Complex biophysical changes and reduced neuronal firing in an SCN8A variant associated with developmental delay and epilepsy.

Mutations in the SCN8A gene, encoding the voltage-gated sodium channel NaV1.6, are associated with a range of neurodevelopmental syndromes. The p.(Gly1625Arg) (G1625R) mutation was identified in a patient diagnosed with developmental epileptic encephalopathy (DEE). While most of the characterized DEE-associated SCN8A mutations were shown to cause a gain-of-channel function, we show that the G1625R variant, positioned within the S4 segment of domain IV, results in complex effects. Voltage-clamp analyses of NaV1.6G1625R demonstrated a mixture of gain- and loss-of-function properties, including reduced current amplitudes, increased time constant of fast voltage-dependent inactivation, a depolarizing shift in the voltage dependence of activation and inactivation, and increased channel availability with high-frequency repeated depolarization. Current-clamp analyses in transfected cultured neurons revealed that these biophysical properties caused a marked reduction in the number of action potentials when firing was driven by the transfected mutant NaV1.6. Accordingly, computational modeling of mature cortical neurons demonstrated a mild decrease in neuronal firing when mimicking the patients' heterozygous SCN8A expression. Structural modeling of NaV1.6G1625R suggested the formation of a cation-π interaction between R1625 and F1588 within domain IV. Double-mutant cycle analysis revealed that this interaction affects the voltage dependence of inactivation in NaV1.6G1625R. Together, our studies demonstrate that the G1625R variant leads to a complex combination of gain and loss of function biophysical changes that result in an overall mild reduction in neuronal firing, related to the perturbed interaction network within the voltage sensor domain, necessitating personalized multi-tiered analysis for SCN8A mutations for optimal treatment selection.

Versatility of reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) from diagnosis of early pathological infection to mutation detection in organisms.

Loop-mediated isothermal amplification (LAMP) is a rapid, state-of-the-art DNA amplification technology, used primarily for the quick diagnosis and early identification of microbial infection, caused by pathogens such as virus, bacteria and malaria. A target DNA can be amplified within 30 min using the LAMP reaction, taking place at a steady temperature. The LAMP method uses four or six primers to bind eight regions of a target DNA and has a very high specificity. The devices used for conducting LAMP are usually simple since the LAMP method is an isothermal process. When LAMP is coupled with Reverse Transcription (RT), it allows direct detection of RNA in a sample. This greatly enhances the efficiency of diagnosis of RNA viruses in a sample. Recently, the rampant spread of COVID-19 demanded such a rapid, simple, and cost-effective Point of Care Test (PoCT) for the accurate diagnosis of this pandemic. Loop-mediated isothermal amplification (LAMP) assays are not only used for the detection of microbial pathogens, but there are various other applications such as detection of genetic mutations in food and various organisms. In this review, various implementations of RT-LAMP techniques would be discussed.

A concise guide to choosing suitable gene expression systems for recombinant protein production.

This overview guides both novices and experienced researchers facing challenging targets to select the most appropriate gene expression system for producing a particular protein. By answering four key questions, readers can determine the most suitable gene expression system following a decision scheme. This guide addresses the most commonly used and accessible systems and provides brief descriptions of the main gene expression systems' key characteristics to assist decision making. Additionally, information has been included for selected less frequently used "exotic" gene expression systems.

Specific quinone reductase 2 inhibitors reduce metabolic burden and reverse Alzheimer's disease phenotype in mice.

Biological aging can be described as accumulative, prolonged metabolic stress and is the major risk factor for cognitive decline and Alzheimer's disease (AD). Recently, we identified and described a quinone reductase 2 (QR2) pathway in the brain, in which QR2 acts as a removable memory constraint and metabolic buffer within neurons. QR2 becomes overexpressed with age, and it is possibly a novel contributing factor to age-related metabolic stress and cognitive deficit. We found that, in human cells, genetic removal of QR2 produced a shift in the proteome opposing that found in AD brains while simultaneously reducing oxidative stress. We therefore created highly specific QR2 inhibitors (QR2is) to enable evaluation of chronic QR2 inhibition as a means to reduce biological age-related metabolic stress and cognitive decline. QR2is replicated results obtained by genetic removal of QR2, while local QR2i microinjection improved hippocampal and cortical-dependent learning in rats and mice. Continuous consumption of QR2is in drinking water improved cognition and reduced pathology in the brains of AD-model mice (5xFAD), with a noticeable between-sex effect on treatment duration. These results demonstrate the importance of QR2 activity and pathway function in the healthy and neurodegenerative brain and what we believe to be the great therapeutic potential of QR2is as first-in-class drugs.

Design of a stable human acid-ß-glucosidase: towards improved Gaucher disease therapy and mutation classification.

Acid-ß-glucosidase (GCase, EC3.2.1.45), the lysosomal enzyme which hydrolyzes the simple glycosphingolipid, glucosylceramide (GlcCer), is encoded by the GBA1 gene. Biallelic mutations in GBA1 cause the human inherited metabolic disorder, Gaucher disease (GD), in which GlcCer accumulates, while heterozygous GBA1 mutations are the highest genetic risk factor for Parkinson's disease (PD). Recombinant GCase (e.g., Cerezyme® ) is produced for use in enzyme replacement therapy for GD and is largely successful in relieving disease symptoms, except for the neurological symptoms observed in a subset of patients. As a first step toward developing an alternative to the recombinant human enzymes used to treat GD, we applied the PROSS stability-design algorithm to generate GCase variants with enhanced stability. One of the designs, containing 55 mutations compared to wild-type human GCase, exhibits improved secretion and thermal stability. Furthermore, the design has higher enzymatic activity than the clinically used human enzyme when incorporated into an AAV vector, resulting in a larger decrease in the accumulation of lipid substrates in cultured cells. Based on stability-design calculations, we also developed a machine learning-based approach to distinguish benign from deleterious (i.e., disease-causing) GBA1 mutations. This approach gave remarkably accurate predictions of the enzymatic activity of single-nucleotide polymorphisms in the GBA1 gene that are not currently associated with GD or PD. This latter approach could be applied to other diseases to determine risk factors in patients carrying rare mutations.

Determining the targeting specificity of the selective peroxisomal targeting factor Pex9.

Accurate and regulated protein targeting is crucial for cellular function and proteostasis. In the yeast Saccharomyces cerevisiae, peroxisomal matrix proteins, which harboring a Peroxisomal Targeting Signal 1 (PTS1), can utilize two paralog targeting factors, Pex5 and Pex9, to target correctly. While both proteins are similar and recognize PTS1 signals, Pex9 targets only a subset of Pex5 cargo proteins. However, what defines this substrate selectivity remains uncovered. Here, we used unbiased screens alongside directed experiments to identify the properties underlying Pex9 targeting specificity. We find that the specificity of Pex9 is largely determined by the hydrophobic nature of the amino acid preceding the PTS1 tripeptide of its cargos. This is explained by structural modeling of the PTS1-binding cavities of the two factors showing differences in their surface hydrophobicity. Our work outlines the mechanism by which targeting specificity is achieved, enabling dynamic rewiring of the peroxisomal proteome in changing metabolic needs.

Systematic multi-level analysis of an organelle proteome reveals new peroxisomal functions.

Seventy years following the discovery of peroxisomes, their complete proteome, the peroxi-ome, remains undefined. Uncovering the peroxi-ome is crucial for understanding peroxisomal activities and cellular metabolism. We used high-content microscopy to uncover peroxisomal proteins in the model eukaryote - Saccharomyces cerevisiae. This strategy enabled us to expand the known peroxi-ome by ~40% and paved the way for performing systematic, whole-organellar proteome assays. By characterizing the sub-organellar localization and protein targeting dependencies into the organelle, we unveiled non-canonical targeting routes. Metabolomic analysis of the peroxi-ome revealed the role of several newly identified resident enzymes. Importantly, we found a regulatory role of peroxisomes during gluconeogenesis, which is fundamental for understanding cellular metabolism. With the current recognition that peroxisomes play a crucial part in organismal physiology, our approach lays the foundation for deep characterization of peroxisome function in health and disease.

Rhodopsin-bestrophin fusion proteins from unicellular algae form gigantic pentameric ion channels.

Many organisms sense light using rhodopsins, photoreceptive proteins containing a retinal chromophore. Here we report the discovery, structure and biophysical characterization of bestrhodopsins, a microbial rhodopsin subfamily from marine unicellular algae, in which one rhodopsin domain of eight transmembrane helices or, more often, two such domains in tandem, are C-terminally fused to a bestrophin channel. Cryo-EM analysis of a rhodopsin-rhodopsin-bestrophin fusion revealed that it forms a pentameric megacomplex (~700 kDa) with five rhodopsin pseudodimers surrounding the channel in the center. Bestrhodopsins are metastable and undergo photoconversion between red- and green-absorbing or green- and UVA-absorbing forms in the different variants. The retinal chromophore, in a unique binding pocket, photoisomerizes from all-trans to 11-cis form. Heterologously expressed bestrhodopsin behaves as a light-modulated anion channel.

Application of Restriction Free (RF) Cloning in Circular Permutation.

The restriction free (RF) cloning has emerged as one of the highly efficient techniques in the area of genetic engineering. RF cloning has wide range of applications in plasmid DNA manipulation including cloning of a single gene, simultaneous assembly of multiple DNA fragments, and mutagenesis from single to multiple simultaneous alterations of a target DNA. Recently, we have developed a new technique of circular permutation using RF cloning. Circular permutation is widely used to investigate the mechanisms of protein folding and function. Previously, restriction enzyme based cloning was used to introduce circular permutation. Our RF cloning method made the protocol faster and more cost-effective. In this chapter, we describe a step-by-step protocol for generating circular permutants using RF methodology.

The identification and characterization of an oxalyl-CoA synthetase from grass pea (Lathyrus sativus L.).

Oxalic acid is a small metabolite found in many plants. It serves as protection from herbivores, a chelator of metal ions, a regulator of calcium levels, and additional tasks. However, it is also a strong di-carboxylic acid that can compromise plant viability by reducing cellular pH. Several metabolic pathways have evolved to control oxalate levels in plants by enzymatic degradation. Among them is the pathway that utilizes oxalyl-CoA synthetase (OCS, EC 6.2.1.8) and ATP to convert oxalate to oxalyl-CoA. Oxalyl-CoA can then be degraded to CO2 or utilized as a precursor for the synthesis of other compounds. In grass pea (Lathyrus sativus L.), a grain legume grown in Asia and Africa for human and animal consumption, the neurotoxic compound ß-N-oxalyl-l-α,ß-diaminopropionic acid (ß-ODAP) is synthesized from oxalyl-CoA and l-α,ß-diaminopropionic acid (l-DAPA). Here, we report on the identification and characterization of oxalyl CoA-synthetase from grass pea (LsOCS). The gene encoding LsOCS was amplified from grass pea, and then expressed and purified from E. coli cells as an untagged, monomeric protein of 56 kDa. Its catalytic efficiency with oxalate, K oxalate M = 71.5 ± 13.3 µM, V max = 8.2 ± 0.8 µmole min-1 mg-1, was similar to that of OCS homologs from Arabidopsis thaliana (AtAAE3) and Medicago truncatula (MtAAE3). The enzyme was crystalized in complex with AMP and is the first OCS whose structure was determined in the thioester-forming conformation. Finally, we propose that substituting LsOCS with an oxalate oxidase or decarboxylase may reduce the levels of ß-ODAP in grass pea.

Identification and characterization of the key enzyme in the biosynthesis of the neurotoxin ß-ODAP in grass pea.

Grass pea (Lathyrus sativus L.) is a grain legume commonly grown in Asia and Africa for food and forage. It is a highly nutritious and robust crop, capable of surviving both droughts and floods. However, it produces a neurotoxic compound, ß-N-oxalyl-L-α,ß-diaminopropionic acid (ß-ODAP), which can cause a severe neurological disorder when consumed as a primary diet component. While the catalytic activity associated with ß-ODAP formation was demonstrated more than 50 years ago, the enzyme responsible for this activity has not been identified. Here, we report on the identity, activity, 3D structure, and phylogenesis of this enzyme-ß-ODAP synthase (BOS). We show that BOS belongs to the benzylalcohol O-acetyltransferase, anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-hydroxycinnamoyl/benzoyltransferase, deacetylvindoline 4-O-acetyltransferase superfamily of acyltransferases and is structurally similar to hydroxycinnamoyl transferase. Using molecular docking, we propose a mechanism for its catalytic activity, and using heterologous expression in tobacco leaves (Nicotiana benthamiana), we demonstrate that expression of BOS in the presence of its substrates is sufficient for ß-ODAP production in vivo. The identification of BOS may pave the way toward engineering ß-ODAP-free grass pea cultivars, which are safe for human and animal consumption.

Computationally designed dual-color MRI reporters for noninvasive imaging of transgene expression.

Imaging of gene-expression patterns in live animals is difficult to achieve with fluorescent proteins because tissues are opaque to visible light. Imaging of transgene expression with magnetic resonance imaging (MRI), which penetrates to deep tissues, has been limited by single reporter visualization capabilities. Moreover, the low-throughput capacity of MRI limits large-scale mutagenesis strategies to improve existing reporters. Here we develop an MRI system, called GeneREFORM, comprising orthogonal reporters for two-color imaging of transgene expression in deep tissues. Starting from two promiscuous deoxyribonucleoside kinases, we computationally designed highly active, orthogonal enzymes ('reporter genes') that specifically phosphorylate two MRI-detectable synthetic deoxyribonucleosides ('reporter probes'). Systemically administered reporter probes exclusively accumulate in cells expressing the designed reporter genes, and their distribution is displayed as pseudo-colored MRI maps based on dynamic proton exchange for noninvasive visualization of transgene expression. We envision that future extensions of GeneREFORM will pave the way to multiplexed deep-tissue mapping of gene expression in live animals.

Origin of life: protoribosome forms peptide bonds and links RNA and protein dominated worlds.

Although the mode of action of the ribosomes, the multi-component universal effective protein-synthesis organelles, has been thoroughly explored, their mere appearance remained elusive. Our earlier comparative structural studies suggested that a universal internal small RNA pocket-like segment called by us the protoribosome, which is still embedded in the contemporary ribosome, is a vestige of the primordial ribosome. Herein, after constructing such pockets, we show using the "fragment reaction" and its analyses by MALDI-TOF and LC-MS mass spectrometry techniques, that several protoribosome constructs are indeed capable of mediating peptide-bond formation. These findings present strong evidence supporting our hypothesis on origin of life and on ribosome's construction, thus suggesting that the protoribosome may be the missing link between the RNA dominated world and the contemporary nucleic acids/proteins life.

De novo developed protein binders mimicking Interferon lambda signaling.

We hereby describe the process of design and selection of nonantibody protein binders mimicking cytokine signaling. We chose to mimic signaling of IFN-λ1, type 3 interferon (also known as IL-29) for its novelty and the importance of its biological functions. All four known interferons λ signal through binding to the extracellular domains of IL-28 receptor 1 (IL-28R1) and IL-10 receptor 2 (IL-10R2). Our binders were therefore trained to bind both receptors simultaneously. The bifunctional binder molecules were developed by yeast display, a method of directed evolution. The signaling capacity of the bivalent binders was tested by measuring phosphorylation of the JAK/STAT signaling pathway and production of mRNA of six selected genes naturally induced by IFN- λ1 in human cell lines. The newly developed bivalent binders offer opportunities to study cytokine-related biological functions and modulation of the cell behavior by receptor activation on the cell surfaces alternative to the use of natural IFN-λ.

Climbing Up and Down Binding Landscapes through Deep Mutational Scanning of Three Homologous Protein-Protein Complexes.

Protein-protein interactions (PPIs) have evolved to display binding affinities that can support their function. As such, cognate and noncognate PPIs could be highly similar structurally but exhibit huge differences in binding affinities. To understand this phenomenon, we study three homologous protease-inhibitor PPIs that span 9 orders of magnitude in binding affinity. Using state-of-the-art methodology that combines protein randomization, affinity sorting, deep sequencing, and data normalization, we report quantitative binding landscapes consisting of ΔΔGbind values for the three PPIs, gleaned from tens of thousands of single and double mutations. We show that binding landscapes of the three complexes are strikingly different and depend on the PPI evolutionary optimality. We observe different patterns of couplings between mutations for the three PPIs with negative and positive epistasis appearing most frequently at hot-spot and cold-spot positions, respectively. The evolutionary trends observed here are likely to be universal to other biological complexes in the cell.

Engineered variants of the Ras effector protein RASSF5 (NORE1A) promote anticancer activities in lung adenocarcinoma.

Within the superfamily of small GTPases, Ras appears to be the master regulator of such processes as cell cycle progression, cell division, and apoptosis. Several oncogenic Ras mutations at amino acid positions 12, 13, and 61 have been identified that lose their ability to hydrolyze GTP, giving rise to constitutive signaling and eventually development of cancer. While disruption of the Ras/effector interface is an attractive strategy for drug design to prevent this constitutive activity, inhibition of this interaction using small molecules is impractical due to the absence of a cavity to which such molecules could bind. However, proteins and especially natural Ras effectors that bind to the Ras/effector interface with high affinity could disrupt Ras/effector interactions and abolish procancer pathways initiated by Ras oncogene. Using a combination of computational design and in vitro evolution, we engineered high-affinity Ras-binding proteins starting from a natural Ras effector, RASSF5 (NORE1A), which is encoded by a tumor suppressor gene. Unlike previously reported Ras oncogene inhibitors, the proteins we designed not only inhibit Ras-regulated procancer pathways, but also stimulate anticancer pathways initiated by RASSF5. We show that upon introduction into A549 lung carcinoma cells, the engineered RASSF5 mutants decreased cell viability and mobility to a significantly greater extent than WT RASSF5. In addition, these mutant proteins induce cellular senescence by increasing acetylation and decreasing phosphorylation of p53. In conclusion, engineered RASSF5 variants provide an attractive therapeutic strategy able to oppose cancer development by means of inhibiting of procancer pathways and stimulating anticancer processes.

Local Mutations Can Serve as a Game Changer for Global Protein Solvent Interaction.

Although it is well-known that limited local mutations of enzymes, such as matrix metalloproteinases (MMPs), may change enzyme activity by orders of magnitude as well as its stability, the completely rational design of proteins is still challenging. These local changes alter the electrostatic potential and thus local electrostatic fields, which impacts the dynamics of water molecules close the protein surface. Here we show by a combined computational design, experimental, and molecular dynamics (MD) study that local mutations have not only a local but also a global effect on the solvent: In the specific case of the matrix metalloprotease MMP14, we found that the nature of local mutations, coupled with surface morphology, have the ability to influence large patches of the water hydrogen-bonding network at the protein surface, which is correlated with stability. The solvent contribution can be experimentally probed via terahertz (THz) spectroscopy, thus opening the door to the exciting perspective of rational protein design in which a systematic tuning of hydration water properties allows manipulation of protein stability and enzymatic activity.

Malaria parasites both repress host CXCL10 and use it as a cue for growth acceleration.

Pathogens are thought to use host molecular cues to control when to initiate life-cycle transitions, but these signals are mostly unknown, particularly for the parasitic disease malaria caused by Plasmodium falciparum. The chemokine CXCL10 is present at high levels in fatal cases of cerebral malaria patients, but is reduced in patients who survive and do not have complications. Here we show a Pf 'decision-sensing-system' controlled by CXCL10 concentration. High CXCL10 expression prompts P. falciparum to initiate a survival strategy via growth acceleration. Remarkably, P. falciparum inhibits CXCL10 synthesis in monocytes by disrupting the association of host ribosomes with CXCL10 transcripts. The underlying inhibition cascade involves RNA cargo delivery into monocytes that triggers RIG-I, which leads to HUR1 binding to an AU-rich domain of the CXCL10 3'UTR. These data indicate that when the parasite can no longer keep CXCL10 at low levels, it can exploit the chemokine as a cue to shift tactics and escape.

Structure reveals the activation mechanism of the MC4 receptor to initiate satiation signaling.

Obesity is a global epidemic that causes morbidity and impaired quality of life. The melanocortin receptor 4 (MC4R) is at the crux of appetite, energy homeostasis, and body-weight control in the central nervous system and is a prime target for anti-obesity drugs. Here, we present the cryo-electron microscopy (cryo-EM) structure of the human MC4R-Gs signaling complex bound to the agonist setmelanotide, a cyclic peptide recently approved for the treatment of obesity. The work reveals the mechanism of MC4R activation, highlighting a molecular switch that initiates satiation signaling. In addition, our findings indicate that calcium (Ca2+) is required for agonist, but not antagonist, efficacy. These results fill a gap in the understanding of MC4R activation and could guide the design of future weight-management drugs.

DNase II mediates a parthanatos-like developmental cell death pathway in Drosophila primordial germ cells.

During Drosophila embryonic development, cell death eliminates 30% of the primordial germ cells (PGCs). Inhibiting apoptosis does not prevent PGC death, suggesting a divergence from the conventional apoptotic program. Here, we demonstrate that PGCs normally activate an intrinsic alternative cell death (ACD) pathway mediated by DNase II release from lysosomes, leading to nuclear translocation and subsequent DNA double-strand breaks (DSBs). DSBs activate the DNA damage-sensing enzyme, Poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) and the ATR/Chk1 branch of the DNA damage response. PARP-1 and DNase II engage in a positive feedback amplification loop mediated by the release of PAR polymers from the nucleus and the nuclear accumulation of DNase II in an AIF- and CypA-dependent manner, ultimately resulting in PGC death. Given the anatomical and molecular similarities with an ACD pathway called parthanatos, these findings reveal a parthanatos-like cell death pathway active during Drosophila development.