Interplay of precision therapeutics and MD study: Calocybe indica's potentials against cervical cancer and its interaction with VEGF via octadecanoic acid.

The evolving landscape of personalized medicine necessitates a shift from traditional therapeutic interventions towards precision-driven approaches. Embracing this paradigm, our research probes the therapeutic efficacy of the aqueous crude extract (ACE) of Calocybe indica in cervical cancer treatment, merging botanical insights with advanced molecular research. We observed that ACE exerts significant influences on nuclear morphology and cell cycle modulation, further inducing early apoptosis and showcasing prebiotic attributes. Characterization of ACE have identified several phytochemicals including significant presence of octadeconoic acid. Simultaneously, utilizing advanced Molecular Dynamics (MD) simulations, we deciphered the intricate molecular interactions between Vascular Endothelial Growth Factor (VEGF) and Octadecanoic acid to establish C.indica's role as an anticancer agent. Our study delineates Octadecanoic acid's potential as a robust binding partner for VEGF, with comprehensive analyses from RMSD and RMSF profiles highlighting the stability and adaptability of the protein-ligand interactions. Further in-depth thermodynamic explorations via MM-GBSA calculations reveal the binding landscape of the VEGF-Octadecanoic acid complex. Emerging therapeutic innovations, encompassing proteolysis-targeting chimeras (PROTACs) and avant-garde nanocarriers, are discussed in the context of their synergy with compounds like Calocybe indica P&C. This convergence underscores the profound therapeutic potential awaiting clinical exploration. This study offers a holistic perspective on the promising therapeutic avenues facilitated by C. indica against cervical cancer, intricately woven with advanced molecular interactions and the prospective integration of precision therapeutics in modern oncology.

AMP-activated protein kinase can be allosterically activated by ADP but AMP remains the key activating ligand.

The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status. When activated by increases in ADP:ATP and/or AMP:ATP ratios (signalling energy deficit), AMPK acts to restore energy balance. Binding of AMP to one or more of three CBS repeats (CBS1, CBS3, CBS4) on the AMPK-γ subunit activates the kinase complex by three complementary mechanisms: (i) promoting α-subunit Thr172 phosphorylation by the upstream kinase LKB1; (ii) protecting against Thr172 dephosphorylation; (iii) allosteric activation. Surprisingly, binding of ADP has been reported to mimic the first two effects, but not the third. We now show that at physiologically relevant concentrations of Mg.ATP2- (above those used in the standard assay) ADP binding does cause allosteric activation. However, ADP causes only a modest activation because (unlike AMP), at concentrations just above those where activation becomes evident, ADP starts to cause competitive inhibition at the catalytic site. Our results cast doubt on the physiological relevance of the effects of ADP and suggest that AMP is the primary activator in vivo. We have also made mutations to hydrophobic residues involved in binding adenine nucleotides at each of the three γ subunit CBS repeats of the human α2ß2γ1 complex and examined their effects on regulation by AMP and ADP. Mutation of the CBS3 site has the largest effects on all three mechanisms of AMP activation, especially at lower ATP concentrations, while mutation of CBS4 reduces the sensitivity to AMP. All three sites appear to be required for allosteric activation by ADP.

Discovery of a potent inhibitor, D-132, targeting AsfvPolX, via protein-DNA complex-guided pharmacophore screening and in vitro molecular characterizations.

The heightened transmissibility and capacity of African swine fever virus (ASFV) induce fatal diseases in domestic pigs and wild boars, posing significant economic repercussions and global threats. Despite extensive research efforts, the development of potent vaccines or treatments for ASFV remains a persistent challenge. Recently, inhibiting the AsfvPolX, a key DNA repair enzyme, emerges as a feasible strategy to disrupt viral replication and control ASFV infections. In this study, a comprehensive approach involving pharmacophore-based inhibitor screening, coupled with biochemical and biophysical analyses, were implemented to identify, characterize, and validate potential inhibitors targeting AsfvPolX. The constructed pharmacophore model, Phar-PolX-S, demonstrated efficacy in identifying a potent inhibitor, D-132 (IC50 = 2.8 ± 0.2 µM), disrupting the formation of the AsfvPolX-DNA complex. Notably, D-132 exhibited strong binding to AsfvPolX (KD = 6.9 ± 2.2 µM) through a slow-on-fast-off binding mechanism. Employing molecular modeling, it was elucidated that D-132 predominantly binds in-between the palm and finger domains of AsfvPolX, with crucial residues (R42, N48, Q98, E100, F102, and F116) identified as hotspots for structure-based inhibitor optimization. Distinctively characterized by a 1,2,5,6-tetrathiocane with modifications at the 3 and 8 positions involving ethanesulfonates, D-132 holds considerable promise as a lead compound for the development of innovative agents to combat ASFV infections.

Inhibitory Efficacy of Main Components of Scutellaria baicalensis on the Interaction between Spike Protein of SARS-CoV-2 and Human Angiotensin-Converting Enzyme II.

Blocking the interaction between the SARS-CoV-2 spike protein and the human angiotensin-converting enzyme II (hACE2) protein serves as a therapeutic strategy for treating COVID-19. Traditional Chinese medicine (TCM) treatments containing bioactive products could alleviate the symptoms of severe COVID-19. However, the emergence of SARS-CoV-2 variants has complicated the process of developing broad-spectrum drugs. As such, the aim of this study was to explore the efficacy of TCM treatments against SARS-CoV-2 variants through targeting the interaction of the viral spike protein with the hACE2 receptor. Antiviral activity was systematically evaluated using a pseudovirus system. Scutellaria baicalensis (S. baicalensis) was found to be effective against SARS-CoV-2 infection, as it mediated the interaction between the viral spike protein and the hACE2 protein. Moreover, the active molecules of S. baicalensis were identified and analyzed. Baicalein and baicalin, a flavone and a flavone glycoside found in S. baicalensis, respectively, exhibited strong inhibitory activities targeting the viral spike protein and the hACE2 protein, respectively. Under optimized conditions, virus infection was inhibited by 98% via baicalein-treated pseudovirus and baicalin-treated hACE2. In summary, we identified the potential SARS-CoV-2 inhibitors from S. baicalensis that mediate the interaction between the Omicron spike protein and the hACE2 receptor. Future studies on the therapeutic application of baicalein and baicalin against SARS-CoV-2 variants are needed.

Native Mass Spectrometry Dissects the Structural Dynamics of an Allosteric Heterodimer of SARS-CoV-2 Nonstructural Proteins.

Structure-based drug design, which relies on precise understanding of the target protein and its interaction with the drug candidate, is dramatically expedited by advances in computational methods for candidate prediction. Yet, the accuracy needs to be improved with more structural data from high throughput experiments, which are challenging to generate, especially for dynamic and weak associations. Herein, we applied native mass spectrometry (native MS) to rapidly characterize ligand binding of an allosteric heterodimeric complex of SARS-CoV-2 nonstructural proteins (nsp) nsp10 and nsp16 (nsp10/16), a complex essential for virus survival in the host and thus a desirable drug target. Native MS showed that the dimer is in equilibrium with monomeric states in solution. Consistent with the literature, well characterized small cosubstrate, RNA substrate, and product bind with high specificity and affinity to the dimer but not the free monomers. Unsuccessfully designed ligands bind indiscriminately to all forms. Using neutral gas collision, the nsp16 monomer with bound cosubstrate can be released from the holo dimer complex, confirming the binding to nsp16 as revealed by the crystal structure. However, we observed an unusual migration of the endogenous zinc ions bound to nsp10 to nsp16 after collisional dissociation. The metal migration can be suppressed by using surface collision with reduced precursor charge states, which presumably resulted in minimal gas-phase structural rearrangement and highlighted the importance of complementary techniques. With minimal sample input (∼µg), native MS can rapidly detect ligand binding affinities and locations in dynamic multisubunit protein complexes, demonstrating the potential of an "all-in-one" native MS assay for rapid structural profiling of protein-to-AI-based compound systems to expedite drug discovery.

The rapid proximity labeling system PhastID identifies ATP6AP1 as an unconventional GEF for Rheb.

Rheb is a small G protein that functions as the direct activator of the mechanistic target of rapamycin complex 1 (mTORC1) to coordinate signaling cascades in response to nutrients and growth factors. Despite extensive studies, the guanine nucleotide exchange factor (GEF) that directly activates Rheb remains unclear, at least in part due to the dynamic and transient nature of protein-protein interactions (PPIs) that are the hallmarks of signal transduction. Here, we report the development of a rapid and robust proximity labeling system named Pyrococcus horikoshii biotin protein ligase (PhBPL)-assisted biotin identification (PhastID) and detail the insulin-stimulated changes in Rheb-proximity protein networks that were identified using PhastID. In particular, we found that the lysosomal V-ATPase subunit ATP6AP1 could dynamically interact with Rheb. ATP6AP1 could directly bind to Rheb through its last 12 amino acids and utilizes a tri-aspartate motif in its highly conserved C-tail to enhance Rheb GTP loading. In fact, targeting the ATP6AP1 C-tail could block Rheb activation and inhibit cancer cell proliferation and migration. Our findings highlight the versatility of PhastID in mapping transient PPIs in live cells, reveal ATP6AP1's role as an unconventional GEF for Rheb, and underscore the importance of ATP6AP1 in integrating mTORC1 activation signals through Rheb, filling in the missing link in Rheb/mTORC1 activation.

Raman spectroscopy study of 7,8-dihydrofolate inhibition on the Wuhan strain SARS-CoV-2 binding to human ACE2 receptor.

Emerging evidence suggests that elevated levels of folic acid in the bloodstream may confer protection against Wuhan-SARS-CoV-2 infection and mitigate its associated symptoms. Notably, two comprehensive studies of COVID-19 patients in Israel and UK uncovered a remarkable trend, wherein individuals with heightened folic acid levels exhibited only mild symptoms and necessitated no ventilatory support. In parallel, research has underscored the potential connection between decreased folic acid levels and the severity of Covid-19 among hospitalized patients. Yet, the underlying mechanisms governing this intriguing inhibition remain elusive. In a quest to elucidate these mechanisms, we conducted a molecular dynamics simulation approach followed by a Raman spectroscopy study to delve into the intricate interplay between the folic acid metabolite, 7,8-dihydrofolate (DHF), and the angiotensin-converting enzyme ACE2 receptor, coupled with its interaction with the receptor-binding domain (RBD) of the Wuhan strain of SARS-CoV-2. Through a meticulous exploration, we scrutinized the transformation of the ACE2 + RBD complex, allowing these reactants to form bonds. This was juxtaposed with a similar investigation where ACE2 was initially permitted to react with DHF, followed by the exposure of the ACE2 + DHF complex to RBD. We find that DHF, when bonded to ACE2, functions as a physical barrier, effectively inhibiting the binding of the Wuhan strain RBD. This physicochemical process offers a cogent explanation for the observed inhibition of host cell infection in subjects receiving supplementary folic acid doses, as epidemiologically substantiated in multiple studies. This study not only sheds light on a potential avenue for mitigating SARS-CoV-2 infection but also underscores the crucial role of folic acid metabolites in host-virus interactions. This research paves the way for novel therapeutic strategies in the battle against COVID-19 and reinforces the significance of investigating the molecular mechanisms underlying the protective effects of folic acid in the context of viral infections.

Inhibition of multiple SARS-CoV-2 variants entry by Lycium barbarum L. polysaccharides through disruption of spike protein-ACE2 interaction.

Viral respiratory infections are major human health concerns. The most striking epidemic disease, COVID-19 is still on going with the emergence of fast mutations and drug resistance of pathogens. A few polysaccharide macromolecules from traditional Chinese medicine (TCM) have been found to have direct anti-SARS-CoV-2 activity but the mechanism remains unclear. In this study, we evaluated the entry inhibition effect of Lycium barbarum polysaccharides (LBP) in vitro and in vivo. We found LBP effectively suppressed multiple SARS-CoV-2 variants entry and protected K18-hACE2 mice from invasion with Omicron pseudovirus (PsV). Moreover, we found LBP interfered with early entry events during infection in time-of-addition (TOA) assay and SEM observation. Further surface plasmon resonance (SPR) study revealed the dual binding of LBP with Spike protein and ACE2, which resulted in the disruption of Spike-ACE2 interaction and subsequently triggered membrane fusion. Therefore, LBP may act as broad-spectrum inhibitors of virus entry and nasal mucosal protective agent against newly emerging respiratory viruses, especially SARS-CoV-2.

Structure and energetics of serum protein complex of tea adulterant dye Bismarck brown Y using experimental and computational methods.

Tea, a widely consumed aromatic beverage, is often adulterated with dyes such as Bismarck brown Y (C.I. 21000) (BBY), Prussian blue, and Plumbago, which pose potential health risks. The objective of this study is to analyze how the food dye BBY interacts with serum protein, bovine serum albumin (BSA). This study investigated the BBY-BSA interaction at the molecular level. Fluorescence spectroscopy results showed that the quenching of BSA by BBY is carried out by dynamic quenching mechanism. The displacement assay and molecular docking studies revealed that BBY binds at the flavanone binding site of BSA with hydrophobic interactions. Circular Dichroism results indicate the structural stability of the protein upon BBY binding. Molecular dynamics simulations demonstrated the stability of the complex in a dynamic solvent system, and quantum mechanics calculations showed slight conformational changes of the diaminophenyl ring due to increased hydrophobic interaction. The energetics of gas phase optimized and stable MD structures of BBY indicated similar values which further confirmed that the conformational changes were minor, and it also exhibited a moderate binding with BSA as shown by the MM/PBSA results. This study enhances our understanding of the molecular-level interactions between BBY and BSA, emphasizing the critical role of hydrophobic interactions.

The interaction on HSA and the antibacterial activity from four polyoxometalate hybrids based on berberine.

Four organic-polyoxometalate hybrids BR4[SiW12O40] (BR-SiW), BR3[PMo12O40] (BR-PMo), BR4K[EuSiW11O40]·2H2O (BR-EuSiW) and BR6Na3[EuW10O36] (BR-EuW) were fabricated by the polyoxometalates (POMs) anions and berberine cations (BR) noted for the alkaloids in traditional Chinese herbal medicine. These hybrids have been characterized and confirmed. The interaction between hybrids and human serum albumin (HSA) was investigated in a buffer solution (pH 7.4) using ultraviolet-visible light absorption and fluorescence techniques. The classical Stern-Volmer equation was used to analyze the fluorescence quenching at three temperatures (296, 303 and 310 K), and the static quenching mechanism for interaction was proposed. The Thermodynamic parameters, enthalpy, entropy change, and Gibbs free energy of hybrids interacting on HSA were calculated by Scatchard equation. The results indicated that therewas one binding site on the protein and BR-POMs all showed stronger binding force than that of raw materials. Synchronous fluorescence results showed that the binding sites of BR-POMs and HSA were not effectively affected the surrounding microenvironment. The following antibacterial experiments implied that inhibitory effect of hybrids were synergistic effect from organic active ingredient and POMs but the simple combination. All these data were prepared for further research on biology.

A New Version of the Tissue Composition-Based Model for Improving the Mechanism-Based Prediction of Volume of Distribution at Steady-State for Neutral Drugs.

In-vitro models are available in the literature for predicting the volume of distribution at steady-state (Vdss) of drugs. The mechanistic model refers to the tissue composition-based model (TCM), which includes important factors that govern Vdss such as drug physiochemistry and physiological data. The recognized TCM published by Rodgers and Rowland (TCM-RR) and a subsequent adjustment made by Simulations Plus Inc. (TCM-SP) have been shown to be generally less accurate with neutral compared to ionized drugs. Therefore, improving these models for neutral drugs becomes necessary. The objective of this study was to propose a new TCM for improving the prediction of Vdss for neutral drugs. The new TCM included two modifications of the published models (i) accentuate the effect of the blood-to-plasma ratio (BPR) that should cover permeated molecules across the biomembranes, which is lacking in these models for neutral compounds, and (ii) use a different approach to estimate the binding in tissues. The new TCM was validated with a large dataset of 202 commercial and proprietary compounds including preclinical and clinical data. All scenario datasets were predicted more accurately with the TCM-New, whereas all statistical parameters indicate that the TCM-New showed significant improvements in terms of accuracy over the TCM-RR and TCM-SP. Predictions of Vdss were frequently more accurate for the TCM-new with 83% within twofold error versus only 50% for the TCM-RR. And more than 95% of the predictions were within threefold error and patient interindividual differences can be predicted with the TCM-New, greatly exceeding the accuracy of the published models. Overall, the new TCM incorporating BPR significantly improved the Vdss predictions in animals and humans for neutral drugs, and, hence, has the potential to better support the drug discovery and facilitate the first-in-human predictions.

Differential molecular mechanisms of substrate recognition by selenium methyltransferases, INMT and TPMT, in selenium detoxification and excretion.

It is known that the recommended dietary allowance of selenium (Se) is dangerously close to its tolerable upper intake level. Se is detoxified and excreted in urine as trimethylselenonium ion (TMSe) when the amount ingested exceeds the nutritional level. Recently, we demonstrated that the production of TMSe requires two methyltransferases: thiopurine S-methyltransferase (TPMT) and indolethylamine N-methyltransferase (INMT). In this study, we investigated the substrate recognition mechanisms of INMT and TPMT in the Se-methylation reaction. Examination of the Se-methyltransferase activities of two paralogs of INMT, namely, nicotinamide N-methyltransferase and phenylethanolamine N-methyltransferase, revealed that only INMT exhibited Se-methyltransferase activity. Consistently, molecular dynamics simulations demonstrated that dimethylselenide was preferentially associated with the active center of INMT. Using the fragment molecular orbital method, we identified hydrophobic residues involved in the binding of dimethylselenide to the active center of INMT. The INMT-L164R mutation resulted in a deficiency in Se- and N-methyltransferase activities. Similarly, TPMT-R152, which occupies the same position as INMT-L164, played a crucial role in the Se-methyltransferase activity of TPMT. Our findings suggest that TPMT recognizes negatively charged substrates, whereas INMT recognizes electrically neutral substrates in the hydrophobic active center embedded within the protein. These observations explain the sequential requirement of the two methyltransferases in producing TMSe.

Flavonoids as G Protein-coupled Receptors Ligands: New Potential Therapeutic Natural Drugs.

G protein coupled receptors (GPCRs) are among the largest family of cell surface receptors found in the human genome. They govern a wide range of physiological responses in both health and diseases, making them one of the potential targeted surface receptors for pharmaceuticals. Flavonoids can modulate GPCRs activity by acting as allosteric ligands. They can either enhance or reduce the GPCR's effect. Emerging research shows that individual flavonoids or mixtures of flavonoids from plant extracts can have relevant pharmacological effects against a number of diseases, particularly by influencing GPCRs. In the present review, we are considering to give a comprehensive overview of flavonoids and related compounds that exhibit GPCRs activity and to further explore which beneficial structural features. Molecular docking was used to strengthen experimental evidence and describe flavonoid-GPCRs interactions at molecular level.

[Determination of plasma protein binding rate of Shuganning Injection using equilibrium dialysis and UPLC-MS/MS].

Traditional Chinese medicine(TCM) compound preparations have complex compositions. As a widely used TCM injection, Shuganning Injection, its in vivo processes are not yet fully understood. Determining the plasma protein binding rate is of great significance for pharmacokinetic and pharmacodynamic studies. In this experiment, the equilibrium dialysis method combined with UPLC-MS/MS technology was used to determine the plasma protein binding rates of 10 components, including p-hydroxyacetophenone, caffeic acid, baicalein, oroxylin A, geniposide, baicalin, cynaroside, oroxylin A-7-O-ß-D-glucuronide, scutellarin, and hyperoside, in Shuganning Injection in rat and human plasma to provide a theoretical basis for further elucidating the in vivo processes of Shuganning Injection and guiding clinical medication. The results showed that, except for baicalein and geniposide, the plasma protein binding rates of the other eight components were higher in human plasma than in rat plasma, and there were interspecies differences. In human plasma, except for geniposide, caffeic acid, and baicalin, the plasma protein binding rates of the remaining seven components were above 80%, with baicalein and oroxylin A exceeding 90%. All components exhibit a high level of binding to plasma proteins, with the exception of geniposide.

[Screening of small molecule inhibitors of IL-15Rα using molecular docking and surface plasmon resonance technology].

The study aims to explore the active molecules of traditional Chinese medicine that specifically bind to interleukin-15 receptor α (IL-15Rα) using molecular docking and surface plasmon resonance (SPR) technology. AutoDock molecular docking software was used to perform simulated docking of more than 3 000 compounds from 48 traditional Chinese medicines at IL-15Rα and screen the specific binding compounds. Then Biocore T200 biomolecular interaction analysis system of SPR was used to confirm the binding specificity of the selected target compounds. Finally, the biological effects of the target compounds on IL-15Rα were verified by cell biological experiments. The results showed that neoprzewaquinone A (Neo) possessed the highest specific binding affinity among the active molecules from traditional Chinese medicine, and the dissociation constant (KD) value was (0.62 ± 0.20) µmol/L. The results of cell experiment showed that Neo significantly inhibited the proliferation of Mo7e cells induced by IL-15, and the IC50 was 1.075 µmol/L, approximately 1/120 of the IC50 of Cefazolin (IL-15 specific antagonist). These results suggest that Neo is a specific inhibitor of IL-15Rα and may be a potential active drug for the treatment of diseases related to the dysfunction of the IL-15Rα signaling.

Dauricine interferes with SARS-CoV-2 variants infection by blocking the interface between RBD and ACE2.

The continued viral evolution results in the emergence of various SARS-CoV-2 variants, such as delta or omicron, that are partially resistant to current vaccines and antiviral medicines, posing an increased risk to global public health and raising the importance of continuous development of antiviral medicines. Inhibitor screening targeting the interactions between the viral spike proteins and their human receptor ACE2 represents a promising approach for drug discovery. Here, we demonstrate that the evolutionary trend of the SARS-CoV-2 variants is associated with increased electrostatic interactions between S proteins and ACE2. Virtual screening based on the ACE2-RBD binding interface identified nine monomers of Traditional Chinese medicine (TCM). Furthermore, live-virus neutralization assays revealed that Dauricine, one of the identified monomers, exhibited an antiviral activity with an IC50 range of 18.2 to 33.3 µM for original strain, Delta, and Omicron strains, respectively. The computational study showed that the polycyclic and methoxy groups of Dauricine adhere to the RBD surface through π-π and electrostatic interactions. The discovery of Dauricine is a successful attempt to target viral entry, which will not only help society to respond quickly to viral variants, but also accelerate variant drug development thereby reducing the pressure on health authorities to respond to outbreaks.

Drug discovery for heart failure targeting myosin-binding protein C.

Cardiac MyBP-C (cMyBP-C) interacts with actin and myosin to fine-tune cardiac muscle contractility. Phosphorylation of cMyBP-C, which reduces the binding of cMyBP-C to actin and myosin, is often decreased in patients with heart failure (HF) and is cardioprotective in model systems of HF. Therefore, cMyBP-C is a potential target for HF drugs that mimic its phosphorylation and/or perturb its interactions with actin or myosin. We labeled actin with fluorescein-5-maleimide (FMAL) and the C0-C2 fragment of cMyBP-C (cC0-C2) with tetramethylrhodamine (TMR). We performed two complementary high-throughput screens (HTS) on an FDA-approved drug library, to discover small molecules that specifically bind to cMyBP-C and affect its interactions with actin or myosin, using fluorescence lifetime (FLT) detection. We first excited FMAL and detected its FLT, to measure changes in fluorescence resonance energy transfer (FRET) from FMAL (donor) to TMR (acceptor), indicating binding. Using the same samples, we then excited TMR directly, using a longer wavelength laser, to detect the effects of compounds on the environmentally sensitive FLT of TMR, to identify compounds that bind directly to cC0-C2. Secondary assays, performed on selected modulators with the most promising effects in the primary HTS assays, characterized the specificity of these compounds for phosphorylated versus unphosphorylated cC0-C2 and for cC0-C2 versus C1-C2 of fast skeletal muscle (fC1-C2). A subset of identified compounds modulated ATPase activity in cardiac and/or skeletal myofibrils. These assays establish the feasibility of the discovery of small-molecule modulators of the cMyBP-C-actin/myosin interaction, with the ultimate goal of developing therapies for HF.

Molecular regulatory mechanism of human myosin-7a.

Myosin-7a is an actin-based motor protein essential for vision and hearing. Mutations of myosin-7a cause type 1 Usher syndrome, the most common and severe form of deafblindness in humans. The molecular mechanisms that govern its mechanochemistry remain poorly understood, primarily because of the difficulty of purifying stable intact protein. Here, we recombinantly produce the complete human myosin-7a holoenzyme in insect cells and characterize its biochemical and motile properties. Unlike the Drosophila ortholog that primarily associates with calmodulin (CaM), we found that human myosin-7a utilizes a unique combination of light chains including regulatory light chain, CaM, and CaM-like protein 4. Our results further reveal that CaM-like protein 4 does not function as a Ca2+ sensor but plays a crucial role in maintaining the lever arm's structural-functional integrity. Using our recombinant protein system, we purified two myosin-7a splicing isoforms that have been shown to be differentially expressed along the cochlear tonotopic axis. We show that they possess distinct mechanoenzymatic properties despite differing by only 11 amino acids at their N termini. Using single-molecule in vitro motility assays, we demonstrate that human myosin-7a exists as an autoinhibited monomer and can move processively along actin when artificially dimerized or bound to cargo adaptor proteins. These results suggest that myosin-7a can serve multiple roles in sensory systems such as acting as a transporter or an anchor/force sensor. Furthermore, our research highlights that human myosin-7a has evolved unique regulatory elements that enable precise tuning of its mechanical properties suitable for mammalian auditory functions.

LTP induction by structural rather than enzymatic functions of CaMKII.

Learning and memory are thought to require hippocampal long-term potentiation (LTP), and one of the few central dogmas of molecular neuroscience that has stood undisputed for more than three decades is that LTP induction requires enzymatic activity of the Ca2+/calmodulin-dependent protein kinase II (CaMKII)1-3. However, as we delineate here, the experimental evidence is surprisingly far from conclusive. All previous interventions inhibiting enzymatic CaMKII activity and LTP4-8 also interfere with structural CaMKII roles, in particular binding to the NMDA-type glutamate receptor subunit GluN2B9-14. Thus, we here characterized and utilized complementary sets of new opto-/pharmaco-genetic tools to distinguish between enzymatic and structural CaMKII functions. Several independent lines of evidence demonstrated LTP induction by a structural function of CaMKII rather than by its enzymatic activity. The sole contribution of kinase activity was autoregulation of this structural role via T286 autophosphorylation, which explains why this distinction has been elusive for decades. Directly initiating the structural function in a manner that circumvented this T286 role was sufficient to elicit robust LTP, even when enzymatic CaMKII activity was blocked.

Site-Specific Covalent Immobilization of SMA-Stabilized ACE2 for SARS-CoV-2 Recognition and Drug Screening.

Membrane protein (MP)-based biomaterials have a wide range of applications in drug screening, antigen detection, and ligand-receptor interaction analysis. Traditional MP immobilization methods have the disadvantage of disordered protein immobilization orientation, leading to the shielded binding domain and unreliable binding pattern. Herein, we describe a site-specific covalent immobilization of MPs, which utilizes the styrene maleic acid (SMA) detergent-free extraction method of MPs as well as the covalent reaction between His-tag and divinyl sulfone (DVS). As an example, we covalently immobilized angiotensin-converting enzyme 2 (ACE2) on a cell membrane chromatography system (ACE2-His-SMALPs/CMC) in a site-specific manner and verified the specificity and stability of this system. This technique significantly improves the service life compared to the physisorption CMC column. The improved protein immobilization strategies of the ACE2-His-SMALPs/CMC system enable it to effectively recognize SARS-CoV-2 pseudoviral particles as well as detect viral particles in ambient air once combined with an aerosol collector; as a powerful ligand biosensor, the ACE2-His-SMALPs/CMC system was used to screen for compounds with anti-SARS-CoV-2 pseudovirus activity. In conclusion, the optimized MP immobilization strategy has been successfully applied to CMC technology, showing enhanced stability and sensitivity, which can provide an efficient and convenient membrane protein immobilization method for biomaterials.