Targeting Motif Research Articles

Investigating the Anticancer Properties of Novel Functionalized Platinum(II)–Terpyridine Complexes

Novel platinum(II) complexes of 4′-substituted terpyridine ligands were synthesized and characterized. Each complex had a different biomolecule (amine, glucose, biotin and hyaluronic acid) as a targeting motif, potentially improving therapeutic outcomes. We demonstrated that complexes can self-assemble in water into about 150 nm nanoparticles. Moreover, the complexes were assayed in vitro toward a panel of human cancer cell lines (ovarian adenocarcinoma A2780, lung cancer A549, breast adenocarcinoma MDA-MB-231, neuroblastoma SHSY5Y) to explore the impact of the pendant moiety on the terpyridine toxicity. The platinum complex of terpyridine amine derivative, [Pt(TpyNH2)Cl]Cl, showed the best antiproliferative effect, which was higher than cisplatin and [Pt(Tpy)Cl]Cl. Selective in vitro antiproliferative activity was achieved in A549 cancer cells with the Pt–HAtpy complex. These findings underline the potential of these novel platinum(II) complexes in cancer therapy and highlight the importance of tailored molecular design for achieving enhanced therapeutic effects.

Mutations in the non-catalytic polyproline motif destabilize TREX1 and amplify cGAS-STING signaling.

The cGAS-STING pathway detects cytosolic DNA and activates a signaling cascade that results in a type I interferon (IFN) response. The endoplasmic reticulum (ER)-associated exonuclease TREX1 suppresses cGAS-STING by eliminating DNA from the cytosol. Mutations that compromise TREX1 function are linked to autoinflammatory disorders, including systemic lupus erythematosus (SLE) and Aicardi-Goutières syndrome (AGS). Despite key roles in regulating cGAS-STING and suppressing excessive inflammation, the impact of many disease-associated TREX1 mutations-particularly those outside of the core catalytic domains-remains poorly understood. Here, we characterize a recessive AGS-linked TREX1 P61Q mutation occurring within the poorly characterized polyproline helix (PPII) motif. In keeping with its position outside of the catalytic core or ER targeting motifs, neither the P61Q mutation, nor aggregate proline-to-alanine PPII mutation, disrupts TREX1 exonuclease activity, subcellular localization, or cGAS-STING regulation in overexpression systems. Introducing targeted mutations into the endogenous TREX1 locus revealed that PPII mutations destabilize the protein, resulting in impaired exonuclease activity and unrestrained cGAS-STING activation. Overall, these results demonstrate that TREX1 PPII mutations, including P61Q, impair proper immune regulation and lead to autoimmune disease through TREX1 destabilization.

Abstract 3106: Targeted Mrna Delivery To The Inflamed Lung For The Treatment Of Ards

Acute respiratory distress syndrome (ARDS), caused by widespread endothelial barrier disruption and uncontrolled cytokine storm, is the major feared complication of pandemic influenza and COVID-19. Currently, there are few pharmacological treatments that directly target ARDS. We have previously discovered that reduction of Krüppel-like Factor 2 (KLF2, a transcription factor) in inflamed endothelium leads to ARDS induced by LPS, influenza, and high-tidal volume ventilation. Recently, we detected a significant reduction of KLF2 in lung autopsies from COVID-19 patients and demonstrated the anti-viral function of endothelial KLF2. Here, we devised a VCAM1-targeting nanomedicine strategy to restore KLF2 in inflamed endothelial cells. We engineered a rationally designed liposome which possesses potent mRNA delivery and transfection capacity. This VCAM1-targeting liposome selectively delivered functional KLF2 mRNA to inflamed vascular endothelium and significantly reduced mouse ARDS induced by influenza virus H1N1 and SARS-CoV-2, by calming local cytokine storm and promoting endothelium health. Moreover, we demonstrated that this targeted liposome serves as a broad platform to effectively deliver various mRNA molecules to targeted tissues of interest by displaying different targeting motifs. Our results demonstrate that this KLF2 mRNA encapsulated, VCAM1-targeting liposome is a promising mRNA therapeutic strategy to treat ARDS and vascular diseases.

Quantum Dot Imaging Agents: Haematopoietic Cell Interactions and Biocompatibility.

Quantum dots (QDs) are semi-conducting nanoparticles that have been developed for a range of biological and non-biological functions. They can be tuned to multiple different emission wavelengths and can have significant benefits over other fluorescent systems. Many studies have utilised QDs with a cadmium-based core; however, these QDs have since been shown to have poor biological compatibility. Therefore, other QDs, such as indium phosphide QDs, have been developed. These QDs retain excellent fluorescent intensity and tunability but are thought to have elevated biological compatibility. Herein we discuss the applicability of a range of QDs to the cardiovascular system. Key disease states such as myocardial infarction and stroke are associated with cardiovascular disease (CVD), and there is an opportunity to improve clinical imaging to aide clinical outcomes for these disease states. QDs offer potential clinical benefits given their ability to perform multiple functions, such as carry an imaging agent, a therapy, and a targeting motif. Two key cell types associated with CVD are platelets and immune cells. Both cell types play key roles in establishing an inflammatory environment within CVD, and as such aid the formation of pathological thrombi. However, it is unclear at present how and with which cell types QDs interact, and if they potentially drive unwanted changes or activation of these cell types. Therefore, although QDs show great promise for boosting imaging capability, further work needs to be completed to fully understand their biological compatibility.

A peptide selectively recognizes Gram-negative bacteria and forms a bacterial extracellular trap (BET) through interfacial self-assembly.

An innate immune system intricately leverages unique mechanisms to inhibit colonization of external invasive Bacteria, for example human defensin-6, through responsive encapsulation of bacteria. Infection and accompanying antibiotic resistance stemming from Gram-negative bacteria aggregation represent an emerging public health crisis, which calls for research into novel anti-bacterial therapeutics. Herein, inspired by naturally found host-defense peptides, we design a defensin-like peptide ligand, bacteria extracellular trap (BET) peptide, with modular design composed of targeting, assembly, and hydrophobic motifs with an aggregation-induced emission feature. The ligand specifically recognizes Gram-negative bacteria via targeting cell wall conserved lipopolysaccharides (LPS) and transforms from nanoparticles to nanofibrous networks in situ to trap bacteria and induce aggregation. Importantly, treatment of the BET peptide was found to have an antibacterial effect on the Pseudomonas aeruginosa strain, which is comparable to neomycin. Animal studies further demonstrate its ability to trigger aggregation of bacteria in vivo. This biomimetic self-assembling BET peptide provides a novel approach to fight against pathogenic Gram-negative bacteria.

A cell-penetrating peptide exerts therapeutic effects against ischemic stroke by mediating the lysosomal degradation of sirtuin 5.

Stroke is a major public health concern worldwide. The lack of effective therapies heightens the need for new therapeutic agents. Previous study identified sirtuin 5 (SIRT5) as a positive regulator of microglia-induced excessive neuroinflammation following ischemic stroke. Interventions targeting SIRT5 should therefore alleviate neuroinflammation and protect against ischemic stroke. Here, we synthesized a membrane-permeable peptide specifically bound to SIRT5 through a chaperone-mediated autophagy targeting motif (Tat-SIRT5-CTM) and examined its therapeutic effect in vitro and in vivo. First, in primary microglia, Tat-SIRT5-CTM suppressed the binding of SIRT5 with annexin-A1 (ANXA1), leading to SIRT5 degradation and thus inhibition of SIRT5-mediated desuccinylation of ANXA1, followed by increased membrane accumulation and secretion of ANXA1. These changes, in turn, alleviated microglia-induced neuroinflammation. Moreover, following intravenous injection, Tat-SIRT5-CTM could efficiently pass through the blood‒brain barrier. Importantly, systemic administration of Tat-SIRT5-CTM reduced the brain infarct area and neuronal loss, mitigated neurological deficit scores, and improved long-term neurological functions in a mouse model of ischemic stroke. Furthermore, no toxicity was observed when high doses Tat-SIRT5-CTM were injected into nonischemic mice. Collectively, our study reveals the promising efficacy of the peptide-directed lysosomal degradation of SIRT5 and suggests it as an effective therapeutic approach for the treatment of ischemic stroke.

Development of a Same-Day Closed CAR-T Cell Manufacturing Process to Generate Lentivector Loaded Lymphocytes Ready for Subcutaneous Injection

Background: Adoptive T-cell therapies with CD19 chimeric antigen receptors (CAR) are now the standard of care for refractory/recurrent hematologic B-cell malignancies. However, challenges remain in delivering a more fit cell product that can be manufactured in a more timely and cost-effective manner. To address these challenges, a rapid same-day fully closed manufacturing process was developed to generate a more fit product for subcutaneous administration and to enable an efficient transition from early-phase clinical trials to commercial-scale production. Methods: A lentivector (LV) was designed with the capability of directly targeting and activating T cells simultaneously to enable manufacturing without prior T-cell selection. Experiments were performed to understand the kinetics of direct lentivector-induced T-cell activation, internalization, reverse transcription, and vector genome integration. After establishing critical process parameters for efficient loading of T cells with LVs, a closed manufacturing process was developed from peripheral blood mononuclear cells (PBMC) starting material derived from whole blood or apheresis. PBMCs were isolated in an automated closed system and exposed to LV for a four-hour incubation period. Cells were washed in an automated closed system to remove residual LV, formulated, and vialed with release testing for identity, potency, purity, and safety. Three full-scale engineering lots were manufactured from donor-derived apheresis in addition to three small-scale analytical lots with patient-derived PBMC. Results: For targeted gene delivery into T cells within PBMC, the CD19 CAR LV was designed to contain a CD3 targeting motif in its envelope to enable T-cell binding, activation, and viral entry into CD3+ T cells. The CD3-directed LV encoding a CD19-CAR and a novel synthetic driver co-stimulatory domain was manufactured in a chemically defined 25L clinical scale suspension-based process and vialed to a target unit potency for ease of cell loading. The purified CD3-directed LV is capable of inducing T-cell receptor internalization and activation through specific bioassays. In surrogate analytical cultures, following LV exposure and automated washing, viral entry into T cells was complete. Analytical cultures demonstrate the ability to successfully generate CAR+ cells with a unique CD3+CD56+CD45RA+CCR7+ T memory-stem cell with NK-like features and a vector copy number of less than 5 vector copies/transduced cell (1.95 +/- 0.58). In preparation for clinical investigation, full-scale runs were performed in GMP facilities with qualified methods for release testing against identity, potency, purity, and safety. Small-scale analytical processes utilizing B-cell lymphoma patient-derived PBMC from whole blood were evaluated for differences relative to donor-derived material. Conclusions: To address current challenges in the manufacturing of autologous cell products, a same-day closed manufacturing process was developed to produce cells with enhanced fitness. Qualification of this novel design included 1) completion of the production of the CD3-directed LV encoding a CD19 CAR with a novel synthetic co-stimulatory driver element at the 25L scale; 2) completion of three full-scale closed engineering runs along with the qualification of QC release testing for identity, potency, purity, and safety and 3) comparability studies utilizing patient-derived PBMC.

Expressing Optogenetic Actuators Fused to N-terminal Mucin Motifs Delivers Targets to Specific Subcellular Compartments in Polarized Cells.

Optogenetics is a powerful approach in neuroscience research. However, other tissues of the body may benefit from controlled ion currents and neuroscience may benefit from more precise optogenetic expression. The present work constructs three subcellularly-targeted optogenetic actuators based on the channelrhodopsin ChR2-XXL, utilizing 5, 10, or 15 tandem repeats (TR) from mucin as N-terminal targeting motifs and evaluates expression in several polarized and non-polarized cell types. The modified channelrhodopsin maintains its electrophysiological properties, which can be used to produce continuous membrane depolarization, despite the expected size of the repeats. This work then shows that these actuators are subcellularly localized in polarized cells. In polarized epithelial cells, all three actuators localize to just the lateral membrane. The TR-tagged constructs also express subcellularly in cortical neurons, where TR5-ChR2XXL and TR10-ChR2XXL mainly target the somatodendrites. Moreover, the transfection efficiencies are shown to be dependent on cell type and tandem repeat length. Overall, this work verifies that the targeting motifs from epithelial cells can be used to localize optogenetic actuators in both epithelia and neurons, opening epithelia processes to optogenetic manipulation and providing new possibilities to target optogenetic tools.

Targeted expression of a designed fusion protein containing BMP2 into the lumen of exosomes

BackgroundExosomes are 30–150 nm membrane vesicles, originating from the endocytic pathway. By acting as natural carriers of biomolecules, they can transfer various materials to recipient cells. Therefore, discovering novel strategies for cargo packaging into exosomes is crucial. MethodsThe fusion constructs, consisting of protein of interest (BMP2) along with the targeting motif, linkers, tracking proteins, and enzyme cleavage sites, were computationally designed. Following the homology modeling, the best structure was selected and subjected to molecular dynamics (MD) simulation and docking analyses. The fusion protein gene was expressed in the HEK-293LTV cell line. The high-efficiency transfected and transduced cells were screened and their exosomes were isolated. Finally, cell and exosome lysates were evaluated for expression of the fusion protein. ResultsA total of 12 constructs with lengths ranging from 483 to 496 were designed. The top three templates, 1REW, 2H5Q, and 2MOF were screened. MD simulation and docking analyses of the structures revealed their stability and functionality. In the protein expression analyses, three bands at sizes of approximately 60, 25, and 12.5 kDa were observed, consistent with the sizes of the complete fusion protein, dimeric, and monomeric BMP2 protein. The presence of a 12.5 kDa band at exosome lysate analysis might suggest that it was loaded and cleaved inside exosomes. ConclusionIn summary, these findings revealed that the proposed idea for cargo sorting within the exosome lumen through incorporating an appropriate cleavage site was effective, thus providing further insight into the potential of exosomes as nano-shuttles bearing therapeutic biomolecules.

2-λ5-Phosphaquinolin-2-ones as Non-cytotoxic, Targetable, and pH-Stable Fluorophores.

Several phosphaquinolinone derivatives have been synthesized and characterized to explore their usefulness in the realm of cell imaging. Solution-state photophysical properties in both aqueous and organic solutions were collected for these derivatives. Additionally, CCK-8 cell viability assays and fluorescent imaging in HeLa cells incubated with the new heterocyclic derivatives show evidence of favorable cell permeability, cell viability, and moderate intracellular localization when appended with the well-known morpholine targeting motif.

Lysosome-Specific Coumarin-Based Fluorescent Bioprobes for in Vivo Polarity Sensing and Cancer Treatment.

About 90% of cancer deaths worldwide are caused by the spread of cancer cells from the primary tumor to distant organs (metastasis). Therefore, there is an urgent need for an early diagnosis and treatment before cancer metastasis occurs. Lysosomes have emerged as attractive targets for cancer diagnosis and treatment because polar defects in lysosomes can induce apoptosis and cell death. Coumarin is a known polar-sensitive dye with good biocompatibility; because of this, we constructed two fluorescent probes of coumarin derivatives with the "D-π-A" structure, CouN-1 and CouN-2, through three simple reactions. In molecular design, due to morpholine's prominent lysosomal targeting characteristics, it was used as both lysosomal targeting motifs and an electron donor (D), while coumarin was used as an electron acceptor (A). The experimental results strongly proved that CouN-1 and CouN-2 have a good linear relationship with the polarity change of Δf = 0.209-0.308. In addition, both in vitro and in vivo imaging results have shown that CouN-1 and CouN-2 can specifically identify and monitor tumor sites. In the cell uptake and apoptosis experiments, the two probes also showed a strong antiproliferation effect on cancer cells. All of these characteristics demonstrated the potential of these two polarity-sensitive biological probes, CouN-1 and CouN-2, in the diagnosis and treatment of cancer.

Abstract PR010: In-“speck”-ting the nucleus: Nuclear speckles are a critical layer of gene regulation that predict outcomes in clear cell renal cell carcinoma

Abstract Nuclear speckles are dynamic substructures within the nucleus that contain a myriad of factors involved in gene activation. While nuclear speckles were discovered over 100 years ago, how they vary in human health and disease and the consequences of such variation remain enigmatic. Using RNA-seq data to estimate nuclear speckle content, we found that nuclear speckle phenotypes vary reproducibly in >20 cancer types. Of these cancers, nuclear speckle variation predicted patient outcomes specifically in clear cell renal cell carcinoma (ccRCC). This ccRCC speckle sensitivity was confirmed in a separate cohort using immunofluorescence to independently assess speckle phenotypes, and was specific to ccRCC with VHL mutation or copy number loss, suggesting involvement of the VHL-degraded HIF stress-responsive transcription factors. Given our previous findings linking nuclear speckles to another stress-responsive transcription factor, p53, we hypothesized that the speckle sensitivities we observed in ccRCC may be a consequence of as-of-yet uncharacterized speckle functions of HIF1A or HIF2A. Previously, we found that p53 drives DNA-speckle contacts of a subset of target genes to boost expression, revealing a novel method of transcription-factor-based gene regulation. However, the extent that DNA-speckle positioning is regulated by transcription factors other than p53, including the HIF transcription factors, is currently unclear. Critically, p53 and HIF2A, but not HIF1A, share a homologous protein motif, now coined the speckle targeting motif, that precisely overlaps with the region where we had mapped p53 DNA-speckle targeting abilities. Using genomic and imaging-based methods, we identified HIF2A as a second transcription factor that drives association between a subset of its target genes and nuclear speckles. Deletion of the HIF2A speckle targeting motifs ablated its DNA-speckle targeting functions, led to lower induction of speckle-associating genes, and resulted in a HIF2A-mediated gene expression program that more closely resembled the better-outcome ccRCC speckle patient group. Beyond p53 and HIF2A, we found that the speckle targeting motif occurs within hundreds of proteins and is specifically enriched among transcriptional regulators. These findings suggest that DNA-speckle targeting by transcription factors is potentially a wide-spread mechanism of gene regulation that may be manipulated by changing speckle-targeting abilities of transcription factors or by altering speckles themselves. For ccRCC, our findings reveal nuclear speckle phenotypes as novel prognostic indicators and illuminate nuclear speckles and HIF2A speckle-targeting abilities as new avenues for therapeutic development. Citation Format: Katherine A. Alexander, Ruofan Yu, Christine Faunce, Nicolas Skuli, Nathan Coffey, Son Nguyen, Nicholas Biddle, Catherine Li, Eric Joyce, Arjun Raj, M. Celeste Simon, Shelley Berger. In-“speck”-ting the nucleus: Nuclear speckles are a critical layer of gene regulation that predict outcomes in clear cell renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr PR010.

DNAi-peptide nanohybrid smart particles target BCL-2 oncogene and induce apoptosis in breast cancer cells

Genomic DNA sequences provide unique target sites, with high druggability value, for treatment of genetically-linked diseases like cancer. B-cell lymphoma protein-2 (BCL-2) prevents Bcl-2-associated X protein (BAX) and Bcl-2 antagonist killer 1 (BAK) oligomerization, which would otherwise lead to the release of several apoptogenic molecules from the mitochondrion. It is also known that BCL-2 binds to and inactivates BAX and other pro-apoptotic proteins, thereby inhibiting apoptosis. BCL-2 protein family, through its role in regulation of apoptotic pathways, is possibly related to chemo-resistance in almost half of all cancer types including breast cancer. Here for the first time, we have developed a nanohybrid using a peptide-based carrier and a Deoxyribonucleic acid inhibitor (DNAi) against BCL-2 oncogene to induce apoptosis in breast cancer cells. The genetically designed nanocarrier was functionalized with an internalizing RGD (iRGD) targeting motif and successfully produced by recombinant DNA technology. Gel retardation assay demonstrated that the peptide-based carrier binds single-stranded DNAi upon simple mixing. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) analyses further revealed the formation of nanohybrid particles with a size of 30 nm and a slightly positive charge. This hemocompatible nanohybrid efficiently delivered its contents into cancer cells using iRGD targeting moiety. Gene expression analysis demonstrated that the nanohybrids, which contained DNAi against BCL-2 proficiently suppressed the expression of this oncogene in a sequence specific manner. In addition, the nanohybrid, triggered release of cytochrome c (cyt c) and caspase3/7 activation with high efficiency. Although the DNAi and free nanocarrier were separately unable to affect the cell viability, the nanohybrid of 20 nM of DNAi showed outstanding antineoplastic potential, which was adjusted by the ratio of the MiRGD nanocarrier to DNAi. It should be noted that, the designed nanohybrid showed a suitable specificity profile and did not affect the viability of normal cells. The results suggest that this nanohybrid may be useful for robust breast cancer treatment through targeting the BCL-2 oncogene without any side effects.

Design of negative-regulating proteins of Rheb/mTORC1 with much-reduced sizes of the tuberous sclerosis protein complex.

The mTORC1 signaling pathway regulates cell growth and metabolism in a variety of organisms from yeast to human, and inhibition of the mTORC1 pathway has the prospect to treat cancer or achieve longevity. The tuberous sclerosis protein complex (TSCC) is a master negative regulator of the mTORC1 signaling pathway through hydrolyzing the GTP loaded on the small GTPase Rheb, which is a key activator of mTOR. However, the large size (~700 kDa) and complex structural organization of TSCC render it vulnerable to degradation and inactivation, thus limiting its potential application. In this work, based on thorough analysis and understanding of the structural mechanism of how the stabilization domain of TSC2 secures the association of TSC2-GAP with Rheb and thus enhances its GAP activity, we designed two proteins, namely SSG-MTM (short stabilization domain and GAP domain-membrane targeting motif) and SSG-TSC1N, which were able to function like TSCC to negatively regulate Rheb and mTORC1, but with much-reduced sizes (~1/15 and ~ 1/9 of the size of TSCC, respectively). Biochemical and cell biological assays demonstrated that these designed proteins indeed could promote the GTPase activity of Rheb to hydrolyze GTP, inhibit the kinase activity of mTORC1, and prevent mTORC1 from down-regulating catabolism and autophagy.

Dynamic display of cell targeting motifs via natural glycopeptide recognition for cancer cell isolation

Reversible binding of cell-targeting motifs by natural glycopeptide recognition technology to achieve cell-specific capture and isolation is of significant importance in the field of single-cell analysis.

Encapsulin cargo loading: progress and potential.

Encapsulins are a recently discovered class of prokaryotic self-assembling icosahedral protein nanocompartments measuring between 24 and 42 nm in diameter, capable of selectively encapsulating dedicated cargo proteins in vivo. They have been classified into four families based on sequence identity and operon structure, and thousands of encapsulin systems have recently been computationally identified across a wide range of bacterial and archaeal phyla. Cargo encapsulation is mediated by the presence of specific targeting motifs found in all native cargo proteins that interact with the interior surface of the encapsulin shell during self-assembly. Short C-terminal targeting peptides (TPs) are well documented in Family 1 encapsulins, while more recently, larger N-terminal targeting domains (TDs) have been discovered in Family 2. The modular nature of TPs and their facile genetic fusion to non-native cargo proteins of interest has made cargo encapsulation, both in vivo and in vitro, readily exploitable and has therefore resulted in a range of rationally engineered nano-compartmentalization systems. This review summarizes current knowledge on cargo protein encapsulation within encapsulins and highlights select studies that utilize TP fusions to non-native cargo in creative and useful ways.

RGD-Coated Polymer Nanoworms for Enriching Cancer Stem Cells

Cancer stem cells (CSCs) are primarily responsible for tumour drug resistance and metastasis; thus, targeting CSCs can be a promising approach to stop cancer recurrence. However, CSCs are small in numbers and readily differentiate into matured cancer cells, making the study of their biological features, including therapeutic targets, difficult. The use of three-dimensional (3D) culture systems to enrich CSCs has some limitations, including low sphere forming efficiency, enzymatic digestion that may damage surface proteins, and more importantly no means to sustain the stem properties. A responsive 3D polymer extracellular matrix (ECM) system coated with RGD was used to enrich CSCs, sustain stemness and avoid enzymatic dissociation. RGD was used as a targeting motif and a ligand to bind integrin receptors. We found that the system was able to increase sphere forming efficiency, promote the growth of spheric cells, and maintain stemness-associated properties compared to the current 3D culture. We showed that continuous culture for three generations of colon tumour spheroid led to the stem marker CD24 gradually increasing. Furthermore, the new system could enhance the cancer cell sphere forming ability for the difficult triple negative breast cancer cells, MBA-MD-231. The key stem gene expression for colon cancer also increased with the new system. Further studies indicated that the concentration of RGD, especially at high doses, could inhibit stemness. Taken together, our data demonstrate that our RGD-based ECM system can facilitate the enrichment of CSCs and now allow for the investigation of new therapeutic approaches for colorectal cancer or other cancers.

A new intracellular targeting motif in the cytoplasmic tail of the spike protein may act as a target to inhibit SARS-CoV-2 assembly

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a threat to global public health, underscoring the urgent need for the development of preventive and therapeutic measures. The spike (S) protein of SARS-CoV-2, which mediates receptor binding and subsequent membrane fusion to promote viral entry, is a major target for current drug development and vaccine design. The S protein comprises a large N-terminal extracellular domain, a transmembrane domain, and a short cytoplasmic tail (CT) at the C-terminus. CT truncation of the S protein has been previously reported to promote the infectivity of SARS-CoV and SARS-CoV-2 pseudoviruses. However, the underlying molecular mechanism has not been precisely elucidated. In addition, the CT of various viral membrane glycoproteins play an essential role in the assembly of virions, yet the role of the S protein CT in SARS-CoV-2 infection remains unclear. In this study, through constructing a series of mutations of the CT of the S protein and analyzing their impact on the packaging of the SARS-CoV-2 pseudovirus and live SARS-CoV-2 virus, we identified V1264L1265 as a new intracellular targeting motif in the CT of the S protein, that regulates the transport and subcellular localization of the spike protein through the interactions with cytoskeleton and vesicular transport-related proteins, ARPC3, SCAMP3, and TUBB8, thereby modulating SARS-CoV-2 pseudovirus and live SARS-CoV-2 virion assembly. Either disrupting the V1264L1265 motif or reducing the expression of ARPC3, SCAMP3, and TUBB8 significantly repressed the assembly of the live SARS-CoV-2 virion, raising the possibility that the V1264L1265 motif and the host responsive pathways involved could be new drug targets for the treatment of SARS-CoV-2 infection. Our results extend the understanding of the role played by the S protein CT in the assembly of pseudoviruses and live SARS-CoV-2 virions, which will facilitate the application of pseudoviruses to the study of SARS-CoV-2 and provide potential strategies for the treatment of SARS-CoV-2 infection.

Targeting motifs in frustule-associated proteins from the centric diatom Thalassiosira pseudonana.

The frustule of diatoms has an exceptional structure composed of inorganic and organic molecules. In the organic fraction, protein families were identified whose members are expected to have a complex cellular targeting to their final location within the frustule. Here we investigated for frustule-targeting signals two representatives of the cingulin family, the proteins CinY2 and CinW2; beside an already known, classical signal peptide, we have identified further regions involved in cellular targeting. By using these regions as a search criteria we were able to identify two new frustule proteins. In addition, we showed that the temporal regulation of the gene expression determines the final location of one cingulin. Our results therefore point to a sophisticated cellular and extracellular targeting of frustule components to build the fascinating frustule structure of a diatom.

Mechanisms of deformation and drug release of targeting polypeptides based on fibronectin induction

Polypeptide nano-carriers with deformation and sustained-release function have gained an attention in anti-tumor treatment. A multifunctional polypeptide with different motifs was discussed and the contribution of each motif to targeted drug release was analyzed by control studies. The transformation and drug release processes of polypeptides were investigated by molecular dynamics method to reveal their dynamics mechanism, and corresponding experiments were performed to verify the simulation results. We observed that the polypeptides could form NPs under the hydrophobic interaction between self-assembly motifs and the electrostatic repulsion between targeting motifs. Affected by the ligand-receptor interaction, the targeting motifs overcame the electrostatic repulsion to approach the ligand proteins, leading to the promotion of the binding of fibrous motifs and the transformation of NPs into NFs for better retention of drugs in the tumor tissues. In addition, the polypeptides with strong hydrophobicity exhibited excellent sustained-release efficiency. These insights allow drawing general conclusions contributed to the design of transformable polypeptide NPs: The decrease in the hydrophobicity of self-assembly motifs is beneficial for the enrichment of doxorubicin in tumor tissues, as well as the similar result can be obtained with the improvement of the hydrophobicity of fibrous motifs and the capability of target.