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Specialized intramembrane proteases, known as iCLiPs, regulate the processing of transmembrane proteins by releasing intracellular domains, which can function as transcriptional regulators. The signal peptide peptidase-like (SPPL) family of iCLiPs, particularly SPPL2b, has roles in immune regulation, neuronal function, and disease pathogenesis. In the brain, SPPL2b localizes mainly in the plasma membrane of neurons and microglia and is abundant in the cortex and hippocampus. Its known substrates regulate neuronal growth, inflammation, and synaptic function, and increased amounts of SPPL2b have been found in postmortem brain tissue from patients with Alzheimer's disease. In this review, we discuss the currently known roles of SPPL2b, its substrates, and its disease implications. Understanding the downstream effects of SPPL2b-cleaved substrates will provide clearer insights into the impact of SPPL2b on cellular homeostasis and disease, potentially leading to new therapeutic strategies.

At the heart of many nucleoside triphosphatases is a conserved phosphate-binding sequence motif. A current model of early enzyme evolution proposes that this six to eight residue motif could have sparked the emergence of the very first nucleoside triphosphatases—a striking example of evolutionary continuity from simple beginnings, if true. To test this provocative model, seven disembodied Walker A-derived peptides were extensively computationally characterized. Although dynamic flickers of nest-like conformations were observed, significant structural similarity between the situated peptide and its disembodied counterpart was not detected. Simulations suggest that phosphate binding is nonspecific, with a preference for GTP over orthophosphate. Control peptides with the same amino acid composition but different sequences and situated conformations behaved similarly to the Walker A peptides, revealing no indication that the Walker A sequence is privileged as a disembodied peptide. We conclude that the evolutionary history of the P-loop NTPase family is unlikely to have started with a disembodied Walker A peptide in an aqueous environment. The limits of evolutionary continuity for this protein family must be reconsidered. Finally, we argue that motifs such as the Walker A motif may represent incomplete or fragmentary molecular fossils—the true nature of which has been eroded by time.

A new, environmentally friendly lactate oxidase (LOX) based biosensor for lactate detection, with unprecedented reuse and storage capabilities at room temperature, has been manufactured using the ambient electrospray deposition (ESD) technique. This technology allows for an efficient, green and easy ambient soft-landing immobilization of the LOX enzyme on a cheap commercial screen-printed Prussian blue/carbon electrode (PB/C-SPE), employing sustainable chemistry. This study shows how ESD can confer the biosensor the ability to be stored at ambient pressure and temperature for long periods without compromising the enzymatic activity. The fabricated biosensor shows a storage capability for up to 90 days, without any particular care under storage conditions, and a reuse performance for up to 24 measurements on both the electrode just prepared and on a three-months-old electrode. The LOX-based biosensor has been tested for lactate detection in the linear range of 0.1–1 mM with a limit of detection of 0.07 ± 0.02 mM and does not show any memory effects. The absence of an entrapment matrix as well as any additional hazardous chemicals during the immobilization phase makes the process competitive in terms of environmental sustainability and toxicity. Moreover, the application of a new electrospray deposition cycle on the used biosensors makes the biosensors work again with performances comparable to those of freshly made ones. This demonstrates that the technique is excellent for recycling and eliminates the waste of disposable devices.

Enzymes are conformationally dynamic, and their dynamical properties play an important role in regulating their specificity and evolvability. In this context, substantial attention has been paid to the role of ligand-gated conformational changes in enzyme catalysis; however, such studies have focused on tremendously proficient enzymes such as triosephosphate isomerase and orotidine 5'-monophosphate decarboxylase, where the rapid (mu s timescale) motion of a single loop dominates the transition between catalytically inactive and active conformations. In contrast, the (beta alpha)(8)-barrels of tryptophan and histidine biosynthesis, such as the specialist isomerase enzymes HisA and TrpF, and the bifunctional isomerase PriA, are decorated by multiple long loops that undergo conformational transitions on the ms (or slower) timescale. Studying the interdependent motions of multiple slow loops, and their role in catalysis, poses a significant computational challenge. This work combines conventional and enhanced molecular dynamics simulations with empirical valence bond simulations to provide rich details of the conformational behavior of the catalytic loops in HisA, PriA, and TrpF, and the role of their plasticity in facilitating bifunctionality in PriA and evolved HisA variants. In addition, we demonstrate that, similar to other enzymes activated by ligand-gated conformational changes, loops 3 and 4 of HisA and PriA act as gripper loops, facilitating the isomerization of the large bulky substrate ProFAR, albeit now on much slower timescales. This hints at convergent evolution on these different (beta alpha)(8)-barrel scaffolds. Finally, our work reemphasizes the potential of engineering loop dynamics as a tool to artificially manipulate the catalytic repertoire of TIM-barrel proteins.

The cooperative interplay between the functional devices of a preorganized active site is fundamental to enzyme catalysis. An in-depth understanding of this phenomenon is central to elucidating the remarkable efficiency of natural enzymes and provides an essential benchmark for enzyme design and engineering. Here, we study the functional interconnectedness of the catalytic nucleophile (His18) in an acid phosphatase by analyzing the consequences of its replacement with aspartate. We present crystallographic, biochemical, and computational evidence for a conserved mechanistic pathway via a phospho-enzyme intermediate on Asp18. Linear free-energy relationships for phosphoryl transfer from phosphomonoester substrates to His18/Asp18 provide evidence for the cooperative interplay between the nucleophilic and general-acid catalytic groups in the wild-type enzyme, and its substantial loss in the H18D variant. As an isolated factor of phosphatase efficiency, the advantage of a histidine compared to an aspartate nucleophile is similar to 10(4)-fold. Cooperativity with the catalytic acid adds >= 10(2)-fold to that advantage. Empirical valence bond simulations of phosphoryl transfer from glucose 1-phosphate to His and Asp in the enzyme explain the loss of activity of the Asp18 enzyme through a combination of impaired substrate positioning in the Michaelis complex, as well as a shift from early to late protonation of the leaving group in the H18D variant. The evidence presented furthermore suggests that the cooperative nature of catalysis distinguishes the enzymatic reaction from the corresponding reaction in solution and is enabled by the electrostatic preorganization of the active site. Our results reveal sophisticated discrimination in multifunctional catalysis of a highly proficient phosphatase active site.

One of the most powerful technologies for single molecule fluorescence spectroscopy at concentrations which are close to biological processes (micro and millimolar) is based on the use of Zero Mode Waveguides (ZMWs). Standard ZMWs are prepared as nanoholes array in Al thin films. Although very efficient to enable single molecule detection, Al-ZMWs are not optimized in term of fluorescence emission. On the contrary an engineered configuration where alternative metals such as Au, Ag, Al, etc. are used can produce significant enhancement in the single molecule detection. Thanks to optimized designs can be possible to enhance the fluorescence emission not only in the visible spectral range but also in the Ultraviolet (UV) and Near-infrared (NIR). Here we discuss the major results on plasmonic ZMWs and their use in single molecule fluorescence.

In the framework of magneto-photonics, the optical properties of a material can controlled by an external magnetic field, providing active functionalities for applications, such as sensing and nonreciprocal optical isolation. For noble metals in particular, the inherently weak magnetooptical coupling of the bulk material can be greatly enhanced via excitation of localized surface plasmons (LSP) in nanostructured samples. Hyperbolic metamaterials therein provide the ideal platform to tune the plasmonic properties via careful design of the effective permittivity tensor. Here, we report on the magnetic circular dichroism of electric and magnetic dipole modes of a type II hyperbolic metasurface. Disk-shaped nanoparticles consist in stacks of alternating dielectric and metallic layers. Using an effective medium theory, we show that the optical properties of the system can be perfectly described by an anisotropic homogenized permittivity. Magnetic circular dichroism spectroscopy experiments are compared with plain gold disk samples and reveal a broadband magneto-optical response across the visible and near infrared spectral range. In particular, derivative-like spectral signatures at the resonances of the nanoparticles prove the induced dichroism for the two modes of the system. Results are interpreted in terms of magnetically induced spatial confinement/broadening of circular currents in the nanoparticles and are compared with a comprehensive numerical model based on the finite elements method using the real dimensions of the nanostructure. Spherical particles are employed as an analytical model system, allowing to generalize the contribution of electric and magnetic modes to the total magneto-optical response. More in detail, interaction cross sections are calculated as a weighted sum of the corresponding Mie coefficients. Utilizing a perturbative approach, we describe the magneto-optical effect in terms of linear changes in the cyclotron frequency of free charge carriers in the metal. By comparing our analytical model with full-wave numerical results, we can identify the contribution of electric and magnetic dipole modes to the spectrum and reproduce the spectral line shape we observe in the experiments for the hyperbolic nanoparticles.

Active nanophotonics can be realized by controlling the optical properties of materials with external magnetic fields. Here, we explore the influence of optical anisotropy on the magneto-optical activity in nonmagnetic hyperbolic nanoparticles. We demonstrate that the magneto-optical response is driven by the hyperbolic dispersion via the coupling of metallic-induced electric and dielectric-induced magnetic dipolar optical modes with static magnetic fields. Magnetic circular dichroism experiments confirm the theoretical predictions and reveal tunable magneto-optical activity across the visible and near infrared spectral range.

Hyperbolic nanoparticles provide a versatile platform to widely tune light-matter interactions. Active nanophotonics can be realized by controlling the optical properties of materials with external magnetic fields. Here, we explore the influence of optical anisotropy on the magneto-optical response of hyperbolic nanoparticles across the visible and near infrared spectral range. By using a perturbative approach, we establish a model where the magneto-optical activity of the system is described in terms of the coupling of fundamental electric and magnetic dipole modes, which are induced by the hyperbolic dispersion, with a static magnetic field. Finally, an analytical model is established in the framework of Mie theory to describe the magneto-optical response and identify the contribution of electric and magnetic modes to the total spectrum.

DNA-binding proteins play an important role in gene regulation and cellular function. The transcription factors MarA and Rob are two homologous members of the AraC/XylS family that regulate multidrug resistance. They share a common DNA-binding domain, and Rob possesses an additional C-terminal domain that permits binding of low-molecular weight effectors. Both proteins possess two helix-turn-helix (HTH) motifs capable of binding DNA; however, while MarA interacts with its promoter through both HTH-motifs, prior studies indicate that Rob binding to DNA via a single HTH-motif is sufficient for tight binding. In the present work, we perform microsecond time scale all-atom simulations of the binding of both transcription factors to different DNA sequences to understand the determinants of DNA recognition and binding. Our simulations characterize sequence-dependent changes in dynamical behavior upon DNA binding, showcasing the role of Arg40 of the N-terminal HTH-motif in allowing for specific tight binding. Finally, our simulations demonstrate that an acidic C-terminal loop of Rob can control the DNA binding mode, facilitating interconversion between the distinct DNA binding modes observed in MarA and Rob. In doing so, we provide detailed molecular insight into DNA binding and recognition by these proteins, which in turn is an important step toward the efficient design of antivirulence agents that target these proteins.