Oral Immunotherapy With Human Secretory Immunoglobulin A Improves Survival in the Hamster Model of Clostridioides difficile Infection.

Coadministration of human secretory IgA (sIgA) together with subtherapeutic vancomycin enhanced survival in the Clostridioides difficile infection (CDI) hamster model. Vancomycin (5 or 10 mg/kg × 5 days) plus healthy donor plasma sIgA/monomeric IgA (TID × 21 days) or hyperimmune sIgA/monomeric IgA (BID × 13 days) enhanced survival. Survival was improved compared to vancomycin alone, P = .018 and .039 by log-rank Mantel-Cox, for healthy and hyperimmune sIgA, respectively. Passive immunization with sIgA (recombinant human secretory component plus IgA dimer/polymer from pooled human plasma) can be administered orally and prevents death in a partially treated CDI hamster model.

Reductive modification of genetically encoded 3-nitrotyrosine sites in alpha synuclein expressed in E.coli.

Tyrosine nitration is a post-translational protein modification relevant to various pathophysiological processes. Chemical nitration procedures have been used to generate and study nitrated proteins, but these methods regularly lead to modifications at other amino acid residues. A novel strategy employs a genetic code modification that allows incorporation of 3-nitrotyrosine (3-NT) during ribosomal protein synthesis to generate a recombinant protein with defined 3-NT-sites, in the absence of other post-translational modifications. This approach was applied to study the generation and stability of the 3-NT moiety in recombinant proteins produced in E.coli. Nitrated alpha-synuclein (ASYN) was selected as exemplary protein, relevant in Parkinson's disease (PD). A procedure was established to obtain pure tyrosine-modified ASYN in mg amounts. However, a rapid (t1/2 = 0.4 h) reduction of 3-NT to 3-aminotyrosine (3-AT) was observed. When screening for potential mechanisms, we found that 3-NT can be reduced enzymatically to 3-AT, whilst biologically relevant low molecular weight reductants, such as NADPH or GSH, did not affect 3-NT. A genetic screen for E.coli proteins, involved in the observed 3-NT reduction, revealed the contribution of several, possibly redundant pathways. Green fluorescent protein was studied as an alternative model protein. These data confirm 3-NT reduction as a broadly-relevant pathway in E.coli. In conclusion, incorporation of 3-NT as a genetically-encoded non-natural amino acid allows for generation of recombinant proteins with specific nitration sites. The potential reduction of the 3-NT moiety by E.coli, however, requires attention to the design of the purification strategy for obtaining pure nitrated protein.

Simultaneous IR-Spectroscopic Observation of α-Synuclein, Lipids, and Solvent Reveals an Alternative Membrane-Induced Oligomerization Pathway.

The intrinsically disordered protein α-synuclein (αS), a known pathogenic factor for Parkinson's disease, can adopt defined secondary structures when interacting with membranes or during fibrillation. The αS-lipid interaction and the implications of this process for aggregation and damage to membranes are still poorly understood. Therefore, we established a label-free infrared (IR) spectroscopic approach to allow simultaneous monitoring of αS conformation and membrane integrity. IR showed its unique sensitivity for identifying distinct ß-structured aggregates. A comparative study of wild-type αS and the naturally occurring splicing variant αS Δexon3 yielded new insights into the membrane's capability for altering aggregation pathways.

Alpha-synuclein binds to the inner membrane of mitochondria in an α-helical conformation.

The human alpha-Synuclein (αS) protein is of significant interest because of its association with Parkinson's disease and related neurodegenerative disorders. The intrinsically disordered protein (140 amino acids) is characterized by the absence of a well-defined structure in solution. It displays remarkable conformational flexibility upon macromolecular interactions, and can associate with mitochondrial membranes. Site-directed spin-labeling in combination with electron paramagnetic resonance spectroscopy enabled us to study the local binding properties of αS on artificial membranes (mimicking the inner and outer mitochondrial membranes), and to evaluate the importance of cardiolipin in this interaction. With pulsed, two-frequency, double-electron electron paramagnetic resonance (DEER) approaches, we examined, to the best of our knowledge for the first time, the conformation of αS bound to isolated mitochondria.

Generation of genetically-modified human differentiated cells for toxicological tests and the study of neurodegenerative diseases.

Human differentiated cell types, such as neurons or hepatocytes, are of limited availability, and their use for experiments requiring ectopic gene expression is challenging. Using the human conditionally-immortalized neuronal precursor line LUHMES, we explored whether genetic modification in the proliferating state could be used for experiments in the differentiated post-mitotic neurons. First, alpha-synuclein (ASYN), a gene associated with the pathology of Parkinson's disease, was overexpressed. Increased amounts of the protein were tolerated without change of phenotype, and this approach now allows further studies on protein variants. Knockdown of ASYN attenuated the toxicity of the parkinsonian toxicant 1-methyl-4-phenylpyridinium (MPP+). Different lentiviral constructs then were tested: cells labeled ubiquitously with green (GFP) or red fluorescent protein (RFP) allowed the quantification of neurite growth and of its disturbance by toxicants; expression of proteins of interest could be targeted to different organelles; production of two different proteins from a single read-through construct was achieved successfully by an expression strategy using a linker peptide between the two proteins, which is cleaved by deubiquitinases; LUHMES, labeled with GFP in the cytosol and RFP in the mitochondria, were used to quantify mitochondrial mobility along the neurites. MPP+ reduced such organelle movement before any other detectable cellular change, and this toxicity was prevented by simultaneous treatment with the antioxidant ascorbic acid. Thus, a strategy has been outlined here to study new functional endpoints, and subtle changes of structure and proteostasis relevant in toxicology and biomedicine in post-mitotic human cells.

Oxidative and nitrative alpha-synuclein modifications and proteostatic stress: implications for disease mechanisms and interventions in synucleinopathies.

Alpha-synuclein (ASYN) is a major constituent of the typical protein aggregates observed in several neurodegenerative diseases that are collectively referred to as synucleinopathies. A causal involvement of ASYN in the initiation and progression of neurological diseases is suggested by observations indicating that single-point (e.g., A30P, A53T) or multiplication mutations of the gene encoding for ASYN cause early onset forms of Parkinson's disease (PD). The relative regional specificity of ASYN pathology is still a riddle that cannot be simply explained by its expression pattern. Also, transgenic over-expression of ASYN in mice does not recapitulate the typical dopaminergic neuronal death observed in PD. Thus, additional factors must contribute to ASYN-related toxicity. For instance, synucleinopathies are usually associated with inflammation and elevated levels of oxidative stress in affected brain areas. In turn, these conditions favor oxidative modifications of ASYN. Among these modifications, nitration of tyrosine residues, formation of covalent ASYN dimers, as well as methionine sulfoxidations are prominent examples that are observed in post-mortem PD brain sections. Oxidative modifications can affect ASYN aggregation, as well as its binding to biological membranes. This would affect neurotransmitter recycling, mitochondrial function and dynamics (fission/fusion), ASYN's degradation within a cell and, possibly, the transfer of modified ASYN to adjacent cells. Here, we propose a model on how covalent modifications of ASYN link energy stress, altered proteostasis, and oxidative stress, three major pathogenic processes involved in PD progression. Moreover, we hypothesize that ASYN may act physiologically as a catalytically regenerated scavenger of oxidants in healthy cells, thus performing an important protective role prior to the onset of disease or during aging.