Evolution of CPEB4 Dynamics Across its Liquid-Liquid Phase Separation Transition.

Knowledge about the structural and dynamic properties of proteins that form membrane-less organelles in cells via liquid-liquid phase separation (LLPS) is required for understanding the process at a molecular level. We used spin labeling and electron paramagnetic resonance (EPR) spectroscopy to investigate the dynamic properties (rotational diffusion) of the low complexity N-terminal domain of cytoplasmic polyadenylation element binding-4 protein (CPEB4NTD) across its LLPS transition, which takes place with increasing temperature. We report the coexistence of three spin labeled CPEB4NTD (CPEB4*) populations with distinct dynamic properties representing different conformational spaces, both before and within the LLPS state. Monomeric CPEB4* exhibiting fast motion defines population I and shows low abundance prior to and following LLPS. Populations II and III are part of CPEB4* assemblies where II corresponds to loose conformations with intermediate range motions and population III represents compact conformations with strongly attenuated motions. As the temperature increased the population of component II increased reversibly at the expense of component III, indicating the existence of an III ⇌ II equilibrium. We correlated the macroscopic LLPS properties with the III ⇌ II exchange process upon varying temperature and CPEB4* and salt concentrations. We hypothesized that weak transient intermolecular interactions facilitated by component II lead to LLPS, with the small assemblies integrated within the droplets. The LLPS transition, however, was not associated with a clear discontinuity in the correlation times and populations of the three components. Importantly, CPEB4NTD exhibits LLPS properties where droplet formation occurs from a preformed microscopic assembly rather than the monomeric protein molecules.

α-Synuclein plasma membrane localization correlates with cellular phosphatidylinositol polyphosphate levels.

The Parkinson's disease protein α-synuclein (αSyn) promotes membrane fusion and fission by interacting with various negatively charged phospholipids. Despite postulated roles in endocytosis and exocytosis, plasma membrane (PM) interactions of αSyn are poorly understood. Here, we show that phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3), two highly acidic components of inner PM leaflets, mediate PM localization of endogenous pools of αSyn in A2780, HeLa, SK-MEL-2, and differentiated and undifferentiated neuronal SH-SY5Y cells. We demonstrate that αSyn binds to reconstituted PIP2 membranes in a helical conformation in vitro and that PIP2 synthesizing kinases and hydrolyzing phosphatases reversibly redistribute αSyn in cells. We further delineate that αSyn-PM targeting follows phosphoinositide-3 kinase (PI3K)-dependent changes of cellular PIP2 and PIP3 levels, which collectively suggests that phosphatidylinositol polyphosphates contribute to αSyn's function(s) at the plasma membrane.

An NMR-Based Biosensor to Measure Stereospecific Methionine Sulfoxide Reductase Activities in Vitro and in Vivo*.

Oxidation of protein methionines to methionine-sulfoxides (MetOx) is associated with several age-related diseases. In healthy cells, MetOx is reduced to methionine by two families of conserved methionine sulfoxide reductase enzymes, MSRA and MSRB that specifically target the S- or R-diastereoisomers of methionine-sulfoxides, respectively. To directly interrogate MSRA and MSRB functions in cellular settings, we developed an NMR-based biosensor that we call CarMetOx to simultaneously measure both enzyme activities in single reaction setups. We demonstrate the suitability of our strategy to delineate MSR functions in complex biological environments, including cell lysates and live zebrafish embryos. Thereby, we establish differences in substrate specificities between prokaryotic and eukaryotic MSRs and introduce CarMetOx as a highly sensitive tool for studying therapeutic targets of oxidative stress-related human diseases and redox regulated signaling pathways.

Protein Nanofibrils as Storage Forms of Peptide Drugs and Hormones.

Amyloids are highly organized cross ß-sheet protein nanofibrils that are associated with both diseases and functions. Thermodynamically amyloids are stable structures as they represent the lowest free energy state that proteins can attain. However, recent studies suggest that amyloid fibrils can be dissociated by a change in environmental parameters such as pH and ionic strength. This reversibility of amyloids can not only be associated with disease, but function as well. In disease-associated amyloids, fibrils can act as reservoirs of cytotoxic oligomers. Recently, in higher organisms such as mammals, hormones were found to be stored in amyloid-like state, where these were reported to act as a reservoir of functional monomers. These hormone amyloids can dissociate to monomers upon release from the secretory granules, and subsequently bind to their respective receptors and perform their functions. In this book chapter, we describe in detail how these protein nanofibrils represent the densest possible peptide packing and are suitable for long-term storage. Thus, mimicking the feature of amyloids to release functional monomers, it is possible to formulate amyloid-based peptide/protein drugs, which can be used for sustained release.

Time-Resolved NMR Analysis of Proteolytic α-Synuclein Processing in vitro and in cellulo.

Targeted proteolysis of the disordered Parkinson's disease protein alpha-synuclein (αSyn) constitutes an important event under physiological and pathological cell conditions. In this work, site-specific αSyn cleavage by different endopeptidases in vitro and by endogenous proteases in extracts of challenged and unchallenged cells was studied by time-resolved NMR spectroscopy. Specifically, proteolytic processing was monitored under neutral and low pH conditions and in response to Rotenone-induced oxidative stress. Further, time-dependent degradation of electroporation-delivered αSyn in intact SH-SY5Y and A2780 cells was analyzed. Results presented here delineate a general framework for NMR-based proteolysis studies in vitro and in cellulo, and confirm earlier reports pertaining to the exceptional proteolytic stability of αSyn under physiological cell conditions. However, experimental findings also reveal altered protease susceptibilities in selected mammalian cell lines and upon induced cell stress.

Atypical profiles and modulations of heme-enzymes catalyzed outcomes by low amounts of diverse additives suggest diffusible radicals' obligatory involvement in such redox reactions.

BACKGROUND: Peroxidations mediated by heme-enzymes have been traditionally studied under a single-site (heme distal pocket), non-sequential (ping-pong), two-substrates binding scheme of Michaelis-Menten paradigm. We had reported unusual modulations of peroxidase and P450 reaction outcomes and explained it invoking diffusible reactive species [Manoj, 2006; Manoj et al., 2010; Andrew et al., 2011, Parashar et al., 2014 & Venkatachalam et al., 2016]. METHODS: A systematic investigation of specific product formation rates was undertaken to probe the hypothesis that involvement of diffusible reactive species could explain undefined substrate specificities and maverick modulations (sponsored by additives) of heme-enzymes. RESULTS: When the rate of specific product formation was studied as a function of reactants' concentration or environmental conditions, we noted marked deviations from normal profiles. We report that heme-enzyme mediated peroxidations of various substrates are inhibited (or activated) by sub-equivalent concentrations of diverse redox-active additives and this is owing to multiple redox equilibriums in the milieu. At low enzyme and peroxide concentrations, the enzyme is seen to recycle via a one-electron (oxidase) cycle, which does not require the substrate to access the heme centre. Schemes are provided that explain the complex mechanistic cycle, kinetics & stoichiometry. CONCLUSION: It is not obligatory for an inhibitor or substrate to interact with the heme centre for influencing overall catalysis. Roles of diffusible reactive species explain catalytic outcomes at low enzyme and reactant concentrations. SIGNIFICANCE: The current work highlights the scope/importance of redox enzyme reactions that could occur "out of the active site" in biological or in situ systems.

Influence of retinoic acid on mesenchymal stem cell differentiation in amyloid hydrogels.

This paper presents data related to the research article "Self healing hydrogels composed of amyloid nano fibrils for cell culture and stem cell differentiation" [1]. Here we probed the collective influence of all-trans retinoic acid (RA) and substrate properties (amyloid hydrogel) on human mesenchymal stem cell (hMSC) differentiation. Stem cells were cultured on soft amyloid hydrogels [1], [2] in the presence and absence of matrix encapsulated RA. The cell morphology was imaged and assessed via quantification of circularity. Further immunostaining and quantitative real time PCR was used to quantify various markers of differentiation in the neuronal lineage.