Junping Yu

α-Synuclein Misfolding: Single Molecule AFM Force Spectroscopy Study

Protein misfolding and aggregation are the very first and critical steps in development of various neurodegenerative disorders, including Parkinsons disease, induced by misfolding of alpha-synuclein. Thus, elucidating properties of proteins in misfolded states and understanding the mechanisms of their assembly into the disease prone aggregates are critical for the development of rational approaches to prevent protein misfolding-mediated pathologies. To accomplish this goal and as a first step to elucidate the mechanism of alpha-synuclein misfolding, we applied single-molecule force spectroscopy capable of detecting protein misfolding. We immobilized alpha-synuclein molecules at their C-termini at the atomic force microscope tips and substrate surfaces, and measured the interaction between the proteins by probing the microscope tip at various locations on the surface. Using this approach, we detected alpha-synuclein misfolded states by enhanced interprotein interaction. We used a dynamics force spectroscopy approach to measure such an important characteristic of dimers of misfolded alpha-synuclein as their lifetimes. We found that the dimer lifetimes are in the range of seconds and these values are much higher than the characteristics for the dynamics of the protein in monomeric state. These data show that compared to highly dynamic monomeric forms, alpha-synuclein dimers are much more stable and thus can serve as stable nuclei for the formation of multimeric and aggregated forms of alpha-synuclein. Importantly, two different lifetimes were observed for the dimers, suggesting that aggregation can follow different pathways that may lead to different aggregated morphologies of alpha-synuclein.

Early Stages for Parkinson’s Development: α-Synuclein Misfolding and Aggregation

Misfolding and aggregation of proteins are common threads linking a number of important human health problems, including various neurodegenerative disorders such as Parkinson’s disease in particular. The first and perhaps most important elements in most neurodegenerative processes are misfolding and aggregation of specific proteins. Despite the crucial importance of protein misfolding and abnormal interactions, very little is currently known about the molecular mechanism underlying these processes. Factors that lead to protein misfolding and aggregation in vitro are poorly understood, in addition to the complexities involved in the formation of protein nanoparticles with different morphologies (e.g. nanopores and other species) in vivo. A clear understanding of the molecular mechanisms of misfolding and aggregation will facilitate rational approaches to prevent protein misfolding mediated pathologies. To accomplish this goal and to elucidate the mechanism of protein misfolding, we developed a novel nanotechnology tool capable of detecting protein misfolding. We applied single molecule probing technique to characterize misfolding and self-assembly of α-synuclein dimers, which is the very first step of the aggregation process. Using AFM force spectroscopy approach, we were able to detect protein misfolding via enhanced interprotein interaction. Moreover, such an important characteristic as the lifetime of dimers formed by misfolded α-synuclein was measured. These data suggest that compared to highly dynamic monomeric forms, α-synuclein dimers are practically static and thus can play a role of aggregation nuclei for the formation of aggregates. Importantly, two different dissociation channels were detected suggesting that aggregation process can follow different pathways. The application of these findings for understanding of the aggregation phenomenon and the development of the disease is discussed.

Nanoimaging for protein misfolding diseases

Misfolding and aggregation of proteins are widespread phenomena leading to the development of numerous neurodegenerative disorders such as Parkinsons, Alzheimers, and Huntingtons diseases. Each of these diseases is linked to structural misfolding and aggregation of a particular protein. The aggregated forms of the protein induce the development of a particular disease at all levels, leading to neuronal dysfunction and loss. Because protein refolding is frequently accompanied by transient association of partially folded intermediates, the propensity to aggregate is considered a general characteristic of the majority of proteins. X-ray crystallography, nuclear magnetic resonance, electron microscopy, and atomic force microscopy have provided important information on the structure of aggregates. However, fundamental questions, such as why the misfolded conformation of the protein is formed, and why this state is important for self-assembly, remain unanswered. Although it is well known that the same protein under pathological conditions can lead to the formation of aggregates with diverse biological consequences, the conditions leading to misfolding and the formation of the disease prone complexes are unclear, complicating any development of efficient prevention of the diseases. Misfolded states exist transiently, so answering these questions requires the use of novel approaches and methods. Progress has been made during the past few years, when recently developed ensemble methods and single-molecule biophysics techniques were applied to the problem of the protein misfolding. In this review, the impacts of these studies on the understanding of the mechanisms of the protein self-assembly into aggregates and on the development of treatments of the diseases are discussed.

Engineered bacteriophage lysins as novel anti-infectives.

Bacteriophage lysins, the highly evolved specific peptidoglycan hydrolases, have long been demonstrated to be effective enzybiotics in various infectious models. The modular structure of lysins makes it possible to design bioengineered lysins that have desired properties, such as higher activity, or broader killing spectrum. Moreover, lysins can even be engineered to kill Gram-negative bacterial pathogens from without, a property that is not present in natural lysins. In this era of ever increasing multidrug resistant pathogens, engineered lysins represent a new class of enzybiotics that are powerful and readily available to fight antimicrobial resistance.

Nanoprobing of α-synuclein misfolding and aggregation with atomic force microscopy

UNLABELLED Atomic force microscopy (AFM) force spectroscopy is a technique with broad nanomedical applications, widely used for the characterization of molecular interactions on the nanoscale. Here we test this technique to evaluate compounds for influencing the protein aggregation process. The results demonstrate that Zn(2+) or Al(3+) cations bring about a dramatic increase of α-synuclein interactions in unfavorable conditions for α-synuclein misfolding (neutral pH). We did not observe the effect of dopamine at favorable conditions for α-synuclein misfolding (acidic pH). We also found that electrostatic interactions do not play a significant role at acidic pH. These findings are generally in line with previous studies by various techniques. The high sensitivity of AFM force spectroscopy as well as its ability to test compounds for the same experimental system makes AFM an efficient nanotool for rapid analysis of compounds inhibiting early protein aggregation studies and quantitative selection of potential therapeutics for neurodegenerative diseases. FROM THE CLINICAL EDITOR Atomic force microscopy is used to interrogate the influence of cations and α-synuclein on the protein aggregation process. The report illustrates an application of AFM to unravel the potential of novel therapeutics on early protein aggregation intrinsic in neurodegenerative diseases.

Novel Chimeric Lysin with High-Level Antimicrobial Activity against Methicillin-Resistant Staphylococcus aureus In Vitro and In Vivo

ABSTRACT The treatment of infections caused by methicillin-resistant Staphylococcus aureus (MRSA) is a challenge worldwide. In our search for novel antimicrobial agents against MRSA, we constructed a chimeric lysin (named as ClyH) by fusing the catalytic domain of Ply187 (Pc) with the non-SH3b-like cell wall binding domain of phiNM3 lysin. Herein, the antimicrobial activity of ClyH against MRSA strains in vitro and in vivo was studied. Our results showed that ClyH could kill all of the tested clinical isolates of MRSA with higher efficacy than lysostaphin as well as its parental enzyme. The MICs of ClyH against clinical S. aureus strains were found to be as low as 0.05 to 1.61 mg/liter. In a mouse model, a single intraperitoneal administration of ClyH protected mice from death caused by MRSA, without obvious harmful effects. The present data suggest that ClyH has the potential to be an alternative therapeutic agent for the treatment of infections caused by MRSA.

Sensitive and rapid detection of staphylococcus aureus in milk via cell binding domain of lysin.

Staphylococcus aureus (S. aureus) is an important food-borne pathogen in dairy products contaminated through raw ingredients or improper food handling. Rapid detection of S. aureus with high sensitivity is of significance for food quality and safety. In this study, a new method was developed for detecting S. aureus in milk by coupling immunomagnetic separation with enzyme linked cell wall binding domain (CBD) of lysin plyV12, which can bind to S. aureus with high affinity. There are millions of binding sites present on the cell surface of S. aureus for the CBD attachment, which greatly improves the detection sensitivity. The method has the overall testing time of only 1.5h with the detection limit of 4 × 10(3)CFU/mL in spiked milk. Because it is simple, rapid and sensitive, this method could be used for the detection of S. aureus in various food samples.

Molecular mechanism of misfolding and aggregation of Aβ(13-23).

The misfolding and self-assembly of the amyloid-beta (Aβ) peptide into aggregates is a molecular signature of the development of Alzheimers disease, but molecular mechanisms of the peptide aggregation remain unknown. Here, we combined Atomic Force Microscopy (AFM) and Molecular Dynamics (MD) simulations to characterize the misfolding process of an Aβ peptide. Dynamic force spectroscopy AFM analysis showed that the peptide forms stable dimers with a lifetime of ∼1 s. During MD simulations, isolated monomers gradually adopt essentially similar nonstructured conformations independent from the initial structure. However, when two monomers approach their structure changes dramatically, and the conformational space for the two monomers become restricted. The arrangement of monomers in antiparallel orientation leads to the cooperative formation of β-sheet conformation. Interactions, including hydrogen bonds, salt bridges, and weakly polar interactions of side chains stabilize the structure of the dimer. Under the applied force, the dimer, as during the AFM experiments, dissociates in a cooperative manner. Thus, misfolding of the Aβ peptide proceeds via the loss of conformational flexibility and formation of stable dimers suggesting their key role in the subsequent Aβ aggregation process.

Existence of Separate Domains in Lysin PlyG for Recognizing Bacillus anthracis Spores and Vegetative Cells

ABSTRACT As a potential antimicrobial, the bacteriophage lysin PlyG has been reported to specifically recognize Bacillus anthracis vegetative cells only and to kill B. anthracis vegetative cells and its germinating spores. However, how PlyG interacts with B. anthracis spores remains unclear. Herein, a 60-amino-acid domain in PlyG (residues 106 to 165), located mainly in the previously identified catalytic domain, was found able to specifically recognize B. anthracis spores but not vegetative cells. The exosporium of the spores was found to be the most probable binding target of this domain. This is the first time that a lysin for spore-forming bacteria has been found to have separate domains to recognize spores and vegetative cells, which might help in understanding the coevolution of phages with spore-forming bacteria. Besides providing new biomarkers for developing better assays for identifying B. anthracis spores, the newly found domain may be helpful in developing PlyG as a preventive antibiotic to reduce the threat of anthrax in suspected exposures to B. anthracis spores.

Construction of a chimeric lysin Ply187N-V12C with extended lytic activity against staphylococci and streptococci

Developing chimeric lysins with a wide lytic spectrum would be important for treating some infections caused by multiple pathogenic bacteria. In the present work, a novel chimeric lysin (Ply187N‐V12C) was constructed by fusing the catalytic domain (Ply187N) of the bacteriophage lysin Ply187 with the cell binding domain (146‐314aa, V12C) of the lysin PlyV12. The results showed that the chimeric lysin Ply187N‐V12C had not only lytic activity similar to Ply187N against staphylococcal strains but also extended its lytic activity to streptococci and enterococci, such as Streptococcus dysgalactiae, Streptococcus agalactiae, Streptococcus pyogenes, Enterococcus faecium and Enterococcus faecalis, which Ply187N could not lyse. Our work demonstrated that generating novel chimeric lysins with an extended lytic spectrum was feasible through fusing a catalytic domain with a cell‐binding domain from lysins with lytic spectra across multiple genera.