The Carboxy Terminal Region on Spike Protein of Porcine Epidemic Diarrhea Virus (PEDV) Is Important for Evaluating Neutralizing Activity

In this study, we evaluated 62 sow sera samples from PED-vaccinated sows to compare the serum neutralizing test (SNT) and enzyme-linked immunosorbent assay (ELISA). We performed protein ELISA (pELISA) using fragments of spike proteins S1, S2, S3 and entire nucleocapsid proteins, and found a correlation between the SNT and ELISA in PEDV-vaccinated sera. Sera with higher neutralizing activity showed higher titers of IgG. In the antibody profiling, the neutralizing activities are correlated with the levels of the spike antibody, especially the S3 region. We confirmed that the carboxy-terminal region, including the endodomain of the S protein, induced stronger neutralizing activity than the ectodomain. This region of the S protein could be useful for evaluating PED vaccine efficacy, and it is a strong neutralizing epitope of PEDV. The S3 protein could be useful for evaluating PED vaccine efficacy, and it is a strong neutralizing epitope of PEDV.

Robust α-synuclein pathology in select brainstem neuronal populations is a potential instigator of multiple system atrophy

Multiple system atrophy (MSA) is an insidious middle age-onset neurodegenerative disease that clinically presents with variable degrees of parkinsonism and cerebellar ataxia. The pathological hallmark of MSA is the progressive accumulation of glial cytoplasmic inclusions (GCIs) in oligodendrocytes that are comprised of α-synuclein (αSyn) aberrantly polymerized into fibrils. Experimentally, MSA brain samples display a high level of seeding activity to induce further αSyn aggregation by a prion-like conformational mechanism. Paradoxically, αSyn is predominantly a neuronal brain protein, with only marginal levels expressed in normal or diseased oligodendrocytes, and αSyn inclusions in other neurodegenerative diseases, including Parkinson’s disease and Dementia with Lewy bodies, are primarily found in neurons. Although GCIs are the hallmark of MSA, using a series of new monoclonal antibodies targeting the carboxy-terminal region of αSyn, we demonstrate that neuronal αSyn pathology in MSA patient brains is remarkably abundant in the pontine nuclei and medullary inferior olivary nucleus. This neuronal αSyn pathology has distinct histological properties compared to GCIs, which allows it to remain concealed to many routine detection methods associated with altered biochemical properties of the carboxy-terminal domain of αSyn. We propose that these previously underappreciated sources of aberrant αSyn could serve as a pool of αSyn prion seeds that can initiate and continue to drive the pathogenesis of MSA.

Variants Affecting the C-Terminal of CSF1R Cause Congenital Vertebral Malformation Through a Gain-of-Function Mechanism

CSF1R encodes the colony-stimulating factor 1 receptor which regulates the proliferation, differentiation, and biological activity of monocyte/macrophage lineages. Pathogenic variants in CSF1R could lead to autosomal dominant adult-onset leukoencephalopathy with axonal spheroids and pigmented glia or autosomal recessive skeletal dysplasia. In this study, we identified three heterozygous deleterious rare variants in CSF1R from a congenital vertebral malformation (CVM) cohort. All of the three variants are located within the carboxy-terminal region of CSF1R protein and could lead to an increased stability of the protein. Therefore, we established a zebrafish model overexpressing CSF1R. The zebrafish model exhibits CVM phenotypes such as hemivertebral and vertebral fusion. Furthermore, overexpression of the mutated CSF1R mRNA depleted of the carboxy-terminus led to a higher proportion of zebrafish with vertebral malformations than wild-type CSF1R mRNA did (p = 0.03452), implicating a gain-of-function effect of the C-terminal variant. In conclusion, variants affecting the C-terminal of CSF1R could cause CVM though a potential gain-of-function mechanism.

UvrD helicase–RNA polymerase interactions are governed by UvrD’s carboxy-terminal Tudor domain

All living organisms have to cope with the constant threat of genome damage by UV light and other toxic reagents. To maintain the integrity of their genomes, organisms developed a variety of DNA repair pathways. One of these, the Transcription Coupled DNA-Repair (TCR) pathway, is triggered by stalled RNA Polymerase (RNAP) complexes at DNA damage sites on actively transcribed genes. A recently elucidated bacterial TCR pathway employs the UvrD helicase pulling back stalled RNAP complexes from the damage, stimulating recruitment of the DNA-repair machinery. However, structural and functional aspects of UvrD’s interaction with RNA Polymerase remain elusive. Here we used advanced solution NMR spectroscopy to investigate UvrD’s role within the TCR, identifying that the carboxy-terminal region of the UvrD helicase facilitates RNAP interactions by adopting a Tudor-domain like fold. Subsequently, we functionally analyzed this domain, identifying it as a crucial component for the UvrD–RNAP interaction besides having nucleic-acid affinity.

UvrD helicase–RNA polymerase interactions are governed by UvrD’s carboxy-terminal Tudor domain

ABSTRACTAll living organisms have to cope with the constant threat of genome damage by UV light and other toxic reagents. To maintain the integrity of their genomes, organisms developed a variety of DNA repair pathways. One of these, the Transcription Coupled DNA-Repair (TCR) pathway, is triggered by stalled RNA Polymerase (RNAP) complexes at DNA damage sites on actively transcribed genes. A recently elucidated bacterial TCR pathway employs the UvrD helicase pulling back stalled RNAP complexes from the damage, stimulating recruitment of the DNA-repair machinery. However, structural and functional aspects of UvrD’s interaction with RNA Polymerase remain elusive. Here we used advanced solution NMR spectroscopy to investigate UvrD’s role within the TCR, identifying that the carboxy-terminal region of the UvrD helicase facilitates RNAP interactions by adopting a Tudor-domain like fold. Subsequently, we functionally analyzed this domain, identifying it as a crucial component for the UvrD–RNAP interaction besides having nucleic-acid affinity.

Crystal structure of the SH3 domain of growth factor receptor-bound protein 2

This study presents the crystal structure of the N-terminal SH3 (SH3N) domain of growth factor receptor-bound protein 2 (Grb2) at 2.5 Å resolution. Grb2 is a small (215-amino-acid) adaptor protein that is widely expressed and involved in signal transduction/cell communication. The crystal structure of full-length Grb2 has previously been reported (PDB entry 1gri). The structure of the isolated SH3N domain is consistent with the full-length structure. The structure of the isolated SH3N domain was solved at a higher resolution (2.5 Å compared with 3.1 Å for the previously deposited structure) and made it possible to resolve some of the loops that were missing in the full-length structure. In addition, interactions between the carboxy-terminal region of the SH3N domain and the Sos1-binding sites were observed in the structure of the isolated domain. Analysis of these interactions provided new information about the ligand-binding properties of the SH3N domain of Grb2.

EXPRESSION OF Mycobacterium tuberculosis RpsA IN Mycobacterium smegmatis INCREASES SUSCEPTIBILITY TO PYRAZINAMIDE

ABSTRACTPyrazinamide (PZA) is one of the most important drugs used in combined antituberculous therapy. After the drug enters Mycobacterium tuberculosis it is hydrolyzed by pyrazinamidase (PZAse) to the bactericidal molecule pyrazinoic acid (POA). Ribosomal protein S1 (RpsA) was recently identified as a possible target of PZA based on its binding activity to POA and capacity to inhibit trans-translation. However, its role is not completely understood.It has been proposed that Mycobacterium smegmatis RpsA is not capable of binding POA, unlike M. tuberculosis RpsA. This may be due to the different amino acid sequence in the carboxy-terminal region of the two molecules: in M. smegmatis RpsA it is much closer to the sites that may interact with POA than in M. tuberculosis RpsA. These differences could be contributing, along with the presence of highly active POA efflux, to the natural resistance to PZA in M. smegmatis.To further understand the mechanisms of action of PZA and the role of RpsA in PZA susceptibility, we evaluated the effect of complementing M. tuberculosis RpsA expression in M. smegmatis using pNIT mycobacterial non-integrative expression vector and then performed a PZA susceptibility test determining the minimum inhibitory concentration (MIC) of PZA. It was expected that chimeric ribosomes comprising M. tuberculosis RpsA may be present and may affect PZA susceptibility.Our results showed a reduction in PZA MIC in M. smegmatis complemented with overexpressed M. tuberculosis RpsA compared to non-overexpressed M. smegmatis (468 µg/mL and >7500 µg/mL respectively).

Prion Replication in the Mammalian Cytosol: Functional Regions within a Prion Domain Driving Induction, Propagation, and Inheritance

ABSTRACTPrions of lower eukaryotes are transmissible protein particles that propagate by converting homotypic soluble proteins into growing protein assemblies. Prion activity is conferred by so-called prion domains, regions of low complexity that are often enriched in glutamines and asparagines (Q/N). The compositional similarity of fungal prion domains with intrinsically disordered domains found in many mammalian proteins raises the question of whether similar sequence elements can drive prion-like phenomena in mammals. Here, we define sequence features of the prototypeSaccharomyces cerevisiaeSup35 prion domain that govern prion activities in mammalian cells by testing the ability of deletion mutants to assemble into self-perpetuating particles. Interestingly, the amino-terminal Q/N-rich tract crucially important for prion induction in yeast was dispensable for the prion life cycle in mammalian cells. Spontaneous and template-assisted prion induction, growth, and maintenance were preferentially driven by the carboxy-terminal region of the prion domain that contains a putative soft amyloid stretch recently proposed to act as a nucleation site for prion assembly. Our data demonstrate that preferred prion nucleation domains can differ between lower and higher eukaryotes, resulting in the formation of prions with strikingly different amyloid cores.