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Targeted cancer therapy is a promising alternative to the currently established cancer treatments, aiming to selectively kill cancer cells while sparing healthy tissues. Hereby, molecular targeting agents, such as monoclonal antibodies, are used to bind to cancer cell surface markers specifically. Although these agents have shown great clinical success, limitations still remain such as low tumor penetration and off-target effects. To overcome this limitation, novel fusion proteins comprised of the two proteins ADAPT6 and Horseradish Peroxidase (HRP) were engineered. Cancer cell targeting is hereby enabled by the small scaffold protein ADAPT6, engineered to specifically bind to human epidermal growth factor receptor 2 (HER2), a cell surface marker overexpressed in various cancer types, while the enzyme HRP oxidizes the nontoxic prodrug indole-3-acetic acid (IAA) which leads to the formation of free radicals and thereby to cytotoxic effects on cancer cells. The high affinity to HER2, as well as the enzymatic activity of HRP, were still present for the ADAPT6-HRP fusion proteins. Further, in vitro cytotoxicity assay using HER2-positive SKOV-3 cells revealed a clear advantage of the fusion proteins over free HRP by association of the fusion proteins directly to the cancer cells and therefore sustained cell killing. This novel strategy of combining ADAPT6 and HRP represents a promising approach and a viable alternative to antibody conjugation for targeted cancer therapy. [Display omitted] • A novel fusion protein for targeted cancer therapeutics is presented. • The fusion protein combines high affinity to HER2 with enzymatic activity. • The therapeutic protein is easily produced in the bacterial host E. coli. • Efficient cancer cell killing through tumor cell-specific binding and activity has been demonstrated. [ABSTRACT FROM AUTHOR]

This study highlights the significance of overexpressing 1-deoxy-d-xylulose-5-phosphate synthase (DXS) from the MEP (methylerythritol 4-phosphate) pathway, in addition to short-chain prenyltransferase fusions for the improved production of the diterpene, taxa-4,11-diene, the first committed intermediate in the production of anti-cancer drug paclitaxel. The results showed that the strain which has (i) the taxadiene synthase (txs) gene integrated into the genome, (ii) the MEP pathway genes overexpressed, (iii) the fpps-crtE prenyltransferases fusion protein and (iv) additional expression of 1-deoxy-d-xylulose-5-phosphate synthase (DXS), yielded the highest production of taxa-4,11-diene at 390 mg/L (26 mg/L/OD 600). This represents a thirteen-fold increase compared to the highest reported concentration in B. subtilis. The focus on additional overexpression of DXS and utilizing short-chain prenyltransferase fusions underscores their pivotal role in achieving significant titer improvements in terpene biosynthesis. [Display omitted] • Utilizing Bacillus subtilis as a cell factory for the production of taxadiene. • Additional expression of DXS from MEP pathway can boost taxadiene production. • Application of fused prenyltransferases can enhance the yield of taxadiene. • The highest taxadiene production so far in B. subtilis has been achieved. [ABSTRACT FROM AUTHOR]

The human epidermal growth factor (hEGF) is a powerful mitogen used in medical treatment and cosmetics. However, the recovery of hEGF from natural sources is insufficient to meet market demands, thus recombinant protein production technology for recombinant hEGF (rhEGF) was developed. Among the heterologous expression systems used for rhEGF production, Escherichia coli is preferred due to its well-established advantages. In this review, we focus on the development of two rhEGF expression strategies (soluble rhEGF and rhEGF inclusion bodies), and discuss the design of fusion rhEGF protein and its cultivation strategies. We also explore downstream processes for rhEGF isolation and purification methods (with or without a column chromatography system), and the recovery of rhEGF from inclusion bodies through solubilisation and refolding. Lastly, methods of quantification, characterisation, and biological activity assays used to quantify and validate purified rhEGF are also listed. [Display omitted] • Recombinant human epidermal growth factor (rhEGF) is used for ulcer treatment and cosmetic applications. • rhEGF can be produced via soluble or insoluble (inclusion bodies) protein strategies. • Exploration on differences in fusion protein, cultivation conditions and purification for both rhEGF production strategies. • Method of analysis of produced rhEGF. [ABSTRACT FROM AUTHOR]

LL-37 is an antimicrobial protein expressed by the CAMP gene in humans. This protein has various antibacterial, antiviral and anticancer effects. Expressing LL-37 as a heterologous protein in E. coli cells has its challenges. LL-37 is a peptide that is so small that it must be engineered with a fusion protein to increase its size solubility and prevent proteolysis of the target protein in cells. Factor Xa is the protease chosen to cleave LL-37 from its fusion protein in this research due to leucine binding factor Xa quite strongly. The aim of this study is, therefore, to express LL-37 as a fusion protein with Histaq_SUMO at the N-terminus linked to LL-37 at the C-terminus through Factor Xa cleavage site. Then, the fusion protein was cleaved by Factor Xa to obtain pure LL-37. In this study, LL-37 was produced by recombinant DNA technology, starting with the construction, expression, purification and cleavage of the fusion protein. The constructed genes consisted of 6xHis, SUMO, the factor X cleavage site, and LL-37, a total of 450 bp inserted in the pD451-SR vector plasmid. The results of this study yielded a SUMO_LL-37 protein with a molecular weight of approximately 17.34 kDa, which could be purified by Ni-NTA under native purification conditions. Based on ImageJ SUMO_LL-37 quantification, it was 1.65 µg/µL. LL-37 can be cleaved by factor Xa from SUMO with an enzyme-to-substrate ratio of 1:12.5 at 37°C with a 24-hour incubation time and results as much as 0.144 µg/µL. [ABSTRACT FROM AUTHOR]

Cervical cancer is the fourth most common cause of cancer and the fourth most common cause of cancer deaths in women. Some reports have shown the effect of Streptococcus agalactiae proteins and capsule products against cancer cell lines. This study aimed to design and produce a fusion of recombinant protein (containing the capsules of Streptococcus agalactiaes with a linker and Antti cancer sequences) CpsA-CpsC-L-ACAN in the plasmid pET-22b (+) vector. Construct pET-22b (+) was designed by researchers, optimized with bioinformatics software, and synthesized by a Biometrics company. For the confirmation of recombinant protein, Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting were performed. Antibacterial activity was performed according to protocol CLSI2020. Based on bioinformatics analysis, the recombinant protein had good spatial structure and stability and half-life. Iterative Threading ASSEmbly Refinement (I-TASSER) results predicted a surfing topology with C-score values (−3.60). The results of Gastro-Oesophageal Reflux 4 (GOR4) analysis showed a little extended strand (30.63%), a random coil with a percentage (50.18%), and an alpha helix with a percentage (19.19%). The recombinant protein was confirmed by SDS-PAGE and western blotting with Anti-his tag. The purpose of peptide fusion design in this article is to help in the development of anti-cervical cancer medicine. Of course, this issue needs to be investigated in animal and human phases. In this article, only bioinformatics investigations and validation of the results in the laboratory have been discussed. [ABSTRACT FROM AUTHOR]

Human and animal health is threatened by Hendra virus (HeV), which has few treatments. This in-silico vaccine design study focuses on HeV G (glycoprotein), F (fusion protein), and M (matrix protein). These proteins were computationally assessed for B and T-cell epitopes after considering HeV strain conservation, immunogenicity, and antigenicity. To improve vaccination immunogenicity, these epitopes were selectively ligated into a multiepitope construct. To improve vaccination longevity and immunological response, adjuvants and linkers were ligated. G, F, and M epitopes were used to create an mRNA HeV vaccine. Cytotoxic, helper, and linear B-lymphocytes' epitopes are targeted by this vaccine. The population coverage analysis demonstrates that multi-epitope vaccination covers 91.81 percent of CTL and 98.55 percent of HTL epitopes worldwide. GRAVY evaluated the vaccine's well-characterized physicochemical properties -0.503, indicating solubility and functional stability. Structure analysis showed well-stabilized 2° and 3° structures in the vaccine, with alpha helix, beta sheet, and coil structures (Ramachandran score of 88.5% and Z score of -3.44). There was a strong affinity as shown by docking tests with TLR-4 (central score of -1139.4 KJ/mol) and TLR-2 (center score of -1277.9 KJ/mol). The coupled V-apo, V-TLR2, and V-TLR4 complexes were tested for binding using molecular dynamics simulation where extremely stable complexes were found. The predicted mRNA structures provided significant stability. Codon optimization for Escherichia. coli synthesis allowed the vaccine to attain a GC content of 46.83% and a CAI score of 1.0, which supports its significant expression. Immunological simulations indicated vaccine-induced innate and adaptive immune reactions. Finally, this potential HeV vaccine needs more studies to prove its efficacy and safety. [ABSTRACT FROM AUTHOR]

CRISPR-associated (Cas) endonucleases and their derivatives are widespread tools for the targeted genetic modification of both prokaryotic and eukaryotic genomes. A critical step of all CRISPR-Cas technologies is the delivery of the Cas endonuclease to the target cell. Here, we investigate the possibility of using bacterial conjugation to translocate Cas proteins into recipient bacteria. Conjugative relaxases are translocated through a type IV secretion system into the recipient cell, covalently attached to the transferred DNA strand. We fused relaxase R388-TrwC with the endonuclease Cas12a and confirmed that it can be transported through a T4SS. The fusion protein maintained its activity upon translocation by conjugation into the recipient cell, as evidenced by the induction of the SOS signal resulting from DNA breaks produced by the endonuclease in the recipient cell, and the detection of mutations at the target position. We further show how a template DNA provided on the transferred DNA can be used to introduce specific mutations. The guide RNA can also be encoded by the transferred DNA, enabling its production in the recipient cells where it can form a complex with the Cas nuclease transferred as a protein. This self-contained setup enables to target wild-type bacterial cells. Finally, we extended this strategy to the delivery of relaxases fused to base editors. Using TrwC and MobA relaxases as drivers, we achieved precise editing of transconjugants. Thus, conjugation provides a delivery system for Cas-derived editing tools, bypassing the need to deliver and express a cas gene in the target cells. [ABSTRACT FROM AUTHOR]

Various techniques using fluorescent reporter probes have been developed, such as GFP transgenic mouse lines that are used to detect spatial–temporal expression levels of genes. Although GFP expression is largely considered non-toxic, recent reports have indicated that under certain conditions GFP can display cellular toxicity. We hereby report the nuclear toxicity of H2B-GFP using a K14 specific Tet-on reporter mouse system. Using this system, GFP accumulates in the nucleus of all K14 expressing cells, such as the ocular surface epithelia and ocular adnexa. Expression of high levels of nuclear GFP during embryonic stages led to an eye open-at-birth (EOB) phenotype and abnormal ocular adnexa development and during adult and aging stages showed notable toxicity to ocular tissues. Other tissues, such as skin, also presented multiple defects associated with H2B-GFP expression. This toxicity was found to be concentration dependent, with homozygous mice presenting extremely high toxicity, while heterozygous mice presented limited toxicity. Upon induction, the accumulation of H2B-GFP in the nucleus of homozygous mice led to apoptosis within 2 weeks. This study therefore shows that although the use of nuclear GFP reporter mice is a valuable tool, at high levels, nuclear GFP can be toxic, leading to cell death and affecting tissue function. [ABSTRACT FROM AUTHOR]

ABSTRACT Objectives Methods Results Conclusions The 24‐month, prospective, non‐interventional, European multicenter A‐SURE study evaluated the real‐world effectiveness of prophylaxis using an extended half‐life recombinant factor VIII (FVIII) Fc fusion protein, efmoroctocog alfa (hereinafter rFVIIIFc), compared with prophylaxis using standard half‐life (SHL) FVIII products in patients with hemophilia A.Primary endpoints were annualized bleeding rate (ABR), annualized injection frequency, and annualized factor consumption. A comparative study design unique for an observational hemophilia study was implemented to reduce potential confounding in effectiveness estimates, wherein each patient prescribed rFVIIIFc was matched with one receiving SHL FVIII. Propensity scores were used for adjustment in statistical analyses.Outcomes for all primary endpoints were significantly better in the rFVIIIFc group (n = 184) compared with the SHL FVIII group (n = 170): mean ABR 1.5 versus 2.3 (difference of −0.8; p = 0.0147); mean annualized injection frequency 114.4 versus 169.2 (difference of −54.8; p < 0.0001); and mean annualized factor consumption 243 024.2 versus 288 718.6 International Units (difference of 45 694.5; p = 0.0003). rFVIIIFc was well tolerated, with no inhibitor development.rFVIIIFc has superior prophylactic effectiveness versus SHL FVIII, providing higher bleed protection with fewer injections and lower factor consumption. [ABSTRACT FROM AUTHOR]

Background: We previously developed a nanobody targeting CTLA-4 and demonstrated that it can boost antitumour T-cell responses in vitro; however, the resulting responses after the injection of T cells into cancer models are usually weak and transient. Here, we explored whether fusing our nanobody to IL-12 would enable it to induce stronger, longer-lasting T-cell immune responses after exposure to immature dendritic cell and tumour cell fusions. Results: The fusion protein enhanced the response of CD8+ T cells to tumour antigens in vitro and led to stronger, more persistent immune responses after the T cells were injected into mice bearing different types of xenografts. Conclusion: Our in vitro and in vivo results suggest the anticancer potential of our nanobody-interleukin fusion system and support the clinical application of this fusion approach for various nanobodies. [ABSTRACT FROM AUTHOR]

Our previous study has found that monoclonal antibodies targeting a conserved epitope peptide spanning from residues 1144 to 1156 of SARS-CoV-2 spike (S) protein, namely S(1144–1156), can broadly neutralize all of the prevalent SARS-CoV-2 strains, including the wild type, Alpha, Epsilon, Delta, and Gamma variants. In the study, S(1144–1156) was conjugated with bovine serum albumin (BSA) and formulated with Montanide ISA 51 adjuvant for inoculation in BALB/c mice to study its potential as a vaccine candidate. Results showed that the titers of S protein-specific IgGs and the neutralizing antibodies in mouse sera against various SARS-CoV-2 variants, including the Omicron sublineages, were largely induced along with three doses of immunization. The significant release of IFN-γ and IL-2 was also observed by ELISpot assays through stimulating vaccinated mouse splenocytes with the S(1144–1156) peptide. Furthermore, the vaccination of the S(1143–1157)- and S(1142–1158)-EGFP fusion proteins can elicit more SARS-CoV-2 neutralizing antibodies in mouse sera than the S(1144–1156)-EGFP fusion protein. Interestingly, the antisera collected from mice inoculated with the S(1144–1156) peptide vaccine exhibited better efficacy for neutralizing Omicron BA.2.86 and JN.1 subvariants than Omicron BA.1, BA.2, and XBB subvariants. Since the amino acid sequences of the S(1144–1156) are highly conserved among various SARS-CoV-2 variants, the immunogen containing the S(1144–1156) core epitope can be designed as a broadly effective COVID-19 vaccine. Key points: • Inoculation of mice with the S(1144–1156) peptide vaccine can induce bnAbs against various SARS-CoV-2 variants. • The S(1144–1156) peptide stimulated significant release of IFN-γ and IL-2 in vaccinated mouse splenocytes. • The S(1143–1157) and S(1142–1158) peptide vaccines can elicit more SARS-CoV-2 nAbs in mice. [ABSTRACT FROM AUTHOR]

The Cas3 nuclease is utilized by canonical type I CRISPR-Cas systems for processive target DNA degradation, while a newly identified type I-F CRISPR variant employs an HNH nuclease domain from the natural fusion Cas8-HNH protein for precise target cleavage both in vitro and in human cells. Here, we report multiple cryo-electron microscopy structures of the type I-F Cas8-HNH system at different functional states. The Cas8-HNH Cascade complex adopts an overall G-shaped architecture, with the HNH domain occupying the C-terminal helical bundle domain (HB) of the Cas8 protein in canonical type I systems. The Linker region connecting Cas8-NTD and HNH domains adopts a rigid conformation and interacts with the Cas7.6 subunit, enabling the HNH domain to be in a functional position. The full R-loop formation displaces the HNH domain away from the Cas6 subunit, thus activating the target DNA cleavage. Importantly, our results demonstrate that precise target cleavage is dictated by a C-terminal helix of the HNH domain. Together, our work not only delineates the structural basis for target recognition and activation of the type I-F Cas8-HNH system, but also guides further developments leveraging this system for precise DNA editing. Synopsis: The newly characterized type I-F Cas8-HNH CRISPR-Cas system features a naturally fused HNH nuclease domain for precise target cleavage. Here, structural and biochemical studies elucidate the molecular mechanisms underlying target recognition and activation of this system. Cryo-EM structures offer insights into the assembly of the Cas8-HNH system. The mechanism of 5'-CN-3' PAM recognition and Cas8-HNH system activation is revealed. Full target validation is essential for Cas8-HNH activation. A C-terminal sensor helix on the HNH domain acts as a checkpoint for full target engagement. Structural and biochemical studies elucidate target recognition and activation mechanisms of a recently characterized CRISPR-Cas system featuring a naturally fused HNH nuclease domain for precise target cleavage. [ABSTRACT FROM AUTHOR]

Monoamine neurotransmitters generated by de novo synthesis are rapidly transported and stored into synaptic vesicles at axon terminals. This transport is essential both for sustaining synaptic transmission and for limiting the toxic effects of monoamines. Here, synthesis of the monoamine histamine by histidine decarboxylase (HDC) and subsequent loading of histamine into synaptic vesicles are shown to be physically and functionally coupled within Drosophila photoreceptor terminals. This process requires HDC anchoring to synaptic vesicles via interactions with N-ethylmaleimide-sensitive fusion protein 1 (NSF1). Disassociating HDC from synaptic vesicles disrupts visual synaptic transmission and causes somatic accumulation of histamine, which leads to retinal degeneration. We further identified a proteasome degradation system mediated by the E3 ubiquitin ligase, purity of essence (POE), which clears mislocalized HDC from the soma, thus eliminating the cytotoxic effects of histamine. Taken together, our results reveal a dual mechanism for translocation and degradation of HDC that ensures restriction of histamine synthesis to axonal terminals and at the same time rapid loading into synaptic vesicles. This is crucial for sustaining neurotransmission and protecting against cytotoxic monoamines. Synopsis: Neurotransmitters are key for synaptic transmission at axon terminals, but their transport modalities remain debated. This study demonstrates that coupling of histamine synthesis with its loading into synaptic vesicles by anchoring histidine decarboxylase (HDC) to synaptic vesicles is critical to maintaining neurotransmission and preventing cytotoxic side effects. Coupling histamine synthesis with synaptic vesicle loading is critical for neurotransmitter homeostasis and synaptic transmission. HDC is co-transported with synaptic vesicles to axonal terminals through binding with vesicle-fusing ATPase NSF1. HDC remaining in soma is degraded by the E3 ubiquitin ligase POE via the proteasome. Aberrant accumulation of histamine in the soma is cytotoxic and causes retinal degeneration. Anchoring of histidine decarboxylase to synaptic vesicles is critical to maintaining neurotransmission and preventing cytotoxic side effects. [ABSTRACT FROM AUTHOR]

Cancer is a global health problem despite the most developed therapeutic modalities. The delivery of specific therapeutic agents to a target increases the effectiveness of cancer treatment by reducing side effects and post‐treatment issues.Our aim in this study was to design a recombinant protein consisting of nanobody molecules and exotoxin that targets the surface GRP78 receptor on tumor cells. Bioinformatics methods make drug design and recombinant protein evaluation much easier before the laboratory steps.Two constructs were designed from a single‐variable domain on heavy chain nanobody domains and PE toxin domains II, Ib, and III. The physicochemical properties, secondary structure, and solubility of the chimeric protein were analyzed using different software. Prostate cancer DU‐145 and breast cancer MDA‐MB‐468 cell lines were used as GRP78‐positive and negative controls, respectively. Accordingly, the cytotoxicity, binding affinity, cell internalization, and apoptosis were evaluated using MTT, enzyme‐linked immunosorbent assay, and western blot. The results showed that in the DU‐145 cell line, the cytotoxicity of two recombinant immunotoxins is dose and time‐dependent. In MDA‐MB‐468 and HEK‐293 cells, such an event does not occur. It is possible that two constructs designed for immunotoxins can attach to GRP78‐positive cancer cells and then eradicate cancer cells by internalization and apoptosis. As our in vitro results were in line with in silico data confirming the Bioinformatics predictions, it can be concluded that the designed recombinant immunotoxins may exhibit therapeutic potential against GRP78‐positive tumor cells. [ABSTRACT FROM AUTHOR]

Paramyxoviruses including measles, Nipah, and parainfluenza viruses are public health threats with pandemic potential. Human parainfluenza virus type 3 (HPIV3) is a leading cause of illness in pediatric, older, and immunocompromised populations. There are no approved vaccines or therapeutics for HPIV3. Neutralizing monoclonal antibodies (mAbs) that target viral fusion are a potential strategy for mitigating paramyxovirus infection, however their utility may be curtailed by viral evolution that leads to resistance. Paramyxoviruses enter cells by fusing with the cell membrane in a process mediated by a complex consisting of a receptor binding protein (HN) and a fusion protein (F). Existing atomic resolution structures fail to reveal physiologically relevant interactions during viral entry. We present cryo-ET structures of pre-fusion HN-F complexes in situ on surfaces of virions that evolved resistance to an anti-HPIV3 F neutralizing mAb. Single mutations in F abolish mAb binding and neutralization. In these complexes, the HN protein that normally restrains F triggering has shifted to uncap the F apex. These complexes are more readily triggered to fuse. These structures shed light on the adaptability of the pre-fusion HN-F complex and mechanisms of paramyxoviral resistance to mAbs, and help define potential barriers to resistance for the design of mAbs. Monoclonal antibodies hold promise for combating serious respiratory virus infections but viruses may evolve to evade them. Here, using structural analysis, the authors show how human parainfluenza virus adapts to escape a powerful antibody by modulating its cell entry mechanism. [ABSTRACT FROM AUTHOR]

Here, we report a magnetogenetic system, based on a single anti-ferritin nanobody-TRPV1 receptor fusion protein, which regulated neuronal activity when exposed to magnetic fields. Adeno-associated virus (AAV)-mediated delivery of a floxed nanobody-TRPV1 into the striatum of adenosine-2a receptor-Cre drivers resulted in motor freezing when placed in a magnetic resonance imaging machine or adjacent to a transcranial magnetic stimulation device. Functional imaging and fiber photometry confirmed activation in response to magnetic fields. Expression of the same construct in the striatum of wild-type mice along with a second injection of an AAVretro expressing Cre into the globus pallidus led to similar circuit specificity and motor responses. Last, a mutation was generated to gate chloride and inhibit neuronal activity. Expression of this variant in the subthalamic nucleus in PitX2-Cre parkinsonian mice resulted in reduced c-fos expression and motor rotational behavior. These data demonstrate that magnetogenetic constructs can bidirectionally regulate activity of specific neuronal circuits noninvasively in vivo using clinically available devices. [ABSTRACT FROM AUTHOR]

Foamy viruses (FVs) constitute a subfamily of retroviruses. Their envelope (Env) glycoprotein drives the merger of viral and cellular membranes during entry into cells. The only available structures of retroviral Envs are those from human and simian immunodeficiency viruses from the subfamily of orthoretroviruses, which are only distantly related to the FVs. We report the cryo-electron microscopy structures of the FV Env ectodomain in the pre-and post-fusion states, which unexpectedly demonstrate structural similarity with the fusion protein (F) of paramyxo- and pneumoviruses, implying an evolutionary link between the viral fusogens. We describe the structural features that are unique to the FV Env and propose a mechanistic model for its conformational change, highlighting how the interplay of its structural elements could drive membrane fusion and viral entry. The structural knowledge on the FV Env now provides a framework for functional investigations, which can benefit the design of FV Env variants with improved features for use as gene therapy vectors. [ABSTRACT FROM AUTHOR]

Backgound Information Results Conclusion and Significance Toxoplasma gondii has a relict plastid, the apicoplast, to which nuclear‐encoded proteins are targeted after synthesis in the cytosol. Proteins exclusively found in the apicoplast use a Golgi‐independent route for trafficking, while dually targeted proteins found in both the apicoplast and the mitochondrion use a Golgi‐dependent route. For apicoplast targeting, N‐terminal signal sequences have been shown to direct the localization of different reporters. In this study, we use chimeric proteins to dissect out the roles of N‐terminal sequences and coding sequences in apicoplast localization and the choice of the trafficking route.We show that when the N‐termini of a dually targeted protein, TgTPx1/2, or of an apicoplast protein, TgACP, are fused with the reporter protein, enhanced green fluorescent protein (eGFP) or endogenous proteins, TgSOD2, TgSOD3, TgACP, or TgTPx1/2, the chimeric proteins exhibit flexibility in apicoplast targeting depending on the coding sequences. Further, the chimeras that are localized to the apicoplast use different trafficking pathways depending on the combination of the N‐terminal signals and the coding sequences.This report shows, for the first time, that in addition to the N‐terminal signal sequences, targeting and trafficking signals also reside within the coding sequences of apicoplast proteins. [ABSTRACT FROM AUTHOR]

Hippocampal neurons can be the first to be impaired with neurodegenerative disorders, including Alzheimer's disease (AD). Most drug candidates for causal therapy of AD cannot either enter the brain or accumulate around hippocampal neurons. Here, we genetically engineered insulin-fusion proteins, called hippocampal neuron-targeting (Ht) proteins, for targeting protein drugs to hippocampal neurons because insulin tends to accumulate in the neuronal cell layers of the hippocampus. In vitro examinations clarified that insulin and Ht proteins were internalized into the cultured hippocampal neurons through insulin receptor-mediated macropinocytosis. Cysteines were key determinants of the delivery of Ht proteins to hippocampal neurons, and insulin B chain mutant was most potent in delivering cargo proteins. In vivo accumulation of Ht proteins to hippocampal neuronal layers occurred after intracerebroventricular administration. Thus, hippocampal neuron-targeting technology can provide great help for developing protein drugs against neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Background: The epigenetic factors KAT6A (MOZ/MYST3) and KMT2A (MLL/MLL1) interact in normal hematopoiesis to regulate progenitors' self-renewal. Both proteins are recurrently translocated in AML, leading to impairment of critical differentiation pathways in these malignant cells. We evaluated the potential of different KAT6A therapeutic targeting strategies to alter the growth of KAT6A and KMT2A rearranged AMLs. Methods: We investigated the action and potential mechanisms of the first-in-class KAT6A inhibitor, WM-1119 in KAT6A and KMT2A rearranged (KAT6Ar and KMT2Ar) AML using cellular (flow cytometry, colony assays, cell growth) and molecular (shRNA knock-down, CRISPR knock-out, bulk and single-cell RNA-seq, ChIP-seq) assays. We also used two novel genetic murine KAT6A models combined with the most common KMT2Ar AML, KMT2A::MLLT3 AML. In these murine models, the catalytic activity of KAT6A, or the whole protein, can be conditionally abrogated or deleted. These models allowed us to compare the effects of specific KAT6A KAT activity inhibition with the complete deletion of the whole protein. Finally, we also tested these therapeutic approaches on human AML cell lines and primary patient AMLs. Results: We found that WM-1119 completely abrogated the proliferative and clonogenic potential of KAT6Ar cells in vitro. WM-1119 treatment was associated with a dramatic increase in myeloid differentiation program. The treatment also decreased stemness and leukemia pathways at the transcriptome level and led to loss of binding of the fusion protein at critical regulators of these pathways. In contrast, our pharmacologic and genetic results indicate that the catalytic activity of KAT6A plays a more limited role in KMT2Ar leukemogenicity, while targeting the whole KAT6A protein dramatically affects leukemic potential in murine KMT2A::MLLT3 AML. Conclusion: Our study indicates that inhibiting KAT6A KAT activity holds compelling promise for KAT6Ar AML patients. In contrast, targeted degradation of KAT6A, and not just its catalytic activity, may represent a more appropriate therapeutic approach for KMT2Ar AMLs. [ABSTRACT FROM AUTHOR]

Loss of function of the RNA-binding protein TDP-43 (TDP-LOF) is a hallmark of amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. Here we describe TDP-REG, which exploits the specificity of cryptic splicing induced by TDP-LOF to drive protein expression when and where the disease process occurs. The SpliceNouveau algorithm combines deep learning with rational design to generate customizable cryptic splicing events within protein-coding sequences. We demonstrate that expression of TDP-REG reporters is tightly coupled to TDP-LOF in vitro and in vivo. TDP-REG enables genomic prime editing to ablate the UNC13A cryptic donor splice site specifically upon TDP-LOF. Finally, we design TDP-REG vectors encoding a TDP-43/Raver1 fusion protein that rescues key pathological cryptic splicing events, paving the way for the development of precision therapies for TDP43-related disorders. [ABSTRACT FROM AUTHOR]

The mRNAs encoding the human papillomavirus type 16 (HPV16) E6 and E7 oncogene mRNAs are subjected to extensive alternative RNA splicing at multiple regulated splice sites. One of the most extensively used 5'-splice sites in the HPV16 genome is named SD880 and is located immediately downstream of the E7 open reading frame. Here, we show that a cluster of three GGG-motifs adjacent to HPV16 SD880 interacts with heterogeneous nuclear ribonucleoprotein (hnRNP) H that cooperates with SD880 to stimulate splicing to the upstream HPV16 3'-splice site SA742. This splice site is located in the E7 coding region and is required for the production of the HPV16 226^742 mRNA that encodes the E6^E7 fusion protein. Enhancement of HPV16 E6^E7 mRNA production by hnRNP H occurred at the expense of the intron-retained E6 mRNAs and the spliced E7 mRNAs, demonstrating that hnRNP H controls the relative levels of E6, E7, and E6^E7 proteins. Unexpectedly, overexpression of hnRNP H also promoted retention of the downstream E1 encoding intron and enhanced E1 protein production. We concluded that hnRNP H plays an important role in the HPV16 gene expression program. [ABSTRACT FROM AUTHOR]

Patients with severe and sometimes moderate hemophilia B are prophylactically treated with factor IX concentrates to prevent bleeding. For some time now, various extended terminal half‐life (EHL) recombinant factor IX concentrates are available allowing less frequent administration during prophylaxis in comparison to standard half‐life recombinant FIX (rFIX). Especially, recombinant FIX‐Fc fusion protein (rFIXFc; Alprolix®) exhibits a rapid distribution phase, potentially due to binding to type IV collagen (Col4) in the extravascular space. Studies suggest that the presence of extravascular rFIXFc is protective against bleeding as without measurable FIX activity in plasma, and no extra bleeding seems to occur. The physiologically based pharmacokinetic (PBPK) model for rFIXFc which we describe in this study, is able to accurately predict the observed concentration‐time profiles of rFIXFc in plasma and is able to quantify the binding of rFIXFc to Col4 in the extravascular space after an intravenous dose of 50 IU/kg rFIXFc in a male population. Our model predicts that the total AUC of rFIXFc bound to Col4 in the extravascular space is approximately 19 times higher compared to the AUC of rFIXFc in plasma. This suggests that rFIXFc present in the extravascular compartment may play an important role in achieving hemostasis after rFIXFc administration. Further studies on extravascular distribution of rFIXFc and the distribution profile of other EHL‐FIX concentrates are needed to evaluate the predictions of our PBPK model and to investigate its clinical relevance. [ABSTRACT FROM AUTHOR]

Cryo-electron tomography enables capture of 3D images of membrane fusion intermediate states with resolution of fusion proteins and membrane leaflet remodeling. Studies of class I, II, and III viral membrane fusion proteins are revealing general features as well as differences in stages of membrane reorganization, leading to complete fusion and pore formation. Viral fusion machinery is comprised of fusion proteins, viral membrane, and internal structural components such as matrix proteins that regulate fusion protein conformation and function. Tightly docked membrane contact zones with closely apposed proximal headgroup layers appear to be a near universal intermediate state along the fusion pathway. Computational modeling that builds upon observed structural information from cryo-electron tomography may offer a path towards a detailed mechanistic understanding of the forces and energetics that drive protein-mediated membrane fusion. Protein-mediated membrane fusion is the dynamic process where specialized protein machinery undergoes dramatic conformational changes that drive two membrane bilayers together, leading to lipid mixing and opening of a fusion pore between previously separate membrane-bound compartments. Membrane fusion is an essential stage of enveloped virus entry that results in viral genome delivery into host cells. Recent studies applying cryo-electron microscopy techniques in a time-resolved fashion provide unprecedented glimpses into the interaction of viral fusion proteins and membranes, revealing fusion intermediate states from the initiation of fusion to release of the viral genome. In combination with complementary structural, biophysical, and computation modeling approaches, these advances are shedding new light on the mechanics and dynamics of protein-mediated membrane fusion. [ABSTRACT FROM AUTHOR]

Numerous gene therapy drugs for cancer have received global approval, yet their efficacy against solid tumors remains inadequate. Our previous research indicated that the fiber protein, a component of the adenovirus capsid, can propagate from infected cells to neighboring cells that express the adenovirus receptor. We hypothesize that merging this fiber protein with an anti-cancer protein could enable the anti-cancer protein to disseminate around the transfected cells, presenting a novel approach to cancer gene therapy. In our study, we discovered that the knob region of the adenovirus type 5 fiber protein is the smallest unit capable of spreading to adjacent cells in a receptor-specific manner. We also showed that the recombinant knob protein infiltrates cells after dispersing to surrounding cells. To assess the potential of the knob protein to augment gene therapy for solid tumors in mice, we expressed a fusion gene of the A subunit of cytotoxic cholera toxin and the knob region in mouse tumors. We found that this fusion protein only inhibited tumor growth in receptor-expressing mouse melanomas, and this inhibitory effect persisted even in mice with anti-knob antibodies. Our study's findings propose a novel cancer gene therapy strategy that enhances therapeutic effects by specifically delivering therapeutic proteins, expressed from in vivo administered genes, to target molecules. This outcome offers a fresh perspective on gene therapy for solid cancers, and we anticipate that knob proteins will serve as a platform for this method. [ABSTRACT FROM AUTHOR]

Background: Receptor tyrosine kinase (RTK) fusions of RET, NTRK1/3, and ALK are enriched among pediatric thyroid cancer patients with metastatic and persistent disease, and their oncoproteins represent attractive drug targets. Methods: We performed RNA-sequencing in a papillary thyroid cancer (PTC) lacking other frequent driver alterations. Results: We report a novel RTK fusion, TG-insulin-like growth factor 1 receptor gene (IGF1R), in a 17-year-old female patient with angioinvasive follicular variant PTC. The in-frame fusion protein preserves the cholinesterase‐like domain of TG with dimerization properties and the transmembrane and kinase domain of IGF1R. The tumor sample shows increased IGF1R mRNA expression and tyrosine kinase phosphorylation, augmentation of Mitogen activated protein kinase (MAPK) transcriptional output genes, and decreased NIS levels. Conclusions: We reveal a novel targetable kinase fusion oncogene in thyroid cancer which is not incorporated in different thyroid-specific sequencing panels. The integration of IGF1R fusion screening in the next versions of thyroid-specific targeted next-generation sequencing panels may be beneficial to thyroid cancer patients. [ABSTRACT FROM AUTHOR]

Objectives: Haemophilia B (HB), characterised by deficient factor IX (FIX), leads to spontaneous bleeds. Severe cases require prophylactic FIX replacement. This post hoc analysis assessed the first spontaneous bleeds among previously untreated patients (PUPs) with HB treated with recombinant FIX Fc fusion protein (rFIXFc) (NCT02234310) to identify factors influencing bleeds. Methods: Subjects included paediatric PUPs with HB (≤2 IU/dL endogenous FIX). Analyses described treatment patterns (on demand [OD] vs. prophylaxis) and prophylaxis type (started on vs. switched to prophylaxis). Kaplan–Meier analyses assessed the time to first spontaneous bleed, including median time to event and fitting models with predictors for treatment regimen and/or baseline age. Results: PUPs B‐LONG enrolled 33 subjects. Baseline age did not influence the time to first spontaneous bleed for any rFIXFc regimen. Those who started on prophylaxis with rFIXFc (n = 11), compared with those treated OD (n = 22), had an extended time to first spontaneous bleed. Starting prophylaxis afforded a 93% reduced risk of first spontaneous bleed versus starting OD (hazard ratio [95% confidence interval]: 0.071 [0.009–0.592]) (p =.015). Conclusion: rFIXFc prophylaxis, particularly starting early, reduced the risk of bleeding and delayed time to first spontaneous bleed compared with rFIXFc OD. Hence, initial treatment regimens impact bleed patterns in paediatric PUPs. [ABSTRACT FROM AUTHOR]

Gas vesicles (GVs) are gas-filled microbial organelles formed by unique 3-nm thick, amphipathic, force-bearing protein shells, which can withstand multiple atmospheric pressures and maintain a physically stable air bubble with megapascal surface tension. However, the molecular process of GV assembly remains elusive. To begin understanding this process, we have devised a high-throughput in vivo assay to determine the interactions of all 11 proteins in the pNL29 GV operon. Complete or partial deletions of the operon establish interdependent relationships among GV proteins during assembly. We also examine the tolerance of the GV assembly process to protein mutations and the cellular burdens caused by GV proteins. Clusters of GV protein interactions are revealed, proposing plausible protein complexes that are important for GV assembly. We anticipate our findings will set the stage for designing GVs that efficiently assemble in heterologous hosts during biomedical applications. Synopsis: Growing interest in biomedical applications for gas vesicles (GVs) demands an understanding of their structure and assembly, but little is known to date on the roles of individual gas vesicle proteins (Gvps) as assembly factors. This study utilizes modular, parallelized protein-protein interaction screening to elucidate key interactions and interdependencies of Gvps during GV assembly. Out of 66 possible Gvp-to-Gvp interactions, 28 formed a dense protein-protein interaction network. Selective inclusion of other Gvps led to gain or loss of protein-protein interactions, revealing interdependence during GV assembly. Specific subnetworks of Gvps are revealed that may be linked to different stages of GV assembly. A number of Gvps show tolerance for fusion proteins during GV assembly, unveiling a degree of plasticity with implications for GV engineering. Iburg at al develop a high-throughput in vivo screen to construct a comprehensive map of the interactions among all 11 proteins of the pNL29 gas vesicle operon. [ABSTRACT FROM AUTHOR]

Fibroblast growth factor 21 (FGF21) is pivotal in regulating energy metabolism, highlighting substantial therapeutic potential for non-alcoholic steatohepatitis (NASH). Previously, we reported a long-acting FGF21 fusion protein, PsTag-FGF21, which was prepared by genetically fusing human FGF21 with a 648-residue polypeptide (PsTag). While this fusion protein demonstrated therapeutic efficacy against NASH, our final product analysis revealed the presence of fixed impurities resistant to effective removal, indicating potential degradation of PsTag-FGF21. Here, we enriched and analyzed the impurities, confirming our hypothesis regarding the C-terminal degradation of PsTag-FGF21. We now describe a new variant developed to eliminate the C-terminal degradation. By introducing one mutation located at the C-terminal of PsTag-FGF21(V169L), we demonstrated that the new molecule, PsTag-FGF21(V169L), exhibits many improved attributes. Compared with PsTag-FGF21, PsTag-FGF21(V169L) displayed elevated bioactivity and stability, along with a twofold enhanced binding affinity to the coreceptor β-Klotho. In vivo, the circulating half-life of PsTag-FGF21(V169L) was further enhanced compared with that of PsTag-FGF21. In NASH mice, PsTag-FGF21(V169L) demonstrated efficacy with sustained improvements in multiple metabolic parameters. Besides, PsTag-FGF21(V169L) demonstrated the ability to alleviate NASH by decreasing hepatocyte apoptosis. The superior biophysical, pharmacokinetic, and pharmacodynamic properties, along with the positive metabolic effects, imply that further clinical development of PsTag-FGF21(V169L) as a metabolic therapy for NASH patients may be warranted. • A novel cleavage site in the C-terminal of FGF21 is reported. • PsTag-FGF21(V169L) is a long-acting FGF21 analog resistant to C-terminal cleavage. • Site-specific mutagenesis is used for enhancing bioactivity and stability. • PsTag-FGF21(V169L) exhibits therapeutic efficacy against NASH. • PsTag-FGF21(V169L) alleviates NASH by reducing hepatocyte apoptosis. [ABSTRACT FROM AUTHOR]

Inhibition of autophagy is one of the hallmarks of the SARS-CoV-2 infection. Recently it was reported that SARS-CoV-2 protein ORF3a inhibits fusion of autophagosomes with lysosomes via interaction with VPS39 thus preventing binding of homotypic fusion and protein sorting (HOPS) complex to RAB7 GTPase. Here we report that myelin basic protein (MBP), a major structural component of the myelin sheath, binds ORF3a and is colocalized with it in mammalian cells. Co-expression of MBP with ORF3a restores autophagy in mammalian cells, inhibited by viral protein. Our data suggest that basic charge of MBP drives suppression of ORF3a-induced autophagy inhibition as its deaminated variants lost ability to bind ORF3a and counteract autophagy blockade. These results together with our recent findings, indicating that MBP interacts with structural components of the vesicle transport machinery-synaptosomal-associated protein 23 (SNAP23), vesicle-associated membrane protein 3 (VAMP3) and Sec1/Munc18-1 family members, may suggest protective role of the MBP in terms of the maintaining of protein traffic and autophagosome–lysosome fusion machinery in oligodendrocytes during SARS-CoV-2 infection. Finally, our data may indicate that deimination of MBP observed in the patients with multiple sclerosis (MS) may contribute to the previously reported worser outcomes of COVID-19 and increase of post-COVID-19 neurologic symptoms in patients with MS. [Display omitted] • MBP interacts with SARS-CoV-2 proteins N, E, nsp9 and nsp16 in mammalian cells. • MBP interacts and colocalizes with SARS-CoV-2 protein ORF3a in mammalian cells. • MBP counteracts ORF3a-mediated failure of the autophagy machinery. • Basic charge of MBP drives suppression of ORF3a-induced autophagy inhibition. [ABSTRACT FROM AUTHOR]

Hemophilia B is an inherited hemorrhagic disorder characterized by a deficiency of blood coagulation factor IX (FIX) that results in abnormal blood coagulation. The blood coagulation is already evident in hemophiliacs at the fetal stage, and thus intracranial hemorrhage and other bleeding complications can occur at birth, leading to sequelae. Therefore, it is important to develop effective treatments for hemophiliacs in utero. In this study, in order to transplacentally deliver FIX from pregnant mice to their fetuses, an improved adenovirus (Ad) vector expressing human FIX fused with the IgG Fc domain (FIX Fc fusion protein), which plays a crucial role in neonatal Fc receptor (FcRn)-mediated transcytosis across the placenta, was intravenously administered to E13.5 pregnant mice. Significant levels of FIX Fc fusion protein were detected in 0-day-old newborn mice whose mothers were administered an Ad vector expressing FIX Fc fusion protein. Wild-type FIX overexpressed in the pregnant mice was not delivered to the fetuses. Plasma FIX levels in the newborn mice were relatively well correlated with those in their mothers, although transplacental delivery efficiencies of FIX Fc fusion protein were slightly reduced when the FIX Fc fusion protein was highly expressed in the mother mice. Plasma FIX levels in the newborn mice were about 3.6–6.4% of those in their mothers, Transplacental delivery of FIX Fc fusion protein to their fetuses successfully improved the blood clotting ability in the newborn mice. [Display omitted] • An improved adenovirus vector mediated efficient expression of FIX Fc fusion protein. • FIX Fc fusion protein, not wild-type FIX, was delivered from pregnant mice to fetuses. • Transplacental delivery of FIX to fetuses improved blood clotting ability in neonates. [ABSTRACT FROM AUTHOR]

Fusing insect derived neurotoxic peptides with Galanthus nivalis agglutinin (GNA) has been shown to enhance the insecticidal activity of the neuropeptides, especially when administered orally. This study produced a recombinant scorpion insect specific neurotoxin BjαIT, GNA, and a fusion protein BjαIT/GNA using Pichia pastoris as an expression host. Recombinant rBjαIT/GNA was found to be easily degraded during expression in yeast which and produced a main protein product with a molecular weight of approximately 14 kDa. Cytotoxicity results showed that rBjαIT, rGNA, and rBjαIT/GNA had no toxicity to mammalian NIH/3T3 cells. Adding rBjαIT or rBjαIT/GNA at a concentration as low as 1 ng/mL to insect cell culture medium inhibited the proliferation of insect Sf9 cells, with rBjαIT exhibiting stronger cytotoxicity, while 20 ng/mL rGNA did not inhibit the proliferation of Sf9 cells. Silkworm larval injection results showed that rBjαIT/GNA was the most toxic of the three proteins, followed by rBjαIT, and rGNA. When rBjαIT/GNA was injected at a concentration of 0.129 nmol/g body weight 46.7% of silkworm died within 48 h. Feeding newly hatched silkworms with rBjαIT/GNA at a leaf surface concentration of 40 µg/cm2 resulted in 76.7% mortality within 24 h. However, rBjαIT/GNA showed almost no oral insecticidal activity against second instar silkworms. The results indicated that rBjαIT/GNA has stronger injection insecticidal activity and feeding insecticidal activity than rBjαIT and rGNA individually, making it more suitable for biological control. [ABSTRACT FROM AUTHOR]

The ability to label proteins by fusion with genetically encoded fluorescent proteins is a powerful tool for understanding dynamic biological processes. However, current approaches for expressing fluorescent protein fusions possess drawbacks, especially at the whole organism level. Expression by transgenesis risks potential overexpression artifacts while fluorescent protein insertion at endogenous loci is technically difficult and, more importantly, does not allow for tissue-specific study of broadly expressed proteins. To overcome these limitations, we have adopted the split fluorescent protein system mNeonGreen2 1-10/11 (split-mNG2) to achieve tissue-specific and endogenous protein labeling in zebrafish. In our approach, mNG2 1-10 is expressed under a tissue-specific promoter using standard transgenesis while mNG2 11 is inserted into protein-coding genes of interest using CRISPR/Cas-directed gene editing. Each mNG2 fragment on its own is not fluorescent, but when co-expressed the fragments self-assemble into a fluorescent complex. Here, we report successful use of split-mNG2 to achieve differential labeling of the cytoskeleton genes tubb4b and krt8 in various tissues. We also demonstrate that by anchoring the mNG2 1-10 component to specific cellular compartments, the split-mNG2 system can be used to manipulate protein localization. Our approach should be broadly useful for a wide range of applications. [Display omitted] • The split-mNG2 system achieves tissue-specific protein labeling while maintaining endogenous expression levels. • mNG2 1-10 is expressed under a tissue-specific promoter using standard transgenesis. • mNG2 11 is fused to proteins of interest using CRISPR/Cas-directed gene editing. • The feasibility of the split-mNG2 approach is demonstrated for the cytoskeletal genes tubb4b and krt8. • The split-mNG2 system can also be used to direct protein localization. [ABSTRACT FROM AUTHOR]

In neurons, macroautophagy/autophagy is a frequent and critical process. In the axon, autophagy begins in the axon terminal, where most nascent autophagosomes form. After formation, autophagosomes must initiate transport to exit the axon terminal and move toward the cell body via retrograde transport. During retrograde transport these autophagosomes mature through repetitive fusion events. Complete lysosomal cargo degradation occurs largely in the cell body. The precipitating events to stimulate retrograde autophagosome transport have been debated but their importance is clear: disrupting neuronal autophagy or autophagosome transport is detrimental to neuronal health and function. We have identified the HOPS complex as essential for early autophagosome maturation and consequent initiation of retrograde transport from the axon terminal. In yeast and mammalian cells, HOPS controls fusion between autophagosomes and late endosomes with lysosomes. Using zebrafish strains with loss-of-function mutations in vps18 and vps41, core components of the HOPS complex, we found that disruption of HOPS eliminates autophagosome maturation and disrupts retrograde autophagosome transport initiation from the axon terminal. We confirmed this phenotype was due to loss of HOPS complex formation using an endogenous deletion of the HOPS binding domain in Vps18. Finally, using pharmacological inhibition of lysosomal proteases, we show that initiation of autophagosome retrograde transport requires autophagosome maturation. Together, our data demonstrate that HOPS-mediated fusion events are critical for retrograde autophagosome transport initiation through promoting autophagosome maturation. This reveals critical roles for the HOPS complex in neuronal autophagy which deepens our understanding of the cellular pathology of HOPS-complex linked neurodegenerative diseases. Abbreviations: CORVET: Class C core vacuole/endosome tethering; gRNA: guide RNA; HOPS: homotypic fusion and protein sorting; pLL: posterior lateral line; Vps18: VPS18 core subunit of CORVET and HOPS complexes; Vps41: VPS41 subunit of HOPS complex. [ABSTRACT FROM AUTHOR]

Microglia are specialized macrophages responsible for the clearance of dead neurons and pathogens by phagocytosis and degradation. The degradation requires phagosome maturation and acidification provided by the vesicular- or vacuolar-type H+-translocating adenosine triphosphatase (V-ATPase), which is composed of the cytoplasmic V1 domain and the membrane-embedded Vo domain. The V-ATPase a subunit, an integral part of the Vo domain, has four isoforms in mammals. The functions of different isoforms on phagosome maturation in different cells/species remain controversial. Here we show that mutations of both the V-ATPase Atp6v0a1 and Tcirg1b/Atp6v0a3 subunits lead to the accumulation of phagosomes in zebrafish microglia. However, their mechanisms are different. The V-ATPase Atp6v0a1 subunit is mainly distributed in early and late phagosomes. Defects of this subunit lead to a defective transition from early phagosomes to late phagosomes. In contrast, The V-ATPase Tcirg1b/Atp6v0a3 subunit is primarily located on lysosomes and regulates late phagosome-lysosomal fusion. Defective Tcirg1b/Atp6v0a3, but not Atp6v0a1 subunit leads to reduced acidification and impaired macroautophagy/autophagy in microglia. We further showed that ATP6V0A1/a1 and TCIRG1/a3 subunits in mouse macrophages preferentially located in endosomes and lysosomes, respectively. Blocking these subunits disrupted early-to-late endosome transition and endosome-to-lysosome fusion, respectively. Taken together, our results highlight the essential and conserved roles played by different V-ATPase subunits in multiple steps of phagocytosis and endocytosis across various species. Abbrevations: Apoe: apolipoprotein E; ANXA5/annexin V: annexin A5; ATP6V0A1/a1: ATPase H+-transporting V0 subunit a1; ATP6V0A2/a2: ATPase H+-transporting V0 subunit a2; ATP6V0A4/a4: ATPase H+-transporting V0 subunit a4; dpf: days post-fertilization; EEA1: early endosome antigen 1; HOPS: homotypic fusion and protein sorting; LAMP1: lysosomal associated membrane protein 1; Lcp1: lymphocyte cytosolic protein 1 (L-plastin); Map1lc3/Lc3: microtubule-associated protein 1 light chain 3; NR: neutral red; PBS: phosphate-buffered saline; PtdIns: phosphatidylinositol; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns(3,5)P2: phosphatidylinositol (3,5)-bisphosphate; RAB4: RAB4, member RAS oncogene family; RAB5: RAB5, member RAS oncogene family; RAB7: RAB7, member RAS oncogene family; TCIRG1/Atp6v0a3/a3: T cell immune regulator 1, ATPase H+-transporting V0 subunit a3; V-ATPase: vacuolar-type H+-translocating adenosine triphosphatase; Xla.Tubb2b/NBT: tubulin beta 2B class IIb. [ABSTRACT FROM AUTHOR]

Xp11.2 translocation renal cell carcinomas (tRCC) are a rare and highly malignant type of renal cancer, lacking efficient diagnostic indicators and therapeutic targets. Through the analysis of public databases and our cohort, we identified NMRK2 as a potential diagnostic marker for distinguishing Xp11.2 tRCC from kidney renal clear cell carcinoma (KIRC) and kidney renal papillary cell carcinoma (KIRP) due to its specific upregulation in Xp11.2 tRCC tissues. Mechanistically, we discovered that TFE3 fusion protein binds to the promoter of the NMRK2 gene, leading to its upregulation. Importantly, we established RNA‐ and protein‐based diagnostic methods for identifying Xp11.2 tRCC based on NMRK2 expression levels, and the diagnostic performance of our methods was comparable to a dual‐color break‐apart fluorescence in situ hybridization assay. Moreover, we successfully identified fresh Xp11.2 tRCC tissues after surgical excision using our diagnostic methods and established an immortalized Xp11.2 tRCC cell line for further research purposes. Functional studies revealed that NMRK2 promotes the progression of Xp11.2 tRCC by upregulating the NAD+/NADH ratio, and supplementation with β‐nicotinamide mononucleotide (NMN) or nicotinamide riboside chloride (NR), effectively rescued the phenotypes induced by the knockdown of NMRK2 in Xp11.2 tRCC. Taken together, these data introduce a new diagnostic indicator capable of accurately distinguishing Xp11.2 tRCC and highlight the possibility of developing novel targeted therapeutics. © 2024 The Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

Ewing sarcoma, an aggressive pediatric cancer, is driven by the EWS::FLI1 fusion protein, which disrupts gene expression by hijacking the BAF chromatin remodeling complex. Central to this mechanism is the formation of biomolecular condensates, mediated by the prion‐like domains (PrLDs) of EWS and ARID1A, a core BAF subunit. ARID1A serves as a critical interface between EWS::FLI1 and the BAF complex, with its condensate‐forming ability essential for the aberrant gene expression that drives tumor growth. The loss of condensate‐competent ARID1A significantly impairs tumor progression, identifying it as a potential therapeutic target. However, targeting condensate formation is challenging due to the transient nature of the interactions involved, complicating the development of effective inhibitors. This work underscores the importance of further investigation into therapeutic strategies aimed at disrupting condensate formation in Ewing sarcoma and other related malignancies. [ABSTRACT FROM AUTHOR]

Cell wall peptidoglycan binding domains (CBDs) of cell lytic enzymes, including bacteriocins, autolysins and bacteriophage endolysins, enable highly selective bacterial binding, and thus, have potential as biorecognition molecules for nondestructive bacterial detection. Here, a novel design for a self-complementing split fluorescent protein (FP) complex is proposed, where a multimeric FP chain fused with specific CBDs ((FP-CBD) n) is assembled inside the cell, to improve sensitivity by enhancing the signal generated upon Staphylococcus aureus or Bacillus anthracis binding. Flow cytometry shows enhanced fluorescence on the cell surface with increasing FP stoichiometry and surface plasmon resonance reveals nanomolar binding affinity to isolated peptidoglycan. The breadth of function of these complexes is demonstrated through the use of CBD modularity and the ability to attach enzymatic detection modalities. Horseradish peroxidase-coupled (FP-CBD) n complexes generate a catalytic amplification, with the degree of amplification increasing as a function of FP length, reaching a limit of detection (LOD) of 103 cells/droplet (approximately 0.1 ng S. aureus or B. anthracis) within 15 min on a polystyrene surface. These fusion proteins can be multiplexed for simultaneous detection. Multimeric split FP-CBD fusions enable use as a biorecognition molecule with enhanced signal for use in bacterial biosensing platforms. [Display omitted] • Multimeric fluorescent protein (FP) chain with cell wall binding domains (CBDs). • Self-complementing split FP complexation with specific CBDs inside the cell. • Enhanced signal generation upon target bacteria binding induced by multimeric CBDs. • Amplified colorimetric signals of HRP-coupled FP-CBD fusions for bacteria sensing. • Multiple FP-CBD fusions for simultaneous detection of different bacteria. [ABSTRACT FROM AUTHOR]

Extracellular Vesicles (EVs) have been proposed as a promising tool for drug delivery because of their natural ability to cross biological barriers, protect their cargo, and target specific cells. Moreover, EVs are not recognized by the immune system as foreign, reducing the risk of an immune response and enhancing biocompatibility. Herein, we proposed an alternative therapeutic strategy to restore STAT3 signaling exploiting STAT3 loaded EVs. This approach could be useful in the treatment of Autosomal Dominant Hyper-IgE Syndrome (AD-HIES), a rare primary immunodeficiency and multisystem disorder due to the presence of mutations in STAT3 gene. These mutations alter the signal transduction of STAT3, thereby impeding Th17 CD4+ cell differentiation that leads to the failure of immune response. We set up a simple and versatile method in which EVs were loaded with fully functional STAT3 protein. Moreover, our method allows to follow the uptake of STAT3 loaded vesicles inside cells due to the presence of EGFP in the EGFP-STAT3 fusion protein construct. Taken together, the data presented in this study could provide the scientific background for the development of new therapeutic strategy aimed to restore STAT3 signaling in STAT3 misfunction associated diseases like AD-HIES. In the future, the administration of fully functional wild type STAT3 to CD4+ T cells of AD-HIES patients might compensate its loss of function and would be beneficial for these patients, lowering the risk of infections, the use of medications, and hospitalizations. [Display omitted] • EVs are a promising tool for drug delivery. • EVs have been loaded with fully functional EGFP-STAT3. • EGFP-STAT3 allows to track EVs and to follow STAT3 trafficking in cells. • EVs loaded with fully functional STAT3 might be useful to restore STAT3 signaling. [ABSTRACT FROM AUTHOR]

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Botulinum neurotoxin (BoNT), produced by Clostridium botulinum, is the most toxic protein known, capable of causing severe paralysis and posing a significant bioterrorism threat due to its extreme lethality even in minute quantities. Despite this, there are currently no FDA-approved vaccines for widespread public use. To address this urgent need, we have developed an innovative vaccine platform by fusing the neuronal binding domain of BoNT/E (Hc/E) with core-streptavidin (CS), resulting in a stable CS-Hc/E vaccine. Mice vaccinated with CS-Hc/E exhibited superior antibody titers compared to those receiving Hc/E alone. To develop a trivalent vaccine against BoNT/A, BoNT/B, and BoNT/E--key contributors to the vast majority of human botulism--we conjugated CS-Hc/E with a biotinylated atoxic chimeric protein incorporating neutralizing epitopes from BoNT/A and BoNT/B. This chimeric protein includes the binding domain of BoNT/A, along with the protease-inactive light chain and translocation domains of BoNT/B. The interaction between CS and biotin formed a stable tetrameric antigen, EBA. Vaccination with EBA in mice elicited robust antibody responses and provided complete protection against lethal doses of BoNT/A, BoNT/B, and BoNT/E. Our findings highlight EBA's potential as a stable and effective broadspectrum vaccine against BoNT. Moreover, our technology offers a versatile platform for developing multivalent, stable vaccines targeting various biological threats by substituting the BoNT domain(s) with neutralizing epitopes from other life-threatening pathogens, thereby enhancing public health preparedness and biodefense strategies. [ABSTRACT FROM AUTHOR]

Introduction: Among hyperthermophilic organisms, in vivo protein localization is challenging due to the high growth temperatures that can disrupt proper folding and function of mostly mesophilic-derived fluorescent proteins. While protein localization in the thermophilic model archaeon S. acidocaldarius has been achieved using antibodies with fluorescent probes in fixed cells, the use of thermostable fluorescent proteins for live imaging in thermophilic archaea has so far been unsuccessful. Given the significance of live protein localization in the field of archaeal cell biology, we aimed to identify fluorescent proteins for use in S. acidocaldarius. Methods: We expressed various previously published and optimized thermostable fluorescent proteins along with fusion proteins of interest and analyzed the cells using flow cytometry and (thermo-) fluorescent microscopy. Results: Of the tested proteins, thermal green protein (TGP) exhibited the brightest fluorescence when expressed in Sulfolobus cells. By optimizing the linker between TGP and a protein of interest, we could additionally successfully fuse proteins with minimal loss of fluorescence. TGP-CdvB and TGP-PCNA1 fusions displayed localization patterns consistent with previous immunolocalization experiments. Discussion: These initial results in live protein localization in S. acidocaldarius at high temperatures, combined with recent advancements in thermomicroscopy, open new avenues in the field of archaeal cell biology. This progress finally enables localization experiments in thermophilic archaea, which have so far been limited to mesophilic organisms. [ABSTRACT FROM AUTHOR]

Background: Mycoplasmal pneumonia of sheep and goats (MPSG) is an important infectious disease that threatens sheep and goat production worldwide, and Mycoplasma ovipneumoniae (Movi) is one of the major aetiological agents causing MPSG. The aim of this study was to investigate the immunological activity of the Hsp70‒P113 fusion protein derived from Movi and to develop a serological assay for the detection of Movi. Methods: This study involved codon optimization of the dominant antigenic regions of Movi heat shock protein 70 (Hsp70) and adhesin P113. Afterwards, the optimized sequences were inserted into the prokaryotic expression vector pET-30a(+) through tandem linking with the aid of a linker. Once a positive recombinant plasmid (pET-30a-rHsp70-P113) was successfully generated, the expression conditions were further refined. The resulting double gene fusion target protein (rHsp70‒P113) was subsequently purified using ProteinIso® Ni–NTA resin, and the reactivity of the protein was confirmed via SDS‒PAGE and Western blot analysis. An indirect enzyme-linked immunosorbent assay (i-ELISA) technique was developed to detect Movi utilizing the fusion protein as the coating antigen. The specificity, sensitivity, and reproducibility of all methods were assessed after each reaction parameter was optimized. Results: The resulting rHsp70-P113 protein had a molecular weight of approximately 51 kDa and was predominantly expressed in the supernatant. Western blot analysis demonstrated its favourable reactivity. The optimal parameters for the i-ELISA technique were as follows: the rHsp70-P113 protein was encapsulated at a concentration of 5 μg/mL; the serum was diluted at a ratio of 1:50; the HRP-labelled donkey anti-goat IgG was diluted at a ratio of 1:6,000. The results of the cross-reactivity assays revealed that the i-ELISA was not cross-reactive with other goat-positive sera against Mycoplasma mycodies subsp. capri (Mmc), Mycoplasma capricolum subsp. capripneumoniae (Mccp), Mycoplasma arginini (Marg), orf virus (ORFV) or enzootic nasal tumour virus of goats (ENTV-2). The sensitivity of this method is high, with a maximum dilution of up to 1:640. The results of the intra- and inter-batch replication tests revealed that the coefficients of variation were both less than 10%, indicating excellent reproducibility. The analysis of 108 clinical serum samples via i-ELISA and indirect haemagglutination techniques yielded significant findings. Among these samples, 43 displayed positive results, whereas 65 presented negative results, resulting in a positivity rate of 39.8% for the i-ELISA method. In contrast, the indirect haemagglutination technique identified 20 positive samples and 88 negative samples, resulting in a positivity rate of 18.5%. Moreover, a comparison between the two methods revealed a conformity rate of 78.7%. Conclusion: The results obtained in this study lay the groundwork for advancements in the use of an Movi antibody detection kit, epidemiological inquiry, and subunit vaccines. [ABSTRACT FROM AUTHOR]

Targeted delivery of therapeutic anticancer chimeric molecules enhances drug efficacy. Numerous studies have focused on developing novel treatments by employing cytokines, particularly interleukins, to inhibit the growth of cancer cells. In the present study, we fused interleukin 24 with the tumor-targeting peptide P20 through a rigid linker to selectively target cancer cells. The secondary structure, tertiary structure, and physicochemical characteristics of the constructed chimeric IL-24-P20 protein were predicted by using bioinformatics tools. In-silico analysis revealed that the fusion construct has a basic nature with 175 amino acids and a molecular weight of 20 kDa. By using the Rampage and ERRAT2 servers, the validity and quality of the fusion protein were evaluated. The results indicated that 93% of the chimeric proteins contained 90.1% of the residues in the favoured region, resulting in a reliable structure. Finally, docking and simulation studies were conducted via ClusPro and Desmond Schrödinger, respectively. Our results indicate that the constructed fusion protein exhibits excellent quality, interaction capabilities, validity, and stability. These findings suggest that the fusion protein is a promising candidate for targeted cancer therapy. [ABSTRACT FROM AUTHOR]

The transition between yeast and hyphae is crucial for regulating the commensalism and pathogenicity in Candida albicans. The mechanisms that affect the invasion of hyphae in solid media, whose deficiency is more related to the pathogenicity of C. albicans, have not been elucidated. Here, we found that the disruption of VAM6 or VPS41 which are components of the homotypic vacuolar fusion and protein sorting (HOPS) complex, or the Rab GTPase YPT72, all responsible for vacuole fusion, led to defects in hyphal growth in both liquid and solid media, but more pronounced on solid agar. The phenotypes of vac8Δ/Δ and GTR1OE-vam6Δ/Δ mutants indicated that these deficiencies are mainly caused by the reduced mechanical forces that drive agar and organs penetration, and confirmed that large vacuoles are required for hyphal mechanical penetration. In summary, our study revealed that large vacuoles generated by vacuolar fusion support hyphal penetration and provided a perspective to refocus attention on the role of solid agar in evaluating C. albicans invasion. The transition between yeast and hyphae is crucial for the pathogenicity of Candida albicans. Here, Liu et al. show that vacuole fusion is active during hyphal extension and that large vacuoles increase the mechanical forces of hyphae penetrating into organs. [ABSTRACT FROM AUTHOR]

Introduction: Cepharanthine (CEP), a bisbenzylisoquinoline alkaloid (bisBIA) extracted from Stephania japonica, has received significant attention for its anti-coronavirus properties. While ethylene response factors (ERFs) have been reported to regulate the biosynthesis of various alkaloids, their role in regulating CEP biosynthesis remains unexplored. Methods: Genome-wide analysis of the ERF genes was performed with bioinformatics technology, and the expression patterns of different tissues, were analyzed by transcriptome sequencing analysis and real-time quantitative PCR verification. The nuclear-localized ERF gene cluster was shown to directly bind to the promoters of several CEP-associated genes, as demonstrated by yeast one-hybrid assays and subcellular localization assays. Results: In this work, 59 SjERF genes were identified in the S. japonica genome and further categorized into ten subfamilies. Notably, a SjERF gene cluster containing three SjERF genes was found on chromosome 2. Yeast one-hybrid assays confirmed that the SjERF gene cluster can directly bind to the promoters of several CEP-associated genes, suggesting their crucial role in CEP metabolism. The SjERFs cluster-YFP fusion proteins were observed exclusively in the nuclei of Nicotiana benthamiana leaves. Tissue expression profiling revealed that 13 SjERFs exhibit high expression levels in the root, and the qRTPCR results of six SjERFs were consistent with the RNA-Seq data. Furthermore, a co-expression network analysis demonstrated that 24 SjERFs were highly positively correlated with the contents of various alkaloids and expression levels of CEP biosynthetic genes. Conclusion: This study provides the first systematic identification and analysis of ERF transcription factors in the S.japonica genome, laying the foundation for the future functional research of SjERFs transcription factors. [ABSTRACT FROM AUTHOR]

Cell membranes are crucial elements in living organisms, serving as protective barriers and providing structural support for cells. They regulate numerous exchange and communication processes between cells and their environment, including interactions with other cells, tissues, ions, xenobiotics, and drugs. However, the complexity and heterogeneity of cell membranes—comprising two asymmetric layers with varying compositions across different cell types and states (e.g., healthy vs. diseased)—along with the challenges of manipulating real cell membranes represent significant obstacles for in vivo studies. To address these challenges, researchers have developed various methodologies to create model cell membranes or membrane fragments, including mono- or bilayers organized in planar systems. These models facilitate fundamental studies on membrane component interactions as well as the interactions of membrane components with external agents, such as drugs, nanoparticles (NPs), or biomarkers. The applications of model cell membranes have extended beyond basic research, encompassing areas such as biosensing and nanoparticle camouflage to evade immune detection. In this review, we highlight advancements in the engineering of planar model cell membranes, focusing on the nanoarchitectonic tools used for their fabrication. We also discuss approaches for incorporating challenging materials, such as proteins and enzymes, into these models. Finally, we present our view on future perspectives in the field of planar model cell membranes. [ABSTRACT FROM AUTHOR]

The centrosome of the amoebozoan model Dictyostelium discoideum provides the best-established model for an acentriolar centrosome outside the Opisthokonta. Dictyostelium exhibits an unusual centrosome cycle, in which duplication is initiated only at the G2/M transition and occurs entirely during the M phase. Little is known about the role of conserved centrosomal kinases in this process. Therefore, we have generated knock-in strains for Aurora (AurK), CDK1, cyclin B, Nek2, and Plk, replacing the endogenous genes with constructs expressing the respective green fluorescent Neon fusion proteins, driven by the endogenous promoters, and studied their behavior in living cells. Our results show that CDK1 and cyclin B arrive at the centrosome first, already during G2, followed by Plk, Nek2, and AurK. Furthermore, CDK1/cyclin B and AurK were dynamically localized at kinetochores, and AurK in addition at nucleoli. The putative roles of all four kinases in centrosome duplication, mitosis, cytokinesis, and nucleolar dynamics are discussed. [ABSTRACT FROM AUTHOR]

Inducible dimerization systems, such as rapamycin‐induced dimerization of FK506 binding protein (FKBP) and FKBP–rapamycin binding (FRB) domain, are widely employed chemical biology tools to manipulate cellular functions. We previously advanced an inducible dimerization system into an inducible oligomerization system by developing a bivalent fusion protein, FRB–FKBP, which forms large oligomers upon rapamycin addition and can be used to manipulate cells. However, the oligomeric structure of FRB–FKBP remains unclear. Here, we report that FRB–FKBP forms a rotationally symmetric trimer in crystals, but a larger oligomer in solution, primarily tetramers and pentamers, which maintain similar inter‐subunit contacts as in the crystal trimer. These findings expand the applications of the FRB–FKBP oligomerization system in diverse biological events. [ABSTRACT FROM AUTHOR]

MET is a receptor tyrosine kinase (RTK) responsible for initiating signaling pathways involved in development and wound repair. MET activation relies on ligand binding to the extracellular receptor, which prompts dimerization, intracellular phosphorylation, and recruitment of associated signaling proteins. Mutations, which are predominantly observed clinically in the intracellular juxtamembrane and kinase domains, can disrupt typical MET regulatory mechanisms. Understanding how juxtamembrane variants, such as exon 14 skipping (METΔEx14), and rare kinase domain mutations can increase signaling, often leading to cancer, remains a challenge. Here, we perform a parallel deep mutational scan (DMS) of the MET intracellular kinase domain in two fusion protein backgrounds: wild-type and METΔEx14. Our comparative approach has revealed a critical hydrophobic interaction between a juxtamembrane segment and the kinase ⍺C-helix, pointing to potential differences in regulatory mechanisms between MET and other RTKs. Additionally, we have uncovered a β5 motif that acts as a structural pivot for the kinase domain in MET and other TAM family of kinases. We also describe a number of previously unknown activating mutations, aiding the effort to annotate driver, passenger, and drug resistance mutations in the MET kinase domain. [ABSTRACT FROM AUTHOR]