Hexasomal particles: consequence or also consequential?

It is long known that an RNA polymerase transcribing through a nucleosome can generate subnucleosomal particles called hexasomes. These particles lack an H2A-H2B dimer, breaking the symmetry of a nucleosome and revealing new interfaces. Whether hexasomes are simply a consequence of RNA polymerase action or they also have a regulatory impact remains an open question. Recent biochemical and structural studies of RNA polymerases and chromatin remodelers with hexasomes motivated us to revisit this question. Here, we build on previous models to discuss how formation of hexasomes can allow sophisticated regulation of transcription and also significantly impact chromatin folding. We anticipate that further cellular and biochemical analysis of these subnucleosomal particles will uncover additional regulatory roles.

Prompt Impact of Muscle Energy Technique on Pectoralis Muscle Tightness in Computer Users: A Quasi-Experimental Study.

Tightness of the pectoralis minor muscle has been a common characteristic of abnormal posture. Prolonged inappropriate posture while using computers/laptops results in musculoskeletal problems, mainly in the upper limb. This study aims to see how the muscular energy technique affected pectoralis minor tightness in computer users right away. This study included 65 individuals aged 20-40 years following the inclusion/exclusion criteria. Participants received muscle energy technique for the pectoralis minor muscle. Pre- and post-assessment included the evaluation of pectoralis minor length, round shoulder posture (RSP), and forward head posture (FHP). We used the Kolmogorov-Smirnov test to assess the normality of data, as this study included > 50 participants. Data analysis was conducted using a paired t-test for within-group analysis. The outcome measures demonstrated significant improvement (p < 0.001). In conclusion, the muscle energy technique is effective in reducing muscle tightness, improving RSP and reducing FHP.

The conformational landscape of a serpin N-terminal subdomain facilitates folding and in-cell quality control.

Many multi-domain proteins including the serpin family of serine protease inhibitors contain non-sequential domains composed of regions that are far apart in sequence. Because proteins are translated vectorially from N- to C-terminus, such domains pose a particular challenge: how to balance the conformational lability necessary to form productive interactions between early and late translated regions while avoiding aggregation. This balance is mediated by the protein sequence properties and the interactions of the folding protein with the cellular quality control machinery. For serpins, particularly α1-antitrypsin (AAT), mutations often lead to polymer accumulation in cells and consequent disease suggesting that the lability/aggregation balance is especially precarious. Therefore, we investigated the properties of progressively longer AAT N-terminal fragments in solution and in cells. The N-terminal subdomain, residues 1-190 (AAT190), is monomeric in solution and efficiently degraded in cells. More ß-rich fragments, 1-290 and 1-323, form small oligomers in solution, but are still efficiently degraded, and even the polymerization promoting Siiyama (S53F) mutation did not significantly affect fragment degradation. In vitro, the AAT190 region is among the last regions incorporated into the final structure. Hydrogen-deuterium exchange mass spectrometry and enhanced sampling molecular dynamics simulations show that AAT190 has a broad, dynamic conformational ensemble that helps protect one particularly aggregation prone ß-strand from solvent. These AAT190 dynamics result in transient exposure of sequences that are buried in folded, full-length AAT, which may provide important recognition sites for the cellular quality control machinery and facilitate degradation and, under favorable conditions, reduce the likelihood of polymerization.

Membrane Interactions of α-Synuclein Probed by Neutrons and Photons.

α-Synuclein (α-syn) is a key protein in the etiology of Parkinson's disease. In a disease state, α-syn accumulates as insoluble amyloid fibrils enriched in ß-sheet structure. However, in its functional state, α-syn adopts an amphipathic helix upon membrane association and plays a role in synaptic vesicle docking, fusion, and clustering. In this Account, we describe our contributions made in the past decade toward developing a molecular understanding of α-syn membrane interactions, which are crucial for function and have pathological implications. Three topics are covered: α-syn membrane binding probed by neutron reflectometry (NR), the effects of membrane on α-syn amyloid formation, and interactions of α-syn with cellular membranes.NR offers a unique perspective by providing direct measurements of protein penetration depth. By the use of segmentally deuterated α-syn generated through native chemical ligation, the spatial resolution of specific membrane-bound polypeptide regions was determined by NR. Additionally, we used NR to characterize the membrane-bound complex of α-syn and glucocerebrosidase, a lysosomal hydrolase whose mutations are a common genetic risk factor for Parkinson's disease. Although phosphatidylcholine (PC) is the most abundant lipid species in mammalian cells, interactions of PC with α-syn have been largely ignored because they are substantially weaker compared with the electrostatically driven binding of negatively charged lipids. We discovered that α-syn tubulates zwitterionic PC membranes, which is likely related to its involvement in synaptic vesicle fusion by stabilization of membrane curvature. Interestingly, PC lipid tubules inhibit amyloid formation, in contrast to anionic phosphatidylglycerol lipid tubules, which stimulate protein aggregation. We also found that membrane fluidity influences the propensity of α-synuclein amyloid formation. Most recently, we obtained direct evidence of binding of α-syn to exocytic sites on intact cellular membranes using a method called cellular unroofing. This method provides direct access to the cytosolic plasma membrane. Importantly, measurements of fluorescence lifetime distributions revealed that α-syn is more conformationally dynamic at the membrane interface than previously appreciated. This exquisite responsiveness to specific lipid composition and membrane topology is important for both its physiological and pathological functions. Collectively, our work has provided insights into the effects of the chemical nature of phospholipid headgroups on the interplay among membrane remodeling, protein structure, and α-syn amyloid formation.

Unroofing site-specific α-synuclein-lipid interactions at the plasma membrane.

Parkinson's disease is associated with α-synuclein (α-syn), a cytosolic protein enriched in presynaptic terminals. The biological function of α-syn remains elusive; however, increasing evidence suggests that the protein is involved in the regulation of synaptic vesicle fusion, signifying the importance of α-syn-lipid interactions. We show that α-syn preferentially binds to GM1-rich, liquid-ordered lipid domains on cytoplasmic membranes by using unroofed cells, which encapsulates lipid complexity and cellular topology. Moreover, proteins (Rab3a, syntaxin-1A, and VAMP2) involved in exocytosis also localize with α-syn, supporting its proposed functional role in exocytosis. To investigate how these lipid/protein interactions influence α-syn at the residue level, α-syn was derivatized with an environmentally sensitive fluorophore (7-nitrobenz-2-oxa-1,3-diazol-4-yl [NBD]) at different N- and C-terminal sites. Measurements of NBD fluorescence lifetime distributions reveal that α-syn adopts a multitude of membrane-bound conformations, which were not recapitulated in simple micelle or vesicle models, indicating an exquisite sensitivity of the protein to the complex lipid environment. Interestingly, these data also suggest the participation of the C terminus in membrane localization, which is generally overlooked and thus emphasize the need to use cellularly derived and biologically relevant membranes for biophysical characterization. Collectively, our results demonstrate that α-syn is more conformationally dynamic at the membrane interface than previously appreciated, which may be important for both its physiological and pathological functions.

Validation of the Applicability of In-Cell Fast Photochemical Oxidation of Proteins across Multiple Eukaryotic Cell Lines.

Fast photochemical oxidation of proteins (FPOP), a hydroxyl radical-based protein footprinting method, coupled to mass spectrometry has been extensively used to study protein structure and protein-protein interactions in vitro. This method utilizes hydroxyl radicals to oxidatively modify solvent-accessible amino acids and has recently been demonstrated to modify proteins within live cells (IC-FPOP) and Caenorhabditis elegans. Here, we have expanded the application of IC-FPOP into a variety of commonly used cell lines to verify the applicability of the method across various cellular systems. IC-FPOP was able to successfully modify proteins in five different cell lines (Vero, HEK 293T, CHO, MCF-10A, and MCF-7). To increase the number of oxidatively modified proteins identified, we have also employed the use of offline high pH reversed-phase liquid chromatography (RPLC) followed by concatenation and online low-pH RPLC. The coupling of IC-FPOP to 2D-LC MS/MS resulted in a 1.7-fold increase in total identifications of oxidatively modified proteins, which expanded the dynamic range of the method. This work demonstrates the efficacy of using IC-FPOP to study protein-protein interactions in cells.

Proteome-Wide Structural Biology: An Emerging Field for the Structural Analysis of Proteins on the Proteomic Scale.

Over the past decade, a suite of new mass-spectrometry-based proteomics methods has been developed that now enables the conformational properties of proteins and protein-ligand complexes to be studied in complex biological mixtures, from cell lysates to intact cells. Highlighted here are seven of the techniques in this new toolbox. These techniques include chemical cross-linking (XL-MS), hydroxyl radical footprinting (HRF), Drug Affinity Responsive Target Stability (DARTS), Limited Proteolysis (LiP), Pulse Proteolysis (PP), Stability of Proteins from Rates of Oxidation (SPROX), and Thermal Proteome Profiling (TPP). The above techniques all rely on conventional bottom-up proteomics strategies for peptide sequencing and protein identification. However, they have required the development of unconventional proteomic data analysis strategies. Discussed here are the current technical challenges associated with these different data analysis strategies as well as the relative analytical capabilities of the different techniques. The new biophysical capabilities that the above techniques bring to bear on proteomic research are also highlighted in the context of several different application areas in which these techniques have been used, including the study of protein ligand binding interactions (e.g., protein target discovery studies and protein interaction network analyses) and the characterization of biological states.