and SP1, both in the absence and presence of nucleic acids, indicate that these regions are intrinsically disordered in the context of CA–SP1–NC.

Fluorescent primuline derivatives inhibit hepatitis C virus NS3-catalyzed RNA unwinding, peptide hydrolysis and viral replicase formation

The hepatitis C virus (HCV) multifunctional nonstructural protein 3 (NS3) is a protease that cleaves viral and host proteins and a helicase that separates DNA and RNA structures in reactions fueled by ATP hydrolysis. Li et al. (2012) recently synthesized a series of new NS3 helicase inhibitors from the benzothiazole dimer component of the fluorescent yellow dye primuline. This study further characterizes a subset of these primuline derivatives with respect to their specificity, mechanism of action, and effect on cells harboring HCV subgenomic replicons. All compounds inhibited DNA and RNA unwinding catalyzed by NS3 from different HCV genotypes, but only some inhibited the NS3 protease function, and few had any effect on HCV NS3 catalyzed ATP hydrolysis. A different subset contained potent inhibitors of RNA stimulated ATP hydrolysis catalyzed by the related NS3 protein from Dengue virus. In assays monitoring intrinsic protein fluorescence in the absence of nucleic acids, the compounds cooperatively bound NS3 with Kds that reflect their potency in assays. The fluorescent properties of the primuline derivatives both in vitro and in cells are also described. The primuline derivative that was the most active against subgenomic replicons in cells caused a 14-fold drop in HCV RNA levels (IC50=5±2μM). In cells, the most effective primuline derivative did not inhibit the cellular activity of NS3 protease but disrupted HCV replicase structures.

Isolation of phosphatidylethanolamine as a solitary cofactor for prion formation in the absence of nucleic acids

Infectious prions containing the pathogenic conformer of the mammalian prion protein (PrP(Sc)) can be produced de novo from a mixture of the normal conformer (PrP(C)) with RNA and lipid molecules. Recent reconstitution studies indicate that nucleic acids are not required for the propagation of mouse prions in vitro, suggesting the existence of an alternative prion propagation cofactor in brain tissue. However, the identity and functional properties of this unique cofactor are unknown. Here, we show by purification and reconstitution that the molecule responsible for the nuclease-resistant cofactor activity in brain is endogenous phosphatidylethanolamine (PE). Synthetic PE alone facilitates conversion of purified recombinant (rec)PrP substrate into infectious recPrP(Sc) molecules. Other phospholipids, including phosphatidylcholine, phosphatidylserine, phosphatidylinositol, and phosphatidylglycerol, were unable to facilitate recPrP(Sc) formation in the absence of RNA. PE facilitated the propagation of PrP(Sc) molecules derived from all four different animal species tested including mouse, suggesting that unlike RNA, PE is a promiscuous cofactor for PrP(Sc) formation in vitro. Phospholipase treatment abolished the ability of brain homogenate to reconstitute the propagation of both mouse and hamster PrP(Sc) molecules. Our results identify a single endogenous cofactor able to facilitate the formation of prions from multiple species in the absence of nucleic acids or other polyanions.

Polymerization and nucleic acid-binding properties of human L1 ORF1 protein

The L1 (LINE 1) retrotransposable element encodes two proteins, ORF1p and ORF2p. ORF2p is the L1 replicase, but the role of ORF1p is unknown. Mouse ORF1p, a coiled-coil-mediated trimer of ∼42-kDa monomers, binds nucleic acids and has nucleic acid chaperone activity. We purified human L1 ORF1p expressed in insect cells and made two findings that significantly advance our knowledge of the protein. First, in the absence of nucleic acids, the protein polymerizes under the very conditions (0.05 M NaCl) that are optimal for high (∼1 nM)-affinity nucleic acid binding. The non-coiled-coil C-terminal half mediates formation of the polymer, an active conformer that is instantly resolved to trimers, or multimers thereof, by nucleic acid. Second, the protein has a biphasic effect on mismatched double-stranded DNA, a proxy chaperone substrate. It protects the duplex from dissociation at 37°C before eventually melting it when largely polymeric. Therefore, polymerization of ORF1p seemingly affects its interaction with nucleic acids. Additionally, polymerization of ORF1p at its translation site could explain the heretofore-inexplicable phenomenon of cis preference—the favored retrotransposition of the actively translated L1 transcript, which is essential for L1 survival.

Purification of M-MLVH- RT on a 9-Aminoethyladenine-(1,6-diamine-hexane)-triazine Selected from a Combinatorial Library of dNTP-Mimetic Ligands

Reverse transcriptase (RT) catalyzes the formation of dsDNA from single-stranded retroviral RNA genome. This enzyme is unique among DNA polymerases in its ability to use either RNA or DNA as a template. Moloney Murine Leukemia virus reverse transcriptase lacking RNase H activity (M-MLVH- RT) especially holds particular interest because of its ability to eliminate the deleterious effect of RNase H, which results in more efficient synthesis of full-length cDNA from mRNA. Therefore, the development of a simple purification method attracts the attention of retroviral drug and enzyme researchers and manufacturers. The present work is the first purification example of a non-tagged (native) RT by affinity chromatography using synthetic affinity ligands. In this study, the ligand was selected from a structure-biased combinatorial library of dNTP-mimetic ligands, and it was evaluated for its ability to bind and purify M-MLVH- RT from inclusion bodies of recombinant E. coli. The selected ligand (AEAd), bearing 9-aminoethyladenine and 1,6-diamine-hexane both linked on the same triazine scaffold, displayed the highest enzyme purifying ability after applying mild desorption conditions (6 mM MnCl(2) in 20 mM Tris-HCl buffer, pH 7.5). The binding capacity of immobilized AEAd with M-MLVH- RT was determined to be equal to approximately 1 mg enzyme/g moist weight gel. Adsorption studies with immobilized AEAd and soluble M-MLVH- RT demonstrated that the formation of the respective complex was perturbed by ATP. Quality control tests of the purified M-MLVH- RT essentially showed a single band (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and absence of nucleic acids and contaminating nuclease activities.

CWD Strain Variation

![Figure][1] CREDIT: ANGERS ET AL. So-called prion diseases are fatal neurogenerative disorders that include chronic wasting disease (CWD) found in deer and other cervids. Prion diseases are thought to be caused by infectious proteins (prions) in the absence of associated infectious DNA. Nevertheless, prion strains have been isolated that can mutate in the absence of nucleic acids, and these strain properties control the ability of prions to cross species barriers. Angers et al. (p. [1154][2], published online 13 May; see the Perspective by [Collinge][3] ) address the issue of strain variation in the context of CWD. Whereas the host range of this contagious disease continues to expand, the prevalence of CWD strains has not been determined. Understanding CWD strain variation may be important in predicting and preventing any future risks to human health. [1]: pending:yes [2]: /lookup/doi/10.1126/science.1187107 [3]: /lookup/doi/10.1126/science.1190815

Prion Strain Mutation Determined by Prion Protein Conformational Compatibility and Primary Structure

Prions are infectious proteins composed of the abnormal disease-causing isoform PrPSc, which induces conformational conversion of the host-encoded normal cellular prion protein PrPC to additional PrPSc. The mechanism underlying prion strain mutation in the absence of nucleic acids remains unresolved. Additionally, the frequency of strains causing chronic wasting disease (CWD), a burgeoning prion epidemic of cervids, is unknown. Using susceptible transgenic mice, we identified two prevalent CWD strains with divergent biological properties but composed of PrPSc with indistinguishable biochemical characteristics. Although CWD transmissions indicated stable, independent strain propagation by elk PrPC, strain coexistence in the brains of deer and transgenic mice demonstrated unstable strain propagation by deer PrPC. The primary structures of deer and elk prion proteins differ at residue 226, which, in concert with PrPSc conformational compatibility, determines prion strain mutation in these cervids.

De Novo Generation of Infectious Prions In Vitro Produces a New Disease Phenotype

Prions are the proteinaceous infectious agents responsible for Transmissible Spongiform Encephalopathies. Compelling evidence supports the hypothesis that prions are composed exclusively of a misfolded version of the prion protein (PrPSc) that replicates in the body in the absence of nucleic acids by inducing the misfolding of the cellular prion protein (PrPC). The most common form of human prion disease is sporadic, which appears to have its origin in a low frequency event of spontaneous misfolding to generate the first PrPSc particle that then propagates as in the infectious form of the disease. The main goal of this study was to mimic an early event in the etiology of sporadic disease by attempting de novo generation of infectious PrPSc in vitro. For this purpose we analyzed in detail the possibility of spontaneous generation of PrPSc by the protein misfolding cyclic amplification (PMCA) procedure. Under standard PMCA conditions, and taking precautions to avoid cross-contamination, de novo generation of PrPSc was never observed, supporting the use of the technology for diagnostic applications. However, we report that PMCA can be modified to generate PrPSc in the absence of pre-existing PrPSc in different animal species at a low and variable rate. De novo generated PrPSc was infectious when inoculated into wild type hamsters, producing a new disease phenotype with unique clinical, neuropathological and biochemical features. Our results represent additional evidence in support of the prion hypothesis and provide a simple model to study the mechanism of sporadic prion disease. The findings also suggest that prion diversity is not restricted to those currently known, and that likely new forms of infectious protein foldings may be produced, resulting in novel disease phenotypes.

Constraining the loop, releasing prion infectivity

Prions are the heretical protein-only agents that propagate biological information in the absence of nucleic acids. In mammals, prions are the infectious agents responsible for transmissible spongiform encephalopathies (TSEs), a group of fatal neurodegenerative diseases, including Creutzfeldt–Jakob disease in humans and bovine spongiform encephalopathy, scrapie, and chronic wasting disease in animals. Prions replicate by propagating the misfolding of the infectious prion protein (termed PrPSc) to the homologous, but natively-folded cellular version of the prion protein (termed PrPC) (1). Despite considerable recent advance in knowledge of the infectious agent's nature and its mechanism of replication, we are still far from understanding the structural requirements for the infectious folding of the prion protein. In this issue of PNAS, Sigurdsson et al. (2) provide an important step forward in elucidating the role of a specific domain of the prion protein in the generation of infectivity.