Background: The molecular details of the effects of DOPAL on ␣-synuclein misfolding and oligomerization are unknown. Results: DOPAL forms Schiff-base and Michael-addition adducts with ␣-synuclein Lys residues. Conclusion: DOPAL modification inhibits aS fibrillization and reduces binding of ␣-synuclein to synaptic-like vesicles. Significance: DOPAL modification may interfere with the normal functions of ␣-synuclein and favor the buildup of potentially toxic oligomers.

The initial line of defense against infection is sustained by the innate immune system. Together, membrane-bound TLR and cytosolic NLR receptors play key roles in the innate immune response by detecting bacterial and viral invaders as well as endogenous stress signals. NLRs are multi-domain proteins with varying N-terminal effector domains that are responsible for regulating downstream signaling events. Here, we report the structure and dynamics of the N-terminal pyrin domain of NLRP12 (NLRP12 PYD) determined using NMR spectroscopy. NLRP12 is a non-inflammasome NLR that has been implicated in the regulation of TLR-dependent NF-κB activation. NLRP12 PYD adopts a typical 6-helical bundle death domain fold. By direct comparison with other PYD structures, we identified hydrophobic residues that are essential for the stable fold of the NLRP PYD family. In addition, we report the first in vitro confirmed non-homotypic PYD interaction between NLRP12 PYD and the pro-apoptotic protein FAF-1, which links the innate immune system to apoptotic signaling. Interestingly, all residues that participate in this protein:protein interaction are confined to the α2-α3 surface, a region of NLRP12 PYD that differs most between currently reported NLRP PYD structures. Finally, we experimentally highlight a significant role for tryptophan 45 in the interaction between NLRP12 PYD and the FAF-1 UBA domain.

Microbial lipases are highly appreciated as biocatalysts due to their peculiar characteristics such as the ability to utilize a wide range of substrates, high activity and stability in organic solvents, and regio- and/or enantioselectivity. These enzymes are currently being applied in a variety of biotechnological processes, including detergent preparation, cosmetics and paper production, food processing, biodiesel and biopolymer synthesis, and the biocatalytic resolution of pharmaceutical derivatives, esters, and amino acids. However, in certain segments of industry, the use of lipases is still limited by their high cost. Thus, there is a great interest in obtaining low-cost, highly active, and stable lipases that can be applied in several different industrial branches. Currently, the design of specific enzymes for each type of process has been used as an important tool to address the limitations of natural enzymes. Nowadays, it is possible to “order” a “customized” enzyme that has ideal properties for the development of the desired bioprocess. This review aims to compile recent advances in the biotechnological application of lipases focusing on various methods of enzyme improvement, such as protein engineering (directed evolution and rational design), as well as the use of structural data for rational modification of lipases in order to create higher active and selective biocatalysts.

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Structural conversion of cellular prion protein (PrP C ) into scrapie PrP (PrP Sc ) and subsequent aggregation are key events associated with the onset of transmissible spongiform encephalopathies (TSEs). Experimental evidence supports the role of nucleic acids (NAs) in assisting this conversion. Here, we asked whether PrP undergoes liquid-liquid phase separation (LLPS) and if this process is modulated by NAs. To this end, two 25-mer DNA aptamers, A1 and A2, were selected against the globular domain of recombinant murine PrP (rPrP 90-231 ) using SELEX methodology. Multiparametric structural analysis of these aptamers revealed that A1 adopts a hairpin conformation. Aptamer binding caused partial unfolding of rPrP 90-231 and modulated its ability to undergo LLPS and fibrillate. In fact, although free rPrP phase separated into large droplets, aptamer binding increased the number of droplets but noticeably reduced their size. Strikingly, a modified A1 aptamer that does not adopt a hairpin structure induced formation of amyloid fibrils on the surface of the droplets. We show here that PrP undergoes LLPS, and that the PrP interaction with NAs modulates phase separation and promotes PrP fibrillation in a NA structure and concentration-dependent manner. These results shed new light on the roles of NAs in PrP misfolding and TSEs. |

NLRP4 is a member of the nucleotide-binding and leucine-rich repeat receptor (NLR) family of cytosolic receptors and a member of an inflammation signaling cascade. Here, we present the crystal structure of the NLRP4 pyrin domain (PYD) at 2.3 Å resolution. The NLRP4 PYD is a member of the death domain (DD) superfamily and adopts a DD fold consisting of six α-helices tightly packed around a hydrophobic core, with a highly charged surface that is typical of PYDs. Importantly, however, we identified several differences between the NLRP4 PYD crystal structure and other PYD structures that are significant enough to affect NLRP4 function and its interactions with binding partners. Notably, the length of helix α3 and the α2−α3 connecting loop in the NLRP4 PYD are unique among PYDs. The apoptosis-associated speck-like protein containing a CARD (ASC) is an adaptor protein whose interactions with a number of distinct PYDs are believed to be critical for activation of the inflammatory response. Here, we use co-immunoprecipitation, yeast two-hybrid, and nuclear magnetic resonance chemical shift perturbation analysis to demonstrate that, despite being important for activation of the inflammatory response and sharing several similarities with other known ASC-interacting PYDs (i.e., ASC2), NLRP4 does not interact with the adaptor protein ASC. Thus, we propose that the factors governing homotypic PYD interactions are more complex than the currently accepted model, which states that complementary charged surfaces are the main determinants of PYD–PYD interaction specificity.

Fibrillization of the protein a-synuclein (a-syn) is a hallmark of Parkinson's disease and other a-synucleinopathies. The well-established idea that a-syn is a natively disordered monomer prone to forming fibrils was recently challenged by data showing that the protein mostly exists in vitro and in vivo as helically folded tetramers that are resistant to fibrillization. These apparently conflicting findings may be reconciled by the idea that a-syn exists as a disordered monomer in equilibrium with variable amounts of dynamic oligomeric species. In this context, varying the approaches used for protein purification, such as the method used to lyse cells or the inclusion of denaturing agents, could dramatically perturb this equilibrium and hence alter the relative abundance of the disordered monomer. In the present study, we investigated how the current methods for a-syn purification affect the structure and oligomeric state of the protein, and we discuss the main pitfalls associated with the production of recombinant a-syn in Escherichia coli. We demonstrate that a-syn was expressed in E. coli as a disordered monomer independent of both the cell lysis method and the use of heating/acidification for protein purification. In addition, we provide convincing evidence that the disordered monomer exists in equilibrium with a dynamic dimer, which is not an artefact of the cross-linking protocol as previously suggested. Unlike the helically folded tetramer, a-syn dimer is prone to fibrillate and thus it may be an interesting target for anti-fibrillogenic molecules. Structured digital abstractaplha-Syn and aplha-Syn bind by cross-linking study (View interaction) aplha-Syn and aplha-Syn bind by detection by mass spectrometry (1, 2) aplha-Syn and aplha-Syn bind by molecular sieving (View interaction) aplha-Syn and aplha-Syn bind by circular dichroism (View interaction)

Muscle relaxation is triggered by the dephosphorylation of Ser19 in the myosin regulatory light chain. This reaction is catalyzed by the holoenzyme myosin phosphatase (MP), which includes the catalytic subunit protein phosphatase 1 (PP1) and the regulatory targeting subunit (MYPT). MYPT1 (myosin phosphatase targeting subunit 1) is responsible for both targeting the holoenzyme to subcellular compartments in the muscle and directing PP1 specificity towards myosin. In order to understand the molecular events leading to the MYPT1:PP1 holoenzyme formation, we used NMR spectroscopy to determine the structural and dynamic characteristics of unbound MYPT1. This allowed the conformations of MYPT1 in the free, unbound state to be directly compared to the PP1-bound state. Our results show that MYPT11-98 behaves like a two-domain protein in solution. The first 40 residues of MYPT11-98, the disordered region, are intrinsically disordered and highly dynamic, whereas residues 41–98, the folded ankyrin-repeat region, are well-structured and rigid. Furthermore, the integrated use of NMR and biophysical data enabled us to calculate an ensemble model for MYPT11-98. The most prominent structural feature of the MYPT11-98 ensemble is a 25% populated transient α-helix in the disordered region of MYPT11-98. This α-helix becomes fully populated when bound to PP1 and, as we show, likely plays a central role in the formation of the MYPT1:PP1 holoenzyme complex. Finally, this combined analysis shows that the structural and dynamic behaviors exhibited by MYPT1 for PP1 are distinct from those of any other previously analyzed PP1 regulatory protein. Collectively, these data enable us to present a new model of the molecular events that drive MYPT1:PP1 holoenzyme formation and demonstrate that there are structural differences in unbound PP1 regulators that have not been previously observed. Thus this work adds significant insights to the currently limited data for molecular structures and dynamics of PP1 regulators.