Protein phosphatases in the RNAPII transcription cycle: erasers, sculptors, gatekeepers, and potential drug targets.

The transcription cycle of RNA polymerase II (RNAPII) is governed at multiple points by opposing actions of cyclin-dependent kinases (CDKs) and protein phosphatases, in a process with similarities to the cell division cycle. While important roles of the kinases have been established, phosphatases have emerged more slowly as key players in transcription, and large gaps remain in understanding of their precise functions and targets. Much of the earlier work focused on the roles and regulation of sui generis and often atypical phosphatases-FCP1, Rtr1/RPAP2, and SSU72-with seemingly dedicated functions in RNAPII transcription. Decisive roles in the transcription cycle have now been uncovered for members of the major phosphoprotein phosphatase (PPP) family, including PP1, PP2A, and PP4-abundant enzymes with pleiotropic roles in cellular signaling pathways. These phosphatases appear to act principally at the transitions between transcription cycle phases, ensuring fine control of elongation and termination. Much is still unknown, however, about the division of labor among the PPP family members, and their possible regulation by or of the transcriptional kinases. CDKs active in transcription have recently drawn attention as potential therapeutic targets in cancer and other diseases, raising the prospect that the phosphatases might also present opportunities for new drug development. Here we review the current knowledge and outstanding questions about phosphatases in the context of the RNAPII transcription cycle.

Crosstalk between RNA Pol II C-Terminal Domain Acetylation and Phosphorylation via RPRD Proteins.

Post-translational modifications of the RNA polymerase II C-terminal domain (CTD) coordinate the transcription cycle. Crosstalk between different modifications is poorly understood. Here, we show how acetylation of lysine residues at position 7 of characteristic heptad repeats (K7ac)-only found in higher eukaryotes-regulates phosphorylation of serines at position 5 (S5p), a conserved mark of polymerases initiating transcription. We identified the regulator of pre-mRNA-domain-containing (RPRD) proteins as reader proteins of K7ac. K7ac enhanced CTD peptide binding to the CTD-interacting domain (CID) of RPRD1A and RPRD1B proteins in isothermal calorimetry and molecular modeling experiments. Deacetylase inhibitors increased K7ac- and decreased S5-phosphorylated polymerases, consistent with acetylation-dependent S5 dephosphorylation by an RPRD-associated S5 phosphatase. Consistent with this model, RPRD1B knockdown increased S5p but enhanced K7ac, indicating that RPRD proteins recruit K7 deacetylases, including HDAC1. We also report autoregulatory crosstalk between K7ac and S5p via RPRD proteins and their interactions with acetyl- and phospho-eraser proteins.

In vitro analysis of RNA polymerase II elongation complex dynamics.

RNA polymerase II elongation complexes (ECs) were assembled from nuclear extract on immobilized DNA templates and analyzed by quantitative mass spectrometry. Time-course experiments showed that initiation factor TFIIF can remain bound to early ECs, while levels of core elongation factors Spt4-Spt5, Paf1C, Spt6-Spn1, and Elf1 remain steady. Importantly, the dynamic phosphorylation patterns of the Rpb1 C-terminal domain (CTD) and the factors that recognize them change as a function of postinitiation time rather than distance elongated. Chemical inhibition of Kin28/Cdk7 in vitro blocks both Ser5 and Ser2 phosphorylation, affects initiation site choice, and inhibits elongation efficiency. EC components dependent on CTD phosphorylation include capping enzyme, cap-binding complex, Set2, and the polymerase-associated factor (PAF1) complex. By recapitulating many known features of in vivo elongation, this system reveals new details that clarify how EC-associated factors change at each step of transcription.

Collaborators or competitors: the communication between RNA polymerase II and the nucleosome during eukaryotic transcription.

Decades of scientific research have been devoted to unraveling the intricacies of eukaryotic transcription since the groundbreaking discovery of eukaryotic RNA polymerases in the late 1960s. RNA polymerase II, the polymerase responsible for mRNA synthesis, has always attracted the most attention. Despite its structural resemblance to its bacterial counterpart, eukaryotic RNA polymerase II faces a unique challenge in progressing transcription due to the presence of nucleosomes that package DNA in the nuclei. In this review, we delve into the impact of RNA polymerase II and histone signaling on the progression of eukaryotic transcription. We explore the pivotal points of interactions that bridge the RNA polymerase II and histone signaling systems. Finally, we present an analysis of recent cryo-electron microscopy structures, which captured RNA polymerase II-nucleosome complexes at different stages of the transcription cycle. The combination of the signaling crosstalk and the direct visualization of RNA polymerase II-nucleosome complexes provides a deeper understanding of the communication between these two major players in eukaryotic transcription.

Yeast PIC-Mediator structure with RNA polymerase II C-terminal domain.

For transcription initiation, RNA polymerase II (Pol II) forms a preinitiation complex (PIC) that associates with the general coactivator Mediator. Whereas atomic models of the human PIC-Mediator structure have been reported, structures for its yeast counterpart remain incomplete. Here, we present an atomic model for the yeast PIC with core Mediator, including the Mediator middle module that was previously poorly resolved and including subunit Med1 that was previously lacking. We observe three peptide regions containing eleven of the 26 heptapeptide repeats of the flexible C-terminal repeat domain (CTD) of Pol II. Two of these CTD regions bind between the Mediator head and middle modules and form defined CTD-Mediator interactions. CTD peptide 1 binds between the Med6 shoulder and Med31 knob domains, whereas CTD peptide 2 forms additional contacts with Med4. The third CTD region (peptide 3) binds in the Mediator cradle and associates with the Mediator hook. Comparisons with the human PIC-Mediator structure show that the central region in peptide 1 is similar and forms conserved contacts with Mediator, whereas peptides 2 and 3 exhibit distinct structures and Mediator interactions.

Combined prediction and design reveals the target recognition mechanism of an intrinsically disordered protein interaction domain.

An increasing number of protein interaction domains and their targets are being found to be intrinsically disordered proteins (IDPs). The corresponding target recognition mechanisms are mostly elusive because of challenges in performing detailed structural analysis of highly dynamic IDP-IDP complexes. Here, we show that by combining recently developed computational approaches with experiments, the structure of the complex between the intrinsically disordered C-terminal domain (CTD) of protein 4.1G and its target IDP region in NuMA can be dissected at high resolution. First, we carry out systematic mutational scanning using dihydrofolate reductase-based protein complementarity analysis to identify essential interaction regions and key residues. The results are found to be highly consistent with an α/ß-type complex structure predicted by AlphaFold2 (AF2). We then design mutants based on the predicted structure using a deep learning protein sequence design method. The solved crystal structure of one mutant presents the same core structure as predicted by AF2. Further computational prediction and experimental assessment indicate that the well-defined core structure is conserved across complexes of 4.1G CTD with other potential targets. Thus, we reveal that an intrinsically disordered protein interaction domain uses an α/ß-type structure module formed through synergistic folding to recognize broad IDP targets. Moreover, we show that computational prediction and experiment can be jointly applied to segregate true IDP regions from the core structural domains of IDP-IDP complexes and to uncover the structure-dependent mechanisms of some otherwise elusive IDP-IDP interactions.

Arabidopsis nicotianamine synthases comprise a common core-NAS domain fused to a variable autoinhibitory C terminus.

Nicotianamine synthase (NAS) catalyzes the biosynthesis of the low-molecular-mass metal chelator nicotianamine (NA) from the 2-aminobutyrate moieties of three SAM molecules. NA has central roles in metal nutrition and metal homeostasis of flowering plants. The enzymatic function of NAS remains poorly understood. Crystal structures are available for archaeal and bacterial NAS-like proteins that carry out simpler aminobutanoyl transferase reactions. Here, we report amino acids essential for the activity of AtNAS1 based on structural modeling and site-directed mutagenesis. Using a newly developed enzyme-coupled continuous activity assay, we compare differing NAS proteins identified through multiple sequence alignments and phylogenetic analyses. In most NAS of dicotyledonous and monocotyledonous plants (class Ia and Ib), the core-NAS domain is fused to a variable C-terminal domain. Compared to fungal and moss NAS that comprise merely a core-NAS domain (class III), NA biosynthetic activities of the four paralogous Arabidopsis thaliana NAS proteins were far lower. C-terminally trimmed core-AtNAS variants exhibited strongly elevated activities. Of 320 amino acids of AtNAS1, twelve, 287-TRGCMFMPCNCS-298, accounted for the autoinhibitory effect of the C terminus, of which approximately one-third was attributed to N296 within a CNCS motif that is fully conserved in Arabidopsis. No detectable NA biosynthesis was mediated by two representative plant NAS proteins that naturally lack the C-terminal domain, class Ia Arabidopsis halleri NAS5 and Medicago truncatula NAS2 of class II which is found in dicots and diverged early during the evolution of flowering plants. Next, we will address a possible posttranslational release of autoinhibition in class I NAS proteins.

Structure of a phosphodiesterase from Streptomyces sanglieri with a novel C-terminal domain.

A glycerophosphoethanolamine ethanolaminephosphodiesterase (GPE-EP) from Streptomyces sanglieri hydrolyzes glycerophosphoethanolamine to phosphoethanolamine and glycerol. The structure of GPE-EP was determined by the molecular replacement method using a search model generated with AlphaFold2. This structure includes the entire length of the mature protein and it is composed of an N-terminal domain and a novel C-terminal domain connected to a flexible linker. The N-terminal domain is the catalytic domain containing calcium ions at the catalytic site. Coordination bonds were observed between five amino acid residues and glycerol. Although the function of the C-terminal domain is currently unknown, inter-domain interactions between the N- and C-terminal domains may contribute to its relatively high thermostability.

Comparison of Xrn1 and Rat1 5' → 3' exoribonucleases in budding yeast supports the specific role of Xrn1 in cotranslational mRNA decay.

The yeast Saccharomyces cerevisiae and most eukaryotes carry two 5' → 3' exoribonuclease paralogs. In yeast, they are called Xrn1, which shuttles between the nucleus and the cytoplasm, and executes major cytoplasmic messenger RNA (mRNA) decay, and Rat1, which carries a strong nuclear localization sequence (NLS) and localizes to the nucleus. Xrn1 is 30% identical to Rat1 but has an extra ~500 amino acids C-terminal extension. In the cytoplasm, Xrn1 can degrade decapped mRNAs during the last round of translation by ribosomes, a process referred to as "cotranslational mRNA decay." The division of labor between the two enzymes is still enigmatic and serves as a paradigm for the subfunctionalization of many other paralogs. Here we show that Rat1 is capable of functioning in cytoplasmic mRNA decay, provided that Rat1 remains cytoplasmic due to its NLS disruption (cRat1). This indicates that the physical segregation of the two paralogs plays roles in their specific functions. However, reversing segregation is not sufficient to fully complement the Xrn1 function. Specifically, cRat1 can partially restore the cell volume, mRNA stability, the proliferation rate, and 5' → 3' decay alterations that characterize xrn1Δ cells. Nevertheless, cotranslational decay is only slightly complemented by cRat1. The use of the AlphaFold prediction for cRat1 and its subsequent docking with the ribosome complex and the sequence conservation between cRat1 and Xrn1 suggest that the tight interaction with the ribosome observed for Xrn1 is not maintained in cRat1. Adding the Xrn1 C-terminal domain to Rat1 does not improve phenotypes, which indicates that lack of the C-terminal is not responsible for partial complementation. Overall, during evolution, it appears that the two paralogs have acquired specific characteristics to make functional partitioning beneficial.

C-TERMINAL DOMAIN PHOSPHATASE-LIKE 3 contributes to GA-mediated growth and flowering by interaction with DELLA proteins.

Gibberellic acid (GA) plays a central role in many plant developmental processes and is crucial for crop improvement. DELLA proteins, the core suppressors in the GA signaling pathway, are degraded by GA via the 26S proteasomal pathway to release the GA response. However, little is known about the phosphorylation-mediated regulation of DELLA proteins. In this study, we combined GA response assays with protein-protein interaction analysis to infer the connection between Arabidopsis thaliana DELLAs and the C-TERMINAL DOMAIN PHOSPHATASE-LIKE 3 (CPL3), a phosphatase involved in the dephosphorylation of RNA polymerase II. We show that CPL3 directly interacts with DELLA proteins and promotes DELLA protein stability by inhibiting its degradation by the 26S proteasome. Consequently, CPL3 negatively modulates multiple GA-mediated processes of plant development, including hypocotyl elongation, flowering time, and anthocyanin accumulation. Taken together, our findings demonstrate that CPL3 serves as a novel regulator that could improve DELLA stability and thereby participate in GA signaling transduction.

SCI1, a flower regulator of cell proliferation, and its partners NtCDKG2 and NtRH35 interact with the splicing machinery.

Successful plant reproduction depends on the adequate development of floral organs controlled by cell proliferation and other processes. The Stigma/style cell-cycle inhibitor 1 (SCI1) gene regulates cell proliferation and affects the final size of the female reproductive organ. To unravel the molecular mechanism exerted by Nicotiana tabacum SCI1 in cell proliferation control, we searched for its interaction partners through semi-in vivo pull-down experiments, uncovering a cyclin-dependent kinase, NtCDKG;2. Bimolecular fluorescence complementation and co-localization experiments showed that SCI1 interacts with NtCDKG;2 and its cognate NtCyclin L in nucleoli and splicing speckles. The screening of a yeast two-hybrid cDNA library using SCI1 as bait revealed a novel DEAD-box RNA helicase (NtRH35). Interaction between the NtCDKG;2-NtCyclin L complex and NtRH35 is also shown. Subcellular localization experiments showed that SCI1, NtRH35, and the NtCDKG;2-NtCyclin L complex associate with each other within splicing speckles. The yeast two-hybrid screening of NtCDKG;2 and NtRH35 identified the conserved spliceosome components U2a', NF-κB activating protein (NKAP), and CACTIN. This work presents SCI1 and its interactors, the NtCDKG;2-NtCyclin L complex and NtRH35, as new spliceosome-associated proteins. Our findings reveal a network of interactions and indicate that SCI1 may regulate cell proliferation through the splicing process, providing new insights into the intricate molecular pathways governing plant development.

Mitochondrial amidoxime-reducing component 1 p.Ala165Thr increases protein degradation mediated by the proteasome.

OBJECTIVE: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a global health concern with no effective and specific drug treatment available. The rs2642438 minor allele in mitochondrial amidoxime-reducing component 1 (MARC1) results in an aminoacidic substitution (p.Ala165Thr) and associates with protection against MASLD. However, the mechanisms behind this protective effect are unknown. In this study, we examined the consequences of this aminoacidic substitution on protein stability and subcellular localization. METHODS: We overexpressed the human MARC1 A165 (wild-type) or 165T (mutant) in vivo in mice and in vitro in human hepatoma cells (HepG2 and HuH-7), generated several mutants at position 165 by in situ mutagenesis and then examined protein levels. We also generated HepG2 cells stably overexpressing MARC1 A165 or 165T to test the effect of this substitution on MARC1 subcellular localization. RESULTS: MARC1 165T overexpression resulted in lower protein levels than A165 both in vivo and in vitro. Similarly, any mutant at position 165 showed lower protein levels compared to the wild-type protein. We showed that the 165T mutant protein is polyubiquitinated and its degradation is accelerated through lysine-48 ubiquitin-mediated proteasomal degradation. We also showed that the 165T substitution does not affect the MARC1 subcellular localization. CONCLUSIONS: This study shows that alanine at position 165 in MARC1 is crucial for protein stability, and the threonine substitution at this position leads to a hypomorphic protein variant due to lower protein levels. Our result supports the notion that lowering hepatic MARC1 protein level may be a successful therapeutic strategy for treating MASLD.

Mediator Undergoes a Compositional Change during Transcriptional Activation.

Mediator is a transcriptional co-activator recruited to enhancers by DNA-binding activators, and it also interacts with RNA polymerase (Pol) II as part of the preinitiation complex (PIC). We demonstrate that a single Mediator complex associates with the enhancer and core promoter in vivo, indicating that it can physically bridge these transcriptional elements. However, the Mediator kinase module associates strongly with the enhancer, but not with the core promoter, and it dissociates from the enhancer upon depletion of the TFIIH kinase. Severing the kinase module from Mediator by removing the connecting subunit Med13 does not affect Mediator association at the core promoter but increases occupancy at enhancers. Thus, Mediator undergoes a compositional change in which the kinase module, recruited via Mediator to the enhancer, dissociates from Mediator to permit association with Pol II and the PIC. As such, Mediator acts as a dynamic bridge between the enhancer and core promoter.

Identification and Analysis of Six Phosphorylation Sites Within the Xenopus laevis Linker Histone H1.0 C-Terminal Domain Indicate Distinct Effects on Nucleosome Structure.

As a key structural component of the chromatin of higher eukaryotes, linker histones (H1s) are involved in stabilizing the folding of extended nucleosome arrays into higher-order chromatin structures and function as a gene-specific regulator of transcription in vivo. The H1 C-terminal domain (CTD) is essential for high-affinity binding of linker histones to chromatin and stabilization of higher-order chromatin structure. Importantly, the H1 CTD is an intrinsically disordered domain that undergoes a drastic condensation upon binding to nucleosomes. Moreover, although phosphorylation is a prevalent post-translational modification within the H1 CTD, exactly where this modification is installed and how phosphorylation influences the structure of the H1 CTD remains unclear for many H1s. Using novel mass spectrometry techniques, we identified six phosphorylation sites within the CTD of the archetypal linker histone Xenopus H1.0. We then analyzed nucleosome-dependent CTD condensation and H1-dependent linker DNA organization for H1.0 in which the phosphorylated serine residues were replaced by glutamic acid residues (phosphomimics) in six independent mutants. We find that phosphomimetics at residues S117E, S155E, S181E, S188E, and S192E resulted in a significant reduction in nucleosome-bound H1.0 CTD condensation compared with unphosphorylated H1.0, whereas S130E did not alter CTD structure. Furthermore, we found distinct effects among the phosphomimetics on H1-dependent linker DNA trajectory, indicating unique mechanisms by which this modification can influence H1 CTD condensation. These results bring to light a novel role for linker histone phosphorylation in directly altering the structure of nucleosome-bound H1 and a potential novel mechanism for its effects on chromatin structure and function.

The intracellular C-terminal domain of mGluR6 contains ER retention motifs.

Metabotropic glutamate receptor 6 (mGluR6) predominantly localizes to the postsynaptic sites of retinal ON-bipolar cells, at which it recognizes glutamate released from photoreceptors. The C-terminal domain (CTD) of mGluR6 contains a cluster of basic amino acids resembling motifs for endoplasmic reticulum (ER) retention. We herein investigated whether these basic residues are involved in regulating the subcellular localization of mGluR6 in 293T cells expressing mGluR6 CTD mutants using immunocytochemistry, immunoprecipitation, and flow cytometry. We showed that full-length mGluR6 localized to the ER and cell surface, whereas mGluR6 mutants with 15- and 20-amino acid deletions from the C terminus localized to the ER, but were deficient at the cell surface. We also demonstrated that the cell surface deficiency of mGluR6 mutants was rescued by introducing an alanine substitution at basic residues within the CTD. The surface-deficient mGluR6 mutant still did not localize to the cell surface and was retained in the ER when co-expressed with surface-expressible constructs, including full-length mGluR6, even though surface-deficient and surface-expressible constructs formed heteromeric complexes. The co-expression of the surface-deficient mGluR6 mutant reduced the surface levels of surface-expressible constructs. These results indicate that basic residues in the mGluR6 CTD served as ER retention signals. We suggest that exposed ER retention motifs in the aberrant assembly containing truncated or misfolded mGluR6 prevent these protein complexes from being transported to the cell surface.

Role of the C-Terminal ß Sandwich of Thermoanaerobacter tengcongensis Thermophilic Esterase in Hydrolysis of Long-Chain Acyl Substrates.

To search for a novel thermostable esterase for optimized industrial applications, esterase from a thermophilic eubacterium species, Thermoanaerobacter tengcongensis MB4, was purified and characterized in this work. Sequence analysis of T. tengcongensis esterase with other homologous esterases of the same family revealed an apparent tail at the C-terminal that is not conserved across the esterase family. Hence, it was hypothesized that the tail is unlikely to have an essential structural or catalytic role. However, there is no documented report of any role for this tail region. We probed the role of the C-terminal domain on the catalytic activity and substrate preference of T. tengcongensis esterase EstA3 with a view to see how it could be engineered for enhanced properties. To achieve this, we cloned, expressed, and purified the wild-type and the truncated versions of the enzyme. In addition, a naturally occurring member of the family (from Brevibacillus brevis) that lacks the C-terminal tail was also made. In vitro characterization of the purified enzymes showed that the C-terminal domain contributes significantly to the catalytic activity and distinct substrate preference of T. tengcongensis esterase EstA3. All three recombinant enzymes showed the highest preference for paranitrophenyl butyrate (pNPC4), which suggests they are true esterases, not lipases. Kinetic data revealed that truncation had a slight effect on the substrate-binding affinity. Thus, the drop in preference towards long-chain substrates might not be a result of substrate binding affinity alone. The findings from this work could form the basis for future protein engineering allowing the modification of esterase catalytic properties through domain swapping or by attaching a modular protein domain.

Role of C-terminal domain of Mycobacterium tuberculosis PE6 (Rv0335c) protein in host mitochondrial stress and macrophage apoptosis.

PE/PPE proteins of Mycobacterium tuberculosis (Mtb) target the host organelles to dictate the outcome of infection. This study investigated the significance of PE6/Rv0335c protein's unique C-terminal in causing host mitochondrial perturbations and apoptosis. In-silico analysis revealed that similar to eukaryotic apoptotic Bcl2 proteins, Rv0335c had disordered, hydrophobic C-terminal and two BH3-like motifs in which one was located at C-terminal. Also, Rv0335c's N terminal had mitochondrial targeting sequence. Since, C-terminal of Bcl2 proteins are crucial for mitochondria targeting and apoptosis; it became relevant to evaluate the role of Rv0335c's C-terminal domain in modulating host mitochondrial functions and apoptosis. To confirm this, in-vitro experiments were conducted with Rv0335c whole protein and Rv0335c∆Cterm (C-terminal domain deleted Rv0335c) protein. Rv0335c∆Cterm caused significant reduction in mitochondrial perturbations and Caspase-mediated apoptosis of THP1 macrophages in comparison to Rv0335c. However, the deletion of C-terminal domain didn't affect Rv0335c's ability to localize to mitochondria. Nine Ca2+ binding residues were predicted within Rv0335c and four of them were at the C-terminal. In-vitro studies confirmed that Rv0335c caused significant increase in intracellular calcium influx whereas Rv0335c∆Cterm had insignificant effect on Ca2+ influx. Rv0335c has been reported to be a TLR4 agonist and, we observed a significant reduction in the expression of TLR4-HLA-DR-TNF-α in response to Rv0335c∆Cterm protein also suggesting the role of Rv0335c's C-terminal domain in host-pathogen interaction. These findings indicate the possibility of Rv0335c as a molecular mimic of eukaryotic Bcl2 proteins which equips it to cause host mitochondrial perturbations and apoptosis that may facilitate pathogen persistence.

A unique C-terminal domain contributes to the molecular function of Restorer-of-fertility proteins in plant mitochondria.

Restorer-of-fertility (Rf) genes encode pentatricopeptide repeat (PPR) proteins that are targeted to mitochondria where they specifically bind to transcripts that induce cytoplasmic male sterility and repress their expression. In searching for a molecular signature unique to this class of proteins, we found that a majority of known Rf proteins have a distinct domain, which we called RfCTD (Restorer-of-fertility C-terminal domain), and its presence correlates with the ability to induce cleavage of the mitochondrial RNA target. A screen of 219 angiosperm genomes from 123 genera using a sequence profile that can quickly and accurately identify RfCTD sequences revealed considerable variation in RFL/RfCTD gene numbers across flowering plants. We observed that plant genera with bisexual flowers have significantly higher numbers of RFL genes compared to those with unisexual flowers, consistent with a role of these proteins in restoration of male fertility. We show that removing the RfCTD from the RFL protein RNA PROCESSING FACTOR 2-nad6 prevented cleavage of its RNA target, the nad6 transcript, in Arabidopsis thaliana mitochondria. We provide a simple way of identifying putative Rf candidates in genome sequences, new insights into the molecular mode of action of Rf proteins and the evolution of fertility restoration in flowering plants.

In vitro and in vivo suppression of SARS-CoV-2 replication by a modified, short, cell-penetrating peptide targeting the C-terminal domain of the viral spike protein.

Peptides are promising therapeutic agents for COVID-19 because of their specificity, easy synthesis, and ability to be fine-tuned. We previously demonstrated that a cell-permeable peptide corresponding to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike C-terminal domain (CD) inhibits the interaction between viral spike and nucleocapsid proteins that results in SARS-CoV-2 replication in vitro. Here, we used docking studies to design R-t-Spike CD(D), a more potent short cell-penetrating peptide composed of all D-form amino acids and evaluated its inhibitory effect against the replication of SARS-CoV-2 S clade and other variants. R-t-Spike CD(D) was internalized into Vero cells and Calu-3 cells and suppressed the replication of SARS-CoV-2 S clade, delta variant, and omicron variant with higher potency than the original peptide. In hemizygous K18-hACE2 mice, intratracheal administration of R-t-Spike CD(D) effectively delivered the peptide to the trachea and lungs, whereas intranasal administration delivered the peptide mostly to the upper respiratory system and stomach, and a small amount to the lungs. Administration by either route reduced viral loads in mouse lungs and turbinates. Furthermore, intranasally administered R-t-Spike CD(D) mitigated pathological change in the lungs and increased the survival of mice after infection with the SARS-CoV-2 S clade or delta variant. Our data suggest that R-t-Spike CD(D) has potential as a therapeutic agent against SARS-CoV-2 infection.

Elabela, a Novel Peptide, Exerts Neuroprotective Effects Against Ischemic Stroke Through the APJ/miR-124-3p/CTDSP1/AKT Pathway.

Elabela (ELA), which is the second endogenous peptide ligand of the apelin receptor (APJ) to be discovered, has been widely studied for potential use as a therapeutic peptide. However, its role in ischemic stroke (IS), which is a leading cause of disability and death worldwide and has limited therapeutic options, is uncertain. The aim of the present study was to investigate the beneficial effects of ELA on neuron survival after ischemia and the underlying molecular mechanisms. Primary cortical neurons were isolated from the cerebral cortex of pregnant C57BL/6J mice. Flow cytometry and immunofluorescence showed that ELA inhibited oxygen-glucose deprivation (OGD) -induced apoptosis and axonal damage in vitro. Additionally, analysis of the Gene Expression Omnibus database revealed that the expression of microRNA-124-3p (miR-124-3p) was decreased in blood samples from patients with IS, while the expression of C-terminal domain small phosphatase 1 (CTDSP1) was increased. These results indicated that miR-124-3p and CTDSP1 were related to ischemic stroke, and there might be a negative regulatory relationship between them. Then, we found that ELA significantly elevated miR-124-3p expression, suppressed CTDSP1 expression, and increased p-AKT expression by binding to the APJ receptor under OGD in vitro. A dual-luciferase reporter assay confirmed that CTDSP1 was a direct target of miR-124-3p. Furthermore, adenovirus-mediated overexpression of CTDSP1 exacerbated neuronal apoptosis and axonal damage and suppressed AKT phosphorylation, while treatment with ELA or miR-124-3p mimics reversed these effects. In conclusion, these results indicated that ELA could alleviate neuronal apoptosis and axonal damage by upregulating miR-124-3p and activating the CTDSP1/AKT signaling pathway. This study, for the first time, verified the protective effect of ELA against neuronal injury after ischemia and revealed the underlying mechanisms. We demonstrated the potential for the use of ELA as a therapeutic agent in the treatment of ischemic stroke.