Inhibition of Porphyromonas gingivalis peptidyl arginine deiminase,a virulence factor,by antioxidant-rich Cratoxylum cochinchinense: In vitro and in silico evaluation

Porphyromonas gingivalis, the cause of periodontitis, is also linked to many systemic disorders due to its citrullination capability from a unique peptidyl arginine deiminase (PPAD). Protein citrullination is able to trigger an autoimmune response, increasing the severity of rheumatoid arthritis. The main objective of this study is to evaluate the inhibitory activity of Cratoxylym cochinchinense leaves extract towards the PPAD in vitro and in silico. Methanolic extract of Cratoxylum cochinchinense (CCM) was tested for total phenolic and flavonoid contents along with antioxidative assays. Inhibition of PPAD activities was conducted thereafter using recombinant PPAD in cell lysate. Phytocompounds postulated present in the CCM such as mangiferin, vismiaquinone A, δ-tocotrienol and α-tocotrienol and canophyllol were used as ligands in a simulated docking study against PPAD. Results obtained indicated high antioxidant potential in CCM while recording abundant phenolic (129.0 ± 2.5495 mg GA/g crude extract) and flavonoid (159.0 ± 2.1529 mg QE/g crude extract) contents. A dose-dependent inhibition of PPAD was observed when CCM was evaluated at various concentrations. CCM at 1 mg/mL exhibited citrulline concentration of 24.37 ± 3.25 mM which was 5 times lower than the negative control (114.23 ± 3.31 mM). Molecular docking simulation revealed that mangiferin and vismiaquinone A engaged in H-bonding and pi-pi interactions with important active site residues (Asp130, Arg152, Arg154 and Trp127) of PPAD and could be the potential phytochemicals that accounted for the inhibitory activities observed in the methanolic leaves extract. As such, CCM could be further explored for its therapeutic properties not only for periodontitis, but also for other systemic diseases like rheumatoid arthritis.     

Designer D-peptides targeting the N-terminal region of α-synuclein to prevent parkinsonian-associated fibrilization and cytotoxicity

The deposition of α-synuclein (αS) aggregates in the gut and the brain is ever present in cases of Parkinson's disease. While the central non-amyloidogenic-component (NAC) region of αS plays a critical role in fibrilization, recent studies have identified a specific sequence from within the N-terminal region (NTR, residues 36–42) as a key modulator of αS fibrilization. Due to the lack of effective therapeutics which specifically target αS aggregates, we have developed a strategy to prevent the aggregation and subsequent toxicity attributed to αS fibrilization utilizing NTR targeting peptides. In this study, L- and D-isoforms of a hexa- (VAQKTV-Aib, 77–82 NAC) and heptapeptide (GVLYVGS-Aib, 36–42 NTR) containing a self-recognition component unique to αS, as well as a C-terminal disruption element, were synthesized to target primary sequence regions of αS that modulate fibrilization. The D-peptide that targets the NTR (NTR-TP-D) was shown by ThT fluorescence assays and TEM to be the most effective at preventing fibril formation and elongation, as well as increasing the abundance of soluble monomeric αS. In addition, NTR-TP-D alters the conformation of destabilised monomers into a less aggregation-prone state and reduces the hydrophobicity of αS fibrils via fibril remodelling. Furthermore, both NTR-TP isoforms alleviate the cytotoxic effects of αS aggregates in both Neuro-2a and Caco-2 cells. Together, this study highlights how targeting the NTR of αS using D-isoform peptide inhibitors may effectively combat the deleterious effects of αS fibrilization and paves the way for future drug design to utilise such an approach to treat Parkinson's disease.     

Past,present, and perspectives of protein N-terminal methylation

The posttranslational methylation of the α-N-terminal amino group of proteins was first documented over 40 years ago, but the functional significance of this modification has been underexplored relative to lysine and arginine methylation. Increasing reports implicates α-N-terminal methylation as a widespread and critical regulator of mitosis, chromatin interactions, DNA repair, and translation fidelity. Here, we summarize advances in the current understanding of protein α-N-terminal methylation biological functions and mechanisms across eukaryotic organisms. Also, we describe the recent literature on substrate recognition and the discovery of potent and selective inhibitors for protein N-terminal methyltransferases. Finally, we summarize the emergent crosstalk between α-N-terminal methylation and other N-terminal modifications.     

Structural Basis of Drug Recognition by the Multidrug Transporter ABCG2

ABCG2 is an ATP-binding cassette (ABC) transporter whose function affects the pharmacokinetics of drugs and contributes to multidrug resistance of cancer cells. While its interaction with the endogenous substrate estrone-3-sulfate (E₁S) has been elucidated at a structural level, the recognition and recruitment of exogenous compounds is not understood at sufficiently high resolution. Here we present three cryo-EM structures of nanodisc-reconstituted, human ABCG2 bound to anticancer drugs tariquidar, topotecan and mitoxantrone. To enable structural insight at high resolution, we used Fab fragments of the ABCG2-specific monoclonal antibody 5D3, which binds to the external side of the transporter but does not interfere with drug-induced stimulation of ATPase activity. We observed that the binding pocket of ABCG2 can accommodate a single tariquidar molecule in a C-shaped conformation, similar to one of the two tariquidar molecules bound to ABCB1, where tariquidar acts as an inhibitor. We also found single copies of topotecan and mitoxantrone bound between key phenylalanine residues. Mutagenesis experiments confirmed the functional importance of two residues in the binding pocket, F439 and N436. Using 3D variability analyses, we found a correlation between substrate binding and reduced dynamics of the nucleotide binding domains (NBDs), suggesting a structural explanation for drug-induced ATPase stimulation. Our findings provide additional insight into how ABCG2 differentiates between inhibitors and substrates and may guide a rational design of new modulators and substrates.     

Cancer stem cell in prostate cancer progression,metastasis and therapy resistance

Prostate cancer (PCa) is the most diagnosed malignancy in the men worldwide. Cancer stem cells (CSCs) are the sub-population of cells present in the tumor which possess unique properties of self-renewal and multilineage differentiation thus thought to be major cause of therapy resistance, disease relapse, and mortality in several malignancies including PCa. CSCs have also been shown positive for the common stem cells markers such as ALDH EZH2, OCT4, SOX2, c-MYC, Nanog etc. Therefore, isolation and characterization of CSCs specific markers which may discriminate CSCs and normal stem cells are critical to selectively eliminate CSCs. Rapid advances in the field offers a theoretical explanation for many of the enduring uncertainties encompassing the etiology and an optimism for the identification of new stem-cell targets, development of reliable and efficient therapies in the future. The emerging reports have also provided unprecedented insights into CSCs plasticity, quiescence, renewal, and therapeutic response. In this review, we discuss the identification of PCa stem cells, their unique properties, stemness-driving pathways, new diagnostics, and therapeutic interventions.     

What does engram encode?: Heterogeneous memory engrams for different aspects of experience

Long-lasting synaptic changes within the neuronal network mediate memory. Neurons bearing such physical traces of memory (memory engram cells) are often equated with neurons expressing immediate early genes (IEGs) during a specific experience. However, past studies observed the expression of different IEGs in non-overlapping neurons or synaptic plasticity in neurons that do not express a particular IEG. Importantly, recent studies revealed that distinct subsets of neurons expressing different IEGs or even IEG negative-(yet active) neurons support different aspects of memory or computation, suggesting a more complex nature of memory engram cells than previously thought. In this short review, we introduce studies revealing such heterogeneous composition of the memory engram and discuss how the memory system benefits from it.     

Molecular Determinants of Human T-cell Leukemia Virus Type 1 Gag Targeting to the Plasma Membrane for Assembly

Assembly of human T-cell leukemia virus type 1 (HTLV-1) particles is initiated by the trafficking of virally encoded Gag polyproteins to the inner leaflet of the plasma membrane (PM). Gag–PM interactions are mediated by the matrix (MA) domain, which contains a myristoyl group (myr) and a basic patch formed by lysine and arginine residues. For many retroviruses, Gag–PM interactions are mediated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂]; however, previous studies suggested that HTLV-1 Gag–PM interactions and therefore virus assembly are less dependent on PI(4,5)P₂. We have recently shown that PI(4,5)P₂ binds directly to HTLV-1 unmyristoylated MA [myr(–)MA] and that myr(–)MA binding to membranes is significantly enhanced by inclusion of phosphatidylserine (PS) and PI(4,5)P₂. Herein, we employed structural, biophysical, biochemical, mutagenesis, and cell-based assays to identify residues involved in MA–membrane interactions. Our data revealed that the lysine-rich motif (Lys47, Lys48, and Lys51) constitutes the primary PI(4,5)P₂–binding site. Furthermore, we show that arginine residues 3, 7, 14 and 17 located in the unstructured N-terminus are essential for MA binding to membranes containing PS and/or PI(4,5)P₂. Substitution of lysine and arginine residues severely attenuated virus-like particle production, but only the lysine residues could be clearly correlated with reduced PM binding. These results support a mechanism by which HTLV-1 Gag targeting to the PM is mediated by a trio engagement of the myr group, Arg-rich and Lys-rich motifs. These findings advance our understanding of a key step in retroviral particle assembly.     

Uncovering post-translational modification-associated protein–protein interactions

In living systems, the chemical space and functional repertoire of proteins are dramatically expanded through the post-translational modification (PTM) of various amino acid residues. These modifications frequently trigger unique protein–protein interactions (PPIs) – for example with reader proteins that directly bind the modified amino acid residue – which leads to downstream functional outcomes. The modification of a protein can also perturb its PPI network indirectly, for example, through altering its conformation or subcellular localization. Uncovering the network of unique PTM-triggered PPIs is essential to fully understand the roles of an ever-expanding list of PTMs in our biology. In this review, we discuss established strategies and current challenges associated with this endeavor.     

Design and engineering of allosteric communications in proteins

Allostery in proteins plays an important role in regulating protein activities and influencing many biological processes such as gene expression, enzyme catalysis, and cell signaling. The process of allostery takes place when a signal detected at a site on a protein is transmitted via a mechanical pathway to a functional site and, thus, influences its activity. The pathway of allosteric communication consists of amino acids that form a network with covalent and non-covalent bonds. By mutating residues in this allosteric network, protein engineers have successfully established novel allosteric pathways to achieve desired properties in the target protein. In this review, we highlight the most recent and state-of-the-art techniques for allosteric communication engineering. We also discuss the challenges that need to be overcome and future directions for engineering protein allostery.     

Directing CAR T cells towards the tumor vasculature for the treatment of solid tumors

For successful application of chimeric antigen receptor (CAR) T cell therapy in solid tumors, major hurdles have to be overcome. CAR T cells have to cross the vascular barrier, which is hampered by the anergic state of the tumor vasculature, characterized by suppressed levels of leukocyte adhesion molecules on the endothelium. Additional immunosuppressive mechanisms in the solid tumor microenvironment can affect infiltration, activity and persistence of CAR T cells. Redirecting CAR T cells towards the tumor vasculature poses a possible solution, as molecular targets of tumor endothelial cells can be directly engaged from within the blood.In this review, we discuss recent advances in CAR T cell therapy against solid tumors, with a focus on targeting the tumor vasculature. Furthermore, we discuss opportunities to overcome challenges and barriers through engineering of CAR T cells to enhance trafficking, safety and efficacy.     

SOD mimics: From the tool box of the chemists to cellular studies

Superoxide dismutases (SODs) are metalloproteins that protect cells against oxidative stress by controlling the concentration of superoxide (O₂⁻) through catalysis of its dismutation. The activity of superoxide dismutases can be mimicked by low-molecular-weight complexes having potential therapeutic applications. This review presents recent strategies for designing efficient SOD mimics, from molecular metal complexes to nanomaterials. Studies of these systems in cells reveal that some SOD mimics, designed to react directly with superoxide, may also indirectly enhance the cellular antioxidant arsenal. Finally, a good understanding of the bioactivity requires information on the cell-penetration, speciation, and subcellular location of the SOD mimics: we will describe recent studies and new techniques that open opportunities for characterizing SOD mimics in biological environments.     

New Insights on Effects of Glucocorticoids in Patients With SARS-CoV-2 Infection

ObjectiveSince January 2020, the highly contagious novel coronavirus SARS-CoV-2 has caused a global pandemic. Severe COVID-19 leads to a massive release of proinflammatory mediators, leading to diffuse damage to the lung parenchyma, and the development of acute respiratory distress syndrome. Treatment with the highly potent glucocorticoid (GC) dexamethasone was found to be effective in reducing mortality in severely affected patients.MethodsTo review the effects of glucocorticoids in the context of COVID-19 we performed a literature search in the PubMed database using the terms COVID-19 and glucocorticoid treatment. We identified 1429 article publications related to COVID-19 and glucocorticoid published from 1.1.2020 to the present including 238 review articles and 36 Randomized Controlled Trials. From these studies, we retrieved 13 Randomized Controlled Trials and 86 review articles that were relevant to our review topics. We focused on the recent literature dealing with glucocorticoid metabolism in critically ill patients and investigating the effects of glucocorticoid therapy on the immune system in COVID-19 patients with severe lung injury.ResultsIn our review, we have discussed the regulation of the hypothalamic-pituitary-adrenal axis in patients with critical illness, selection of a specific GC for critical illness-related GC insufficiency, and recent studies that investigated hypothalamic-pituitary-adrenal dysfunction in patients with COVID-19. We have also addressed the specific activation of the immune system with chronic endogenous glucocorticoid excess, as seen in patients with Cushing syndrome, and, finally, we have discussed immune activation due to coronavirus infection and the possible mechanisms leading to improved outcomes in patients with COVID-19 treated with GCs.ConclusionFor clinical endocrinologists prescribing GCs for their patients, a precise understanding of both the molecular- and cellular-level mechanisms of endogenous and exogenous GCs is imperative, including timing of administration, dosage, duration of treatment, and specific formulations of GCs.     

Cholesterol metabolism: from lipidomics to immunology

Oxysterols, the oxidized forms of cholesterol or of its precursors, are formed in the first steps of cholesterol metabolism. Oxysterols have interested chemists, biologists, and physicians for many decades, but their exact biological relevance in vivo, other than as intermediates in bile acid biosynthesis, has long been debated. However, in the first quarter of this century, a role for side-chain oxysterols and their C-7 oxidized metabolites has been convincingly established in the immune system. 25-Hydroxycholesterol has been shown to be synthesized by macrophages in response to the activation of Toll-like receptors and to offer protection against microbial pathogens, whereas 7α,25-dihydroxycholesterol has been shown to act as a chemoattractant to lymphocytes expressing the G protein-coupled receptor Epstein-Barr virus-induced gene 2 and to be important in coordinating the action of B cells, T cells, and dendritic cells in secondary lymphoid tissue. There is a growing body of evidence that not only these two oxysterols but also many of their isomers are of importance to the proper function of the immune system. Here, we review recent findings related to the roles of oxysterols in immunology.     

Development of an efficient semisynthetic modification of FR901459 via a novel regioselective N,O-acyl migration

HCV utilizes cellular protein cyclophilins in the virus replication cycle and cyclophilin inhibitors have become a new class of anti-HCV agents. In our screening of natural products, we identified a unique cyclosporin analogue, FR901459, as a cyclophilin inhibitor with potent anti-HCV activity. In this work, we developed an efficient synthetic methodology to prepare FR901459 derivatives via an N, O-acyl migration reaction. This method allows us to efficiently manipulate the amino acid residues at the 3 position while avoiding lengthy total synthesis for each compound. By using this methodology, we discovered 4, which has superior anti-HCV activity and decreased immunosuppressive activity compared to FR901459.     

Oligodendrocyte-derived transcellular signaling regulates axonal energy metabolism

The unique morphology and functionality of central nervous system (CNS) neurons necessitate specialized mechanisms to maintain energy metabolism throughout long axons and extensive terminals. Oligodendrocytes (OLs) enwrap CNS axons with myelin sheaths in a multilamellar fashion. Apart from their well-established function in action potential propagation, OLs also provide intercellular metabolic support to axons by transferring energy metabolites and delivering exosomes consisting of proteins, lipids, and RNAs. OL-derived metabolic support is crucial for the maintenance of axonal integrity; its dysfunction has emerged as an important player in neurological disorders that are associated with axonal energy deficits and degeneration. In this review, we discuss recent advances in how these transcellular signaling pathways maintain axonal energy metabolism in health and neurological disorders.     

Mechanobiology of neural development

As the brain develops, proliferating cells organize into structures, differentiate, migrate, extrude long processes, and connect with other cells. These biological processes produce mechanical forces that further shape cellular dynamics and organ patterning. A major unanswered question in developmental biology is how the mechanical forces produced during development are detected and transduced by cells to impact biochemical and genetic programs of development. This gap in knowledge stems from a lack of understanding of the molecular players of cellular mechanics and an absence of techniques for measuring and manipulating mechanical forces in tissue. In this review article, we examine recent advances that are beginning to clear these bottlenecks and highlight results from new approaches that reveal the role of mechanical forces in neurodevelopmental processes.     

Cellular phosphatidic acid sensor, α-synuclein N-terminal domain,detects endogenous phosphatidic acid in macrophagic phagosomes and neuronal growth cones

Phosphatidic acid (PA) is the simplest phospholipid and is involved in the regulation of various cellular events. Recently, we developed a new PA sensor, the N-terminal region of α-synuclein (α-Syn-N). However, whether α-Syn-N can sense physiologically produced, endogenous PA remains unclear. We first established an inactive PA sensor (α-Syn-N-KQ) as a negative control by replacing all eleven lysine residues with glutamine residues. Using confocal microscopy, we next verified that α-Syn-N, but not α-Syn-N-KQ, detected PA in macrophagic phagosomes in which PA is known to be enriched, further indicating that α-Syn-N can be used as a reliable PA sensor in cells. Finally, because PA generated during neuronal differentiation is critical for neurite outgrowth, we investigated the subcellular distribution of PA using α-Syn-N. We found that α-Syn-N, but not α-Syn-N-KQ, accumulated at the peripheral regions (close to the plasma membrane) of neuronal growth cones. Experiments using a phospholipase D (PLD) inhibitor strongly suggested that PA in the peripheral regions of the growth cone was primarily produced by PLD. Our findings provide a reliable sensor of endogenous PA and novel insights into the distribution of PA during neuronal differentiation.     

Targeting protein kinases benefits cancer immunotherapy

Small-molecule kinase inhibitors have been well established and successfully developed in the last decades for cancer target therapies. However, intrinsic or acquired drug resistance is becoming the major barrier for their clinical application. With the development of immunotherapies, in particular the discovery of immune checkpoint inhibitors (ICIs), the combination of ICIs with other therapies have recently been extensively explored, among which combination of ICIs with kinase inhibitors achieves promising clinical outcome in a plethora of cancer types. Here we comprehensively summarize the potent roles of protein kinases in modulating immune checkpoints both in tumor and immune cells, and reshaping tumor immune microenvironments by evoking innate immune response and neoantigen generation or presentation. Moreover, the clinical trial and approval of combined administration of kinase inhibitors with ICIs are collected, highlighting the precise strategies to benefit cancer immune therapies.