Intracellular Protein Research Articles

Validation of GCN5L1/BLOC1S1/BLOS1 antibodies using knockout cells and tissue.

GCN5L1, also known as BLOC1S1 and BLOS1, is a small intracellular protein involved in many key biological processes. Over the last decade, GCN5L1 has been implicated in the regulation of protein lysine acetylation, energy metabolism, endo-lysosomal function, and cellular immune pathways. An increasing number of published papers have used commercially-available reagents to interrogate GCN5L1 function. However, in many cases these reagents have not been rigorously validated, leading to potentially misleading results. In this report we tested several commercially-available antibodies for GCN5L1, and found that two-thirds of those available did not unambiguously detect the protein by western blot in cultured mouse cells or ex vivo liver tissue. These data suggest that previously published studies which used these unverified antibodies to measure GCN5L1 protein abundance, in the absence of other independent methods of corroboration, should be interpreted with appropriate caution.

Compartmental analysis: a new approach to estimate protein breakdown and meal response in health and critical illness

Purpose of reviewThis study aimed to discuss the use of the pulse stable isotope tracer approach to study changes in metabolism in healthy individuals and critically ill patients.Recent findings and conclusionWe found that in the postabsorptive state and healthy condition, intracellular protein breakdown and net intracellular protein breakdown, when calculated using the pulse tracer approach, are about double what has previously been reported using the more traditional primed-constant and continuous stable isotope approaches (600 versus 300 grams of protein/day). In critically ill patients, protein breakdown is even higher and calculated to be approximately 900 grams of protein/day, using the pulse tracer approach. Based on these data, we hypothesize that reducing protein breakdown in the postabsorptive state is key when trying to improve the condition of critically ill patients. Moreover, we also used the pulse tracer approach during feeding to better estimate the intracellular metabolic response to feeding. Our first observation is that endogenous protein breakdown does not seem to be reduced during feeding. We also have shown that when consuming a meal with a certain amount of protein, the biological value of that protein meal can be calculated with the pulse tracer approach. In conclusion, using the pulse stable isotope tracer approach to study protein kinetics in the postabsorptive state and during feeding expands our understanding of how dietary proteins can affect human protein metabolism. The intracellular protein synthesis stimulatory effect of a meal is an important factor to consider when calculating the exact protein requirements and needs, particularly in critical illness.

Modulation of the Blood–Brain Barrier by Sigma-1R Activation

Sigma non-opioid intracellular receptor 1 (Sigma-1R) is an intracellular chaperone protein residing on the endoplasmic reticulum at the mitochondrial-associated membrane (MAM) region. Sigma-1R is abundant in the brain and is involved in several physiological processes as well as in various disease states. The role of Sigma-1R at the blood–brain barrier (BBB) is incompletely characterized. In this study, the effect of Sigma-1R activation was investigated in vitro on rat brain microvascular endothelial cells (RBMVEC), an important component of the blood–brain barrier (BBB), and in vivo on BBB permeability in rats. The Sigma-1R agonist PRE-084 produced a dose-dependent increase in mitochondrial calcium, and mitochondrial and cytosolic reactive oxygen species (ROS) in RBMVEC. PRE-084 decreased the electrical resistance of the RBMVEC monolayer, measured with the electric cell-substrate impedance sensing (ECIS) method, indicating barrier disruption. These effects were reduced by pretreatment with Sigma-1R antagonists, BD 1047 and NE 100. In vivo assessment of BBB permeability in rats indicates that PRE-084 produced a dose-dependent increase in brain extravasation of Evans Blue and sodium fluorescein brain; the effect was reduced by the Sigma-1R antagonists. Immunocytochemistry studies indicate that PRE-084 produced a disruption of tight and adherens junctions and actin cytoskeleton. The brain microcirculation was directly visualized in vivo in the prefrontal cortex of awake rats with a miniature integrated fluorescence microscope (aka, miniscope; Doric Lenses Inc.). Miniscope studies indicate that PRE-084 increased sodium fluorescein extravasation in vivo. Taken together, these results indicate that Sigma-1R activation promoted oxidative stress and increased BBB permeability.

Causal relationship between inflammatory proteins and glioblastoma: a two-sample bi‑directional mendelian randomization study

Background: Observational studies have indicated a potential correlation between glioblastoma and circulating inflammatory proteins. Further investigation is required to establish a causal relationship between these two factors.Methods: We performed a Mendelian randomization (MR) analysis using genome-wide association study (GWAS) summary of 91 circulating inflammation-related proteins (N = 14,824) to assess their causal impact on glioblastoma. The GWAS summary data for glioblastoma included 243 cases and 287,137 controls. The inverse variance weighted (IVW) method was used as the primary analytical method to assess causality. Four additional MR methods [simple mode, MR-Egger, weighted median, and weighted mode] were used to supplement the IVW results. Furthermore, several sensitivity analyses were performed to assess heterogeneity, horizontal pleiotropy, and stability. Reverse MR analysis was also performed. glioblastoma transcriptomic data from The Cancer Genome Atlas (TCGA) were analyzed to validate the findings obtained through MR, while pathway and functional enrichment analyses were conducted to predict the potential underlying mechanisms.Results: Our findings from employing the inverse variance weighted method in our forward MR analysis provide robust evidence supporting a potential association between glioblastoma and elevated levels of Cystatin D, as well as decreased levels of fibroblast growth factor 21 (FGF21) in the circulation. Moreover, our reverse MR analysis revealed that glioblastoma may contribute to increased concentrations of C-X-C motif chemokine 9 (CXCL9) and Interleukin-33 (IL-33) in the bloodstream. Transcriptomic analysis showed that FGF21 expression was inversely associated with the risk of developing glioblastoma, whereas an increased risk was linked to elevated levels of CXCL9 and IL-33. Pathway and functional enrichment analyses suggested that Cystatin D might exert its effects on glioblastoma through intracellular protein transport, whereas FGF21 might affect glioblastoma via glucose response mechanisms.Conclusion: These results indicate that FGF21 is a significant factor in glioblastoma susceptibility. Glioblastoma also affects the expression of inflammatory proteins such as C-X-C motif chemokine 9 and Interleukin-33, providing new insights into the mechanisms of glioblastoma genesis and clinical research.

Enhanced biodegradation of phenol under Cr(VI) stress by microbial collaboration and potential application of machine learning for phenol biodegradation

ABSTRACT Cr(VI) and phenol commonly coexist in wastewater, posing a great threat to the environment and human health. However, it is still a challenge for microorganisms to degrade phenol under high Cr(VI) stress. In this study, the phenol-degrading strain Bacillus cereus ZWB3 was co-cultured with the Cr(VI)-reducing strain Bacillus licheniformis MZ-1 to enhance phenol biodegradation under Cr(Ⅵ) stress. Compared with phenol-degrading strain ZWB3, which has weak tolerance to Cr(Ⅵ), and Cr(Ⅵ)-reducing strain MZ-1, which has no phenol-degrading ability, the co-culture of two strains could significantly increase the degraded rate and capacity of phenol. In addition, the co-cultured strains exhibited phenol degradation ability over a wide pH range (7–10). The reduced content of intracellular proteins and polysaccharides produced by the co-cultured strains contributed to the enhancement of phenol degradation and Cr(Ⅵ) tolerance. The determination coefficients R2, RMSE, and MAPE showed that the BP-ANN model could predict the degradation of phenol under various conditions, which saved time and economic cost. The metabolic pathway of microbial degradation of phenol was deduced by metabolic analysis. This study provides a valuable strategy for wastewater treatment containing Cr(Ⅵ) and phenol.

A TME-enlightened protein-binding photodynamic nanoinhibitor for highly effective oncology treatment

The efficiency of photodynamic therapy (PDT) is greatly dependent on intrinsic features of photosensitizers (PSs), but most PSs suffer from narrow diffusion distances and short life span of singlet oxygen (1O2). Here, to conquer this issue, we propose a strategy for in situ formation of complexes between PSs and proteins to deactivate proteins, leading to highly effective PDT. The tetrafluorophenyl bacteriochlorin (FBC), a strong near-infrared absorbing photosensitizer, can tightly bind to intracellular proteins to form stable complexes, which breaks through the space-time constraints of PSs and proteins. The generated singlet oxygen directly causes the protein dysfunction, leading to high efficiency of PSs. To enable efficient delivery of PSs, a charge-conversional and redox-responsive block copolymer POEGMA-b-(PAEMA/DMMA-co-BMA) (PB) was designed to construct a protein-binding photodynamic nanoinhibitor (FBC@PB), which not only prolongs blood circulation and enhances cellular uptake but also releases FBC on demand in tumor microenvironment (TME). Meanwhile, PDT-induced destruction of cancer cells could produce tumor-associated antigens which were capable to trigger robust antitumor immune responses, facilitating the eradication of residual cancer cells. A series of experiments in vitro and in vivo demonstrated that this multifunctional nanoinhibitor provides a promising strategy to extend photodynamic immunotherapy.

Cathepsin K deficiency prevented stress-related thrombosis in a mouse FeCl3 model

BackgroundExposure to chronic psychological stress (CPS) is a risk factor for thrombotic cardiocerebrovascular diseases (CCVDs). The expression and activity of the cysteine cathepsin K (CTSK) are upregulated in stressed cardiovascular tissues, and we investigated whether CTSK is involved in chronic stress-related thrombosis, focusing on stress serum-induced endothelial apoptosis.Methods and resultsEight-week-old wild-type male mice (CTSK+/+) randomly divided to non-stress and 3-week restraint stress groups received a left carotid artery iron chloride3 (FeCl3)-induced thrombosis injury for biological and morphological evaluations at specific timepoints. On day 21 post-stress/injury, the stress had enhanced the arterial thrombi weights and lengths, in addition to harmful alterations of plasma ADAMTS13, von Willebrand factor, and plasminogen activation inhibitor-1, plus injured-artery endothelial loss and CTSK protein/mRNA expression. The stressed CTSK+/+ mice had increased levels of injured arterial cleaved Notch1, Hes1, cleaved caspase8, matrix metalloproteinase-9/-2, angiotensin type 1 receptor, galactin3, p16IN4A, p22phox, gp91phox, intracellular adhesion molecule-1, TNF-α, MCP-1, and TLR-4 proteins and/or genes. Pharmacological and genetic inhibitions of CTSK ameliorated the stress-induced thrombus formation and the observed molecular and morphological changes. In cultured HUVECs, CTSK overexpression and silencing respectively increased and mitigated stressed-serum- and H2O2-induced apoptosis associated with apoptosis-related protein changes. Recombinant human CTSK degraded γ-secretase substrate in a dose-dependent manor and activated Notch1 and Hes1 expression upregulation.ConclusionsCTSK appeared to contribute to stress-related thrombosis in mice subjected to FeCl3 stress, possibly via the modulation of vascular inflammation, oxidative production and apoptosis, suggesting that CTSK could be an effective therapeutic target for CPS-related thrombotic events in patients with CCVDs.

Retrograde AAV-mediated gene modulation reveals chloride intracellular channel proteins as potent regulators of retinal ganglion cell death

Most projection neurons, including retinal ganglion cells (RGCs), undergo cell death after axotomy proximal to the cell body. Specific RGC subtypes, such as ON-OFF direction selective RGCs (ooDSGCs) are particularly vulnerable, whereas intrinsically photosensitive RGCs (ipRGCs) exhibit resilience to axonal injury. Through the application of RNA sequencing and fluorescent in situ hybridization, we show that the expression of chloride intracellular channel protein 1 and 4 (Clic1 and Clic4) are highly increased in the ooDSGCs after axonal injury. Toward determining a gene's role in RGCs, we optimized the utility and efficacy of adenovirus associated virus (AAV)-retro expressing short hairpin RNA (shRNA). Injection of AAV2-retro into the superior colliculus results in efficient shRNA expression in RGCs. Incorporating histone H2B gene fused with mGreenLantern results in bright nuclear reporter expression, thereby enhancing single RGC identification and cell quantitation in live retinas. Lastly, we demonstrate that AAV2-retro mediated knockdown of both Clic1 and Clic4 promotes RGC survival after injury. Our findings establish an integrated use of AAV2-retro-shRNA and real-time fundus imaging and reveals CLICs' contribution to RGC death.

Development of a new caged intein for multi-input conditional translation of synthetic mRNA.

mRNA medicines can be used to express therapeutic proteins, but the production of such proteins in non-target cells has a risk of adverse effects. To accurately distinguish between therapeutic target and nontarget cells, it is desirable to utilize multiple proteins expressed in each cell as indicators. To achieve such multi-input translational regulation of mRNA medicines, in this study, we engineered Rhodothermus marinus (Rma) DnaB intein to develop "caged Rma DnaB intein" that enables conditional reconstitution of full-length translational regulator protein from split fragments. By combining the caged Rma DnaB intein, the split translational regulator protein, and target protein-binding domains, we succeeded in target protein-dependent translational repression of mRNA in human cells. In addition, the caged Rma intein showed orthogonality to the previously reported Nostoc punctiforme (Npu) DnaE-based caged intein. Finally, by combining these two orthogonal caged inteins, we developed an mRNA-based logic gate that regulates translation based on the expression of multiple intracellular proteins. This study provides important information to develop safer mRNA medicines.

Metabolic effects of phospholipids extracted from capelin (Mallotus villosus) roe on HepG2 cells

This study aimed to evaluate the effects of capelin roe phospholipid (CPL) on lipid accumulation, fatty acid profile, oxidative stress, and changes in target macromolecular structure in HepG2 cells using multidimensional hyperspectral technique. The results showed that CPL significantly inhibited oleic acid-induced accumulation of intracellular lipids and increased the activity of intracellular antioxidant enzymes (CAT, GSH-Px, SOD). The control and CPL-treated groups promoted accumulation of unsaturated fatty acids and reduced deposition of saturated fatty acids. The therapeutic effects of CPL positively correlated with its concentration. Fourier transform infrared spectroscopy and two-dimensional correlation infrared analysis indicated that CPL decreased the relative contents of intracellular carbohydrate, nucleic acid, -CH2-, and ν(C=O)/νas (CH2+CH3), and maintained the order of intracellular protein structure. Fourier transform infrared spectroscopy could be used for label-free cell sorting. This work provided a direction for the subsequent development of functional lipids in aquaculture.

Ras-Related Protein Rab24 is a Predictor for Hepatocellular Carcinoma Prognosis and Promotes Tumor Growth

Ras-related protein Rab24, a member of the small GTPase family, plays a vital role in regulating intracellular protein trafficking. Recent research has uncovered dysregulation of Rab24 in hepatocellular carcinoma (HCC), but its clinical implications and tumor-related effects require further investigation. We aimed to investigate Rab24’s expression patterns and its role in HCC progression. We analyzed Rab24 expression in HCC and adjacent tissues at the transcriptional, mRNA, and protein levels. The prognostic significance of Rab24 in HCC was assessed through univariate and multivariate analyses, along with Kaplan–Meier survival analysis. Rab24’s impact on cell proliferation was investigated through cellular and xenograft experiments. Our findings revealed elevated Rab24 expression in HCC tissues compared to adjacent liver tissues. High Rab24 expression correlated with larger tumor size and advanced tumor stage. Additionally, HCC patients with high Rab24 expression experienced poorer overall survival, with Rab24 identified as an independent prognostic factor. Manipulating Rab24 expression in HCC cell lines demonstrated its role in promoting tumor proliferation. Silencing Rab24 significantly reduced xenograft growth in vivo. This study highlights the significant association between high Rab24 expression and poorer HCC prognosis, suggesting Rab24’s potential as a novel clinical biomarker and therapeutic target.

Evaluating The Cytotoxic Activity of Lactobacillus plantarum IIA-1A5 Against MCF-7 Human Breast Cancer Cells and Identifying Its Surface Layer Protein Gene

Breast cancer is a serious global health concern, with a high mortality rate worldwide. Using natural compounds as potential cancer therapies is a promising approach to address this issue. Previous research has shown that probiotic lactic acid bacteria (LAB) and their metabolites, such as surface layer protein (slp), have a positive impact on a variety of health disorders, including cancer. The purpose of this study was to evaluate the ability of Lactobacillus plantarum IIA-1A5 to suppress the growth of the MCF-7 breast cancer cell line and detect the presence of the slp gene. Intracellular and extracellular protein fractions were isolated from L. plantarum IIA-1A5 cultures. The protein concentrations and molecular weights of the extracts were measured. The anticancer activity of the extracts was assessed using the MTT cytotoxicity test, and IC50 values were calculated. The slp gene was identified through polymerase chain reaction (PCR) amplification and nucleotide sequencing. The results demonstrated that L. plantarum IIA-1A5 had a concentration-dependent inhibitory effect on MCF-7 breast cancer cells, with IC50 values of 6.831 and 12.35 μg/mL for intracellular extracts and extracellular extracts, respectively. Additionally, PCR amplification and nucleotide sequencing confirmed the presence of the slp gene, which may contribute to the strain’s anticancer abilities. These findings suggest the potential of L. plantarum IIA-1A5 as a natural anticancer agent against MCF-7 breast cancer cells. Further research is warranted to elucidate the underlying mechanisms of L. plantarum IIA-1A5 in breast cancer treatment.

Interlaboratory comparison of high-throughput protein biomarker assay quantifications for radiation exposure classification.

In the event of a widespread radiological incident, thousands of individuals will require rapid assessment of exposure using validated biodosimetry assays to inform clinical triage. In this scenario, multiple biodosimetry laboratories may be necessary for large-volume sample processing. To meet this need, we have developed a high-throughput assay for the rapid measurement of intracellular protein biomarkers in human peripheral blood samples using an Imaging Flow Cytometry (IFC) platform. The objective of this work was to harmonize and validate the reproducibility of our blood biomarker assay for radiation exposure across three IFC instruments, two located at Columbia University (CU) and the third at Health Canada. The Center for Radiological Research (CRR) at CU served as the central laboratory and reference instrument, where samples were prepared in triplicate, labeled with two radiation responsive leukocyte biomarkers (BAX and phosphor-p53 (Ser37)), and distributed for simultaneous interrogation by each IFC. Initial tests showed that significantly different baseline biomarker measurements were generated on each instrument when using the same acquisition settings, suggesting that harmonization of signal intensities is necessary. Subsequent tests harmonized biomarker measurements after irradiation by modulating laser intensity using two reference materials: unstained samples and standardized rainbow beads. Both methods generated measurements on each instrument without significant differences between the new and references instruments, allowing for the use of one master template to quantify biomarker expression across multiple instruments. Deming regression analyses of 0-5 Gy dose-response curves showed overall good correlation of BAX and p53 values across new and reference instruments. While Bland-Altman analyses indicated low to moderate instrument biases, ROC Curve analyses ultimately show successful discrimination between exposed and unexposed samples on each instrument (AUC values > 0.85).

Zymolyase Treatment of Saccharomyces cerevisiae Affects Cellular Proteins and Degrades Tyrosyl-DNA Phosphodiesterase I.

Saccharomyces cerevisiae is a genetically tractable, affordable, and extensively documented eukaryotic single-cell model organism. This budding yeast is amenable for the development of genetic and biochemical experiments and is frequently used to investigate the function, activity, and mechanism of mammalian proteins. However, yeast contains a cell wall that hinders select assays including organelle isolation. Lytic enzymes, with Zymolyase as the most effective and frequently used tool, are utilized to weaken the yeast cell wall resulting in yeast spheroplasts. Spheroplasts are easily lysed by, for example, osmotic-shock conditions to isolate yeast nuclei or mitochondria. However, during our studies of the DNA repair enzyme tyrosyl-DNA phosphodiesterase I (Tdp1), we encountered a negative effect of Zymolyase. We observed that Zymolyase treatment affected the steady-state protein levels of Tdp1. This was revealed by inconsistencies in technical and biological replicate lysates of plasmid-born galactose-induced expression of Tdp1. This off-target effect of Zymolyase is rarely discussed in articles and affects a select number of intracellular proteins, including transcription factors and assays such as chromatin immunoprecipitations. Following extensive troubleshooting, we concluded that the culprit is the Ser-protease, Zymolyase B, component of the Zymolyase enzyme mixture that causes the degradation of Tdp1. In this study, we report the protocols we have used, and our final protocol with an easy, affordable adaptation to any assay/protocol involving Zymolyase.

Antimicrobial Activity and Mechanisms of Punicalagin against Vibrio parahaemolyticus.

This study sought to explore the antimicrobial activity of punicalagin against V. parahaemolyticus and its potential modes of action. V. parahaemolyticus ATCC 17802 and RIMD 2210633Sm were exposed to punicalagin, and the energy production, membrane potential, and envelope permeability, as well as the interaction with cell biomolecules, were measured using a variety of fluorescent probes combined with electrophoresis and Raman spectroscopy. Punicalagin treatment disrupted the envelope integrity and induced a decrease in intracellular ATP and pH. The uptake of 1-N-phenyl-naphtylamine (NPN) demonstrated that punicalagin weakened the outer membrane. Punicalagin damaged the cytoplasmic membrane, as indicated by the membrane depolarization and the leakage of intracellular potassium ions, proteins, and nucleic acids. Electronic microscopy observation visualized the cell damage caused by punicalagin. Further, gel electrophoresis coupled with the Raman spectrum assay revealed that punicalagin affected the protein expression of V. parahaemolyticus, and there was no effect on the integrity of genomic DNA. Therefore, the cell envelope and proteins of V. parahaemolyticus were the assailable targets of punicalagin treatment. These findings suggested that punicalagin may be promising as a natural bacteriostatic agent to control the growth of V. parahaemolyticus.

Enhanced Spine Stability and Survival Lead to Increases in Dendritic Spine Density as an Early Response to Local Alpha-Synuclein Overexpression in Mouse Prefrontal Cortex

Lewy Body Dementias (LBD), including Parkinson’s disease dementia and Dementia with Lewy Bodies, are characterized by widespread accumulation of intracellular alpha-Synuclein protein deposits in regions beyond the brainstem, including in the cortex. However, the impact of local pathology in the cortex is unknown. To investigate this, we employed viral overexpression of human alpha-Synuclein protein targeting the mouse prefrontal cortex (PFC). We then used in vivo 2-photon microscopy to image awake head-fixed mice via an implanted chronic cranial window to assess the early consequences of alpha-Synuclein overexpression in the weeks following overexpression. We imaged apical tufts of Layer V pyramidal neurons in the PFC of Thy1-YFP transgenic mice at 1-week intervals from 1 to 2 weeks before and 9 weeks following viral overexpression, allowing analysis of dynamic changes in dendritic spines. We found an increase in the relative dendritic spine density following local overexpression of alpha-Synuclein, beginning at 5 weeks post-injection, and persisting for the remainder of the study. We found that alpha-Synuclein overexpression led to an increased percentage and longevity of newly-persistent spines, without significant changes in the total density of newly formed or eliminated spines. A follow-up study utilizing confocal microscopy revealed that the increased spine density is found in cortical cells within the alpha-Synuclein injection site, but negative for alpha-Synuclein phosphorylation at Serine-129, highlighting the potential for effects of dose and local circuits on spine survival. These findings have important implications for the physiological role and early pathological stages of alpha-Synuclein in the cortex.Graphical

Chemical Plasma Membrane Perforation Generated by a Microfluidic Probe for Single-Cell Intracellular Protein Delivery.

Efficient and convenient delivery of exogenous molecules into cells is important for cell biology research. However, many intracellular delivery methods require carrier-mediated or physical field assistance, complicating the delivery process. Here, a general, simple, and effective method for in situ single-cell intracellular delivery is reported. A solution containing digitonin and cargo is precisely applied to single cells using a microfluidic probe. Digitonin binds to cholesterol in the plasma membrane to induce perforation, and the cargo enters the cell through the pore. By optimizing parameters, propidium iodide (0.67 kDa) and FITC-dextran (10, 40, and 150 kDa) can be successfully introduced into single cells within 3 min while maintaining cell viability. To prove the potential of this method for cell research, we delivered cytochrome C (13 kDa) and cyclophilin A (18 kDa) into cells by this method. The delivered cytochrome C successfully induces cell apoptosis by activating the caspase pathway, and cyclophilin A performs an antioxidant effect in the cells, which may enhance the drug resistance of glioma cells. It is believed that this method will be an attractive tool for single-cell intracellular delivery.

PKIB, a Novel Target for Cancer Therapy.

The serine-threonine kinase protein kinase A (PKA) is a cyclic AMP (cAMP)-dependent intracellular protein with multiple roles in cellular biology including metabolic and transcription regulation functions. The cAMP-dependent protein kinase inhibitor β (PKIB) is one of three known endogenous protein kinase inhibitors of PKA. The role of PKIB is not yet fully understood. Hormonal signaling is correlated with increased PKIB expression through genetic regulation, and increasing PKIB expression is associated with decreased cancer patient prognosis. Additionally, PKIB impacts cancer cell behavior through two mechanisms; the first is the nuclear modulation of transcriptional activation and the second is the regulation of oncogenic AKT signaling. The limited research into PKIB indicates the oncogenic potential of PKIB in various cancers. However, some studies suggest a role of PKIB in non-cancerous disease states. This review aims to summarize the current literature and background of PKIB regarding cancer and related issues. In particular, we will focus on cancer development and therapeutic possibilities, which are of paramount interest in PKIB oncology research.

Hypoxia-Responsive Hydrogen-Bonded Organic Framework-Mediated Protein Delivery for Cancer Therapy.

The efficient delivery of therapeutic proteins to tumor sites is a promising cancer treatment modality. Hydrogen-bonded organic frameworks (HOFs) have been successfully used for the protective encapsulation of proteins; however, easy precipitation and lack of controlled release of existing HOFs limit their further application for protein delivery in vivo. In this study, a hypoxia-responsive HOF, self-assembled from azobenzenedicarboxylate/polyethylene glycol-conjugated azobenzenedicarboxylate and tetrakis(4-amidiniumphenyl)methane through charge-assisted hydrogen-bonding, is developed for systemic protein delivery to tumor cells. The newly generated HOF platform efficiently encapsulates representative cytochrome C, demonstrating good dispersibility under physiological conditions. Moreover, it can respond to overexpressed reductases in the cytoplasm under hypoxic conditions, inducing fast intracellular protein release to exert therapeutic effects. The strategy presented herein can be applied to other therapeutic proteins and can be expanded to encompass more intrinsic tumor microenvironment stimuli. This offers a novel avenue for utilizing HOFs in protein-based cancer therapy. This article is protected by copyright. All rights reserved.

Flotillins affect LPS-induced TLR4 signaling by modulating the trafficking and abundance of CD14

Lipopolysaccharide (LPS) induces a strong pro-inflammatory reaction of macrophages upon activation of Toll-like receptor 4 (TLR4) with the assistance of CD14 protein. Considering a key role of plasma membrane rafts in CD14 and TLR4 activity and the significant impact exerted on that activity by endocytosis and intracellular trafficking of the both LPS acceptors, it seemed likely that the pro-inflammatory reaction could be modulated by flotillins. Flotillin-1 and -2 are scaffolding proteins associated with the plasma membrane and also with endo-membranes, affecting both the plasma membrane dynamics and intracellular protein trafficking. To verify the above hypothesis, a set of shRNA was used to down-regulate flotillin-2 in Raw264 cells, which were found to also become deficient in flotillin-1. The flotillin deficiency inhibited strongly the TRIF-dependent endosomal signaling of LPS-activated TLR4, and to a lower extent also the MyD88-dependent one, without affecting the cellular level of TLR4. The flotillin depletion also inhibited the pro-inflammatory activity of TLR2/TLR1 and TLR2/TLR6 but not TLR3. In agreement with those effects, the depletion of flotillins down-regulated the CD14 mRNA level and the cellular content of CD14 protein, and also inhibited constitutive CD14 endocytosis thereby facilitating its shedding. Ultimately, the cell-surface level of CD14 was markedly diminished. Concomitantly, CD14 recycling was enhanced via EEA1-positive early endosomes and golgin-97-positive trans-Golgi network, likely to compensate for the depletion of the cell-surface CD14. We propose that the paucity of surface CD14 is the reason for the down-regulated signaling of TLR4 and the other TLRs depending on CD14 for ligand binding.Graphical abstract