Search

Quantitative targeted absolute proteomics (QTAP) quantifies proteins by measuring the signature peptides produced from target proteins by trypsin digestion. The selection of signature peptides is critical for reliable peptide quantification. The purpose of this study was to comprehensively assess the digestion efficiency and stability of tryptic peptides and to identify optimal signature peptides for human hepatic transporters and membrane marker proteins.The plasma membrane fraction of the human liver was digested at different time points and the peptides were comprehensively quantified using quantitative proteomics. Transporters and membrane markers were quantified using the signature peptides by QTAP.Tryptic peptides were classified into clusters with low digestion efficiency, low stability, and high digestion efficiency and stability. Using the cluster information, we found that a proline residue next to the digestion site or the peptide position in or close to the transmembrane domains lowers digestion efficiency. A peptide containing cysteine at the N-terminus or arginine-glycine lowers peptide stability. Based on this information and the time course of peptide quantification, optimal signature peptides were identified for human hepatic transporters and membrane markers. The quantification of transporters with multiple signature peptides yielded consistent absolute values with less than 30% of coefficient variants in human liver microsomes and homogenates.The signature peptides selected in the present study enabled the reliable quantification of human hepatic transporters. The QTAP protocol using these optimal signature peptides provides quantitative data on hepatic transporters usable for integrated pharmacokinetic studies.

P-glycoprotein, the encoded product of the MDR1 / ABCB1 gene in humans, is expressed in numerous tissues including brain capillary endothelial cells and restricts the distribution of xenobiotics into the brain as an efflux pump. Although a large number of single nucleotide polymorphisms in the MDR1 gene have been identified, the influence of the nonsynonymous 2677GT/A single nucleotide polymorphism on P-glycoprotein at the blood-brain barrier has remained unclear. In the present study, we developed a novel P-glycoprotein humanized mouse line carrying the 2677GT mutation by utilizing a mouse artificial chromosome vector constructed by genetic engineering technology and we evaluated the influence of 2677GT on the expression and function of P-glycoprotein at the blood-brain barrier in vivo . The results of this study showed that the introduction of the 2677GT mutation does not alter the expression levels of P-glycoprotein protein in the brain capillary fraction. On the other hand, the brain penetration of verapamil, a representative substrate of P-glycoprotein, was increased by the introduction of the 2677GT mutation. These results suggested that the 2677GT single nucleotide polymorphism may attenuate the function of P-glycoprotein, resulting in increased brain penetration of P-glycoprotein substrates, without altering the expression levels of P-glycoprotein protein in the blood-brain barrier. This mutant mouse line is a useful model for elucidating the influence of an MDR1 gene single nucleotide polymorphism on the expression and function of P-glycoprotein at the blood-brain barrier.

Circadian rhythms influence the transport function of the blood-brain barrier (BBB) and peripheral organs. However, the influence of circadian rhythms on protein expression in the BBB remains to be completely elucidated. Therefore, we aimed to investigate diurnal changes in protein expression in the mouse BBB using quantitative proteomics. Quantitative proteomics showed that the expression of 67, 10, and 20 proteins in the isolated mouse brain capillary fraction changed significantly at zeitgeber time (ZT) 6, 12, and 18, respectively, compared to ZT0. Among them, the levels of 44 proteins were significantly increased at ZT6 and then returned to the same level as ZT0 at ZT12 and ZT18. Gene ontology analysis indicated that the proteins significantly increased at ZT6 were majorly related to translation. The brain capillary endothelial cell-selective proteins sepiapterin reductase and vascular endothelial growth factor receptor 2 showed diurnal variation. In contrast, the expression of ABC transporters, SLC transporters, and receptors associated with receptor-mediated transcytosis, and tight junction proteins did not change within a day. The present findings demonstrated that protein expression related to transport function and physical barrier at the BBB was maintained throughout the day, although the proteins involved in some biological processes exhibited diurnal variation at the BBB.

Supplementary Figure S2 shows the selectivity of JQKD82 against KDM5 in biochemical assay and in MM cells

Lysine demethylase 5A (KDM5A) is a negative regulator of histone H3 lysine 4 trimethy­lation (H3K4me3), a histone mark associated with activate gene transcription. We identify that KDM5A interacts with the P-TEFb complex and cooperates with MYC to control MYC-targeted genes in multiple myeloma cells. We develop a cell-permeable and selective KDM5 inhibitor, JQKD82, that increases H3K4me3 but paradoxically inhibits downstream MYC-driven transcriptional output in vitro and in vivo. Using genetic ablation together with our inhibitor, we establish that KDM5A supports MYC target gene transcription independent of MYC itself by supporting TFIIH (CDK7)- and P-TEFb (CDK9)–mediated phosphorylation of RNAPII. These data identify KDM5A as a unique vulnerability in multiple myeloma functioning through regulation of MYC target gene transcription and establish JQKD82 as a tool compound to block KDM5A function as a potential therapeutic strategy for multiple myeloma.Significance:We delineate the function of KDM5A in activating the MYC-driven transcriptional landscape. We develop a cell-permeable KDM5 inhibitor to define the activating role of KDM5A on MYC target gene expression and implicate the therapeutic potential of this compound in mouse models and multiple myeloma patient samples.See related video from the AACR Annual Meeting 2021: https://vimeo.com/554896826

Supplementary Table S2 shows the ChIP-seq data with the peaks significantly overlap with KDM5A ChIP-seq peaks MM1S

Supplementary Methods, compound synthesis, Supplementary Materials and Supplementary References

Figure S1. Overview of the CRISPR/Cas9 screen performed in EGFR mutation positive NSCLC PC9 cells. Figure S2. Cell viability assay of cytotoxic agents or ALK-TKI in SHOC2-depleted cells. Figure S3. Supplementary data for domain analysis. Figure S4. Supplementary data for phospho-proteomics analysis. Figure S5. Cell viability assay of celastrol in PC9 and H1975 cells. Figure S6. Supplementary data for xenograft model. Supplemental Table Legends 1-3

EGFR mutation-positive patients with non–small cell lung cancer (NSCLC) respond well to treatment with EGFR–tyrosine kinase inhibitors (EGFR–TKI); however, treatment with EGFR–TKIs is not curative, owing to the presence of residual cancer cells with intrinsic or acquired resistance to this class of drugs. Additional treatment targets that may enhance the efficacy of EGFR–TKIs remain elusive. Using a CRISPR/Cas9-based screen, we identified the leucine-rich repeat scaffold protein SHOC2 as a key modulator of sensitivity to EGFR–TKI treatment. On the basis of in vitro assays, we demonstrated that SHOC2 expression levels strongly correlate with the sensitivity to EGFR–TKIs and that SHOC2 affects the sensitivity to EGFR–TKIs in NSCLC cells via SHOC2/MRAS/PP1c and SHOC2/SCRIB signaling. The potential SHOC2 inhibitor celastrol phenocopied SHOC2 depletion. In addition, we confirmed that SHOC2 expression levels were important for the sensitivity to EGFR–TKIs in vivo. Furthermore, IHC showed the accumulation of cancer cells that express high levels of SHOC2 in lung cancer tissues obtained from patients with NSCLC who experienced acquired resistance to EGFR–TKIs. These data indicate that SHOC2 may be a therapeutic target for patients with NSCLC or a biomarker to predict sensitivity to EGFR–TKI therapy in EGFR mutation-positive patients with NSCLC. Our findings may help improve treatment strategies for patients with NSCLC harboring EGFR mutations.Implications:This study showed that SHOC2 works as a modulator of sensitivity to EGFR–TKIs and the expression levels of SHOC2 can be used as a biomarker for sensitivity to EGFR–TKIs.

A list of sgRNAs in a small-scale custom library

The insulin receptor (INSR) mediates insulin signaling to modulate cellular functions. Although INSR is expressed at the blood–brain barrier (BBB), its role in the modulation of BBB function is poorly understood. Therefore, in this study, we aimed to analyze the effect of INSR knockdown on the expression levels of functional proteins at the BBB. We established the INSR-knockdown cell line (shINSR) using human cerebral microvascular endothelial cells (hCMEC/D3). The cellular proteome was analyzed using quantitative proteomics. INSR mRNA and protein expressions were decreased in shINSR cells. The suppression of INSR-mediated signaling in shINSR cells was evaluated. The proteins involved in glycolysis and glycogenolysis were suppressed in shINSR cells. As amyloid-β peptide-related proteins, the expressions of presenilin-1 was increased, and those of the insulin-degrading enzyme and neprilysin were decreased. The expressions of BBB transporters, including the ABCB1/MDR1, ABCG2/BCRP, and SLCO2A1/OATP2A1 were significantly decreased by more than 50% in shINSR cells. The efflux activity of ABCB1/MDR1 was also suppressed. The expressions of the low-density lipoprotein receptor-related protein 1 were significantly increased, and those of the transferrin receptor were significantly decreased in shINSR cells. The expression of claudin-5 was also suppressed in shINSR cells. The present study suggests that INSR-mediated signaling is involved in the regulation of functional protein expression at the BBB and contributes to the maintenance of BBB function. Changes in the expressions of amyloid-β peptide-related proteins may contribute to the development of cerebral amyloid angiopathy via the suppression of INSR-mediated signaling.

Here we examined the effect of nicotine on angiogenesis in the brain after intracerebral hemorrhage (ICH), as angiogenesis is considered to provide beneficial effects on brain tissues during recovery from injury after stroke. Nicotine was administered to C57BL/6 mice suffering from collagenase-induced ICH in the striatum, either by inclusion in drinking water or by daily intraperitoneal injection. Nicotine administration by both routes enhanced angiogenesis within the hematoma-affected regions, as revealed by increased CD31-immunopositive area at 7 and 14 d after ICH. Double immunofluorescence histochemistry against CD31 and proliferating cell nuclear antigen revealed that nicotine increased the number of newly generated vascular endothelial cells within the hematoma. In spite of enhanced angiogenesis, nicotine did not worsen vascular permeability after ICH, as assessed by Evans Blue extravasation. These effects of nicotine were accompanied by an increased number of surviving neurons in the hematoma at 7 d after ICH. Unexpectedly, nicotine did not increase expression of vascular endothelial growth factor mRNA in the brain and did not enhance recruitment of endothelial progenitor cells from the bone marrow. These results suggest that nicotine enhances angiogenesis in the brain after ICH, via mechanisms distinct from those involved in its action on angiogenesis in peripheral tissues.

Prototypic antidepressants, such as tricyclic/tetracyclic antidepressants (TCAs), have multiple pharmacological properties and have been considered to be more effective than newer antidepressants, such as selective serotonin reuptake inhibitors (SSRIs), in treating severe depression. However, the molecular mechanisms underlying the high efficacy of TCAs have not been completely understood. Herein, we found that lysophosphatidic acid receptor 1 (LPAR1), a G protein-coupled receptor, mediates the antidepressant effects of amitriptyline, a typical TCA. Amitriptyline directly bound to LPAR1 and activated downstream G protein signaling without affecting β-arrestin signaling, which implied that amitriptyline could act as a G protein-biased agonist of LPAR1. This biased agonism is unique to TCAs and has not been observed in other antidepressants, such as SSRIs. Long-term infusion of mouse hippocampus with 1-oleoyl-2-O-methyl-glycerophosphothionate (OMPT), a potent G protein-biased LPAR1 agonist, induced behavior similar to that induced by antidepressants. In contrast, LPA, a non-biased agonist of LPAR1, induced anxious behavior, indicating that LPAR1 may regulate conflicting emotional behaviors because of the downstream signaling bias. Furthermore, RNA-seq analysis revealed that LPA and OMPT have opposite patterns of gene expression changes in hippocampus. Ingenuity pathway analysis indicated that chronic intrahippocampal administration of OMPT could activate LPAR1 downstream signaling (Rho and MAPK), whereas LPA suppressed LPAR1 signaling. The results reveal the unique antidepressant effects of TCAs and indicate the potential of G protein-biased agonists of LPAR1 as targets for novel antidepressants.

The post-translational acetylation of lysine residues is found in many nonhistone proteins and is involved in a wide range of biological processes. Recently, we showed that the nucleoprotein of the influenza A virus is acetylated by histone acetyltransferases (HATs), a phenomenon that affects viral transcription. Here, we report that the PA subunit of influenza A virus RNA-dependent RNA polymerase is acetylated by the HATs, P300/CREB-binding protein-associated factor (PCAF), and general control nonderepressible 5 (GCN5), resulting in accelerated endonuclease activity. Specifically, the full-length PA subunit expressed in cultured 293T cells was found to be strongly acetylated. Moreover, the partial recombinant protein of the PA N-terminal region containing the endonuclease domain was also acetylated by PCAF and GCN5 in vitro, which facilitated its endonuclease activity. Mass spectrometry analyses identified K19 as a candidate acetylation target in the PA N-terminal region. Notably, the substitution of the lysine residue at position 19 with glutamine, a mimic of the acetyl-lysine residue, enhanced its endonuclease activity in vitro; this point mutation also accelerated influenza A virus RNA-dependent RNA polymerase activity in the cell. Our findings suggest that PA acetylation is important for the regulation of the endonuclease and RNA polymerase activities of the influenza A virus.

Recent advances in single-cell proteomics highlight the promise of sensitive analyses in limited cell populations. However, technical challenges remain for sample recovery, throughput, and versatility. Here, we first report a water droplet-in-oil digestion (WinO) method based on carboxyl-coated beads and phase transfer surfactants for proteomic analysis using limited sample amounts. This method was developed to minimize the contact area between the sample solution and the container to reduce the loss of proteins and peptides by adsorption. This method increased protein and peptide recovery 10-fold. The proteome profiles obtained from 100 cells using the WinO method highly correlated with those from 10,000 cells using the in-solution digestion method. We successfully applied the WinO method to single-cell proteomics and quantified 462 proteins. Using the WinO method, samples can be easily prepared in a multi-well plate, making it a widely applicable and suitable method for single-cell proteomics.

Blood-brain barrier (BBB)-permeable middle- or macromolecules (middle/macromolecules) have recently attracted significant attention as new drug delivery carriers into the human brain via receptor-mediated transcytosis (RMT). During the development process of such carriers, it is necessary to thoroughly evaluate their human BBB permeability levels. In such evaluations, our recently established human immortalized cell-based multicellular spheroidal BBB models (hiMCS-BBB models) have shown high potential. However, the specifics of those capabilities have yet to be elucidated. Therefore, in this study, we characterize the ability of the hiMCS-BBB models to evaluate RMT-mediated BBB penetration properties of middle/macromolecules. More specifically, we began by validating transferrin receptor (TfR)-mediated RMT functionalities using transferrin in the hiMCS-BBB models and then examined the BBB permeability levels of MEM189 antibodies (known BBB-permeable anti-TfR antibodies). The obtained results showed that, as with the case of transferrin, temperature-dependent uptake of MEM189 antibodies was observed in the hiMCS-BBB models, and the extent of that uptake increased in a time-dependent manner until reaching a plateau after around 2 h. To further expand the evaluation applicability of the models, we also examined the BBB permeability levels of the recently developed SLS cyclic peptide and observed that peptide uptake was also temperature-dependent. To summarize, our results show that the hiMCS-BBB models possess the ability to evaluate the RMT-mediated BBB-permeable properties of antibodies and peptides and thus have the potential to provide valuable tools for use in the exploration and identification of middle/macromolecules showing excellent BBB permeability levels, thereby contributing powerfully to the development of new drug delivery carriers for transporting drugs into the human brain.

Metformin is widely used for the treatment of type 2 diabetes, and increasing numbers of studies have shown that metformin also ameliorates tumor progression, inflammatory disease, and fibrosis. However, the ability of metformin to improve non-diabetic glomerular disease and chronic kidney disease (CKD) has not been explored. To investigate the effect of metformin on non-diabetic glomerular disease, we used a mouse model of Alport syndrome (Col4a5 G5X) which were treated with metformin or losartan, used as a control treatment. We also investigated the effect of metformin on adriamycin-induced glomerulosclerosis model. Pathological and biochemical analysis showed that metformin or losartan suppressed proteinuria, renal inflammation, fibrosis, and glomerular injury and extended the lifespan in Alport syndrome mice. Transcriptome analysis showed that metformin and losartan influenced molecular pathways-related to metabolism and inflammation. Metformin altered multiple genes including metabolic genes not affected by losartan. Metformin also suppressed proteinuria and glomerular injury in the adriamycin-induced glomerulosclerosis mouse model. Our results showed that metformin ameliorates the glomerular sclerosis and CKD phenotype in non-diabetic chronic glomerular diseases. Metformin may have therapeutic potential for not only diabetic nephropathy but also non-diabetic glomerular disease including Alport syndrome.

In predicting the hepatic elimination of compounds, the extended clearance concept has proven useful. Yet, its experimental proof was scarce partly due to the lack of models with the controlled expression of transporters. Here, the uptake and efflux transporters [NTCP (SLC10A1) and BSEP (ABCB11), respectively] were doubly and transiently expressed in MDCKII cells by electroporation-based transfection (with the BSEP plasmid amount varied and with the NTCP plasmid fixed), achieving the activity levels of NTCP and BSEP comparable to those of sandwich cultured human hepatocytes. The biliary excretion clearance for taurocholate increased proportionally to the BSEP expression level. Under the same conditions, the basal-to-apical transcellular clearance of taurocholate displayed an initial increase, and a subsequent plateau, indicating that the basolateral uptake of taurocholate became rate-limiting. The doubly transfected MDCKII cells were also used to kinetically analyze the inhibitory effects of rifampicin on BSEP and NTCP. The obtained results showed a bell-shaped profile for cell-to-medium concentration ratios over a range of rifampicin concentrations, which were quantitatively captured by kinetic modeling based on the extended clearance concept. The present study highlights the utility of the transient, tunable transporter expression system in delineating the rate-determining process and providing mechanistic insights into intracellular substrate accumulation.

Targeted proteomics is a method that measures the amount of target proteins via liquid chromatography-tandem mass spectrometry and is used to verify and validate the candidate cancer biomarker proteins. Compared with antibody-based quantification methods such as ELISA, targeted proteomics enables rapid method development, simultaneous measurement of multiple proteins, and high-specificity detection of modifications. Moreover, by spiking the internal standard peptide, targeted proteomics detects the absolute amounts of marker proteins, which is essential for determining the cut-off values for diagnosis and thus for multi-institutional validation. With these unique features, targeted proteomics can seamlessly transfer cancer biomarker candidate proteins from the discovery phase to the verification and validation phases, thereby resulting in an accelerated cancer biomarker pipeline. Furthermore, understanding the basic principles, advantages, and disadvantages is necessary to effectively utilize targeted proteomics in cancer biomarker pipelines. This review aimed to introduce the technical principles of targeted proteomics for cancer biomarker verification and validation.

Hemoglobin-vesicles (Hb-V), hemoglobin encapsulated within a liposome, were developed as an artificial red blood cell (RBC). When Hb-V becomes clinically available in the future, patients would presumably be co-administered with one or more drugs. Since drug-drug interactions can cause serious adverse effects and impede overall curative effects, evidence regarding the risk associated with drug-drug interactions between Hb-V and such simultaneously administered drugs is needed. Therefore, we report on cytochrome P450 (CYP)-based drug interactions with Hb-V in healthy rats. At 1 day after the saline, Hb-V or packed RBC (PRBC) administration, the blood retention of CYP-metabolizing drugs (caffeine, chlorzoxazone, tolbutamide and midazolam) were moderately prolonged in the case of the Hb-V group, but not the PRBC group, compared to saline group. The results of a proteome analysis revealed that the Hb-V administration had only negligible effects on the protein expression of CYPs in the liver. Hb-V administration, however, clearly suppressed the CYP metabolic activity of the four target CYP isoforms compared with the saline and PRBC group. However, these alterations were nearly recovered at 7 day after the Hb-V administration. Taken together, these results suggest that the administration of Hb-V slightly and transiently affects the CYP-based metabolism of the above drugs.

Quantifying the protein levels of drug transporters in plasma membrane fraction helps elucidate the function of these transporters. In this study, we conducted a proteomic evaluation of enriched drug-related transporter proteins in plasma membrane fraction prepared from mouse liver and kidney tissues using the membrane protein extraction kit and a bead homogenizer. Crude and plasma membrane fractions were prepared using either the Dounce or bead homogenizer, and protein levels were determined using quantitative proteomics. In liver tissues, the plasma membrane fractions were more enriched in transporter proteins than the crude membrane fractions; the average enrichment ratios of plasma-to-crude membrane fractions were 3.31 and 6.93 using the Dounce and bead homogenizers, respectively. The concentrations of transporter proteins in plasma membrane fractions determined using the bead homogenizer were higher than those determined using the Dounce homogenizer. Meanwhile, in kidney tissues, the plasma membrane fractions were enriched in transporters localized in the brush-border membrane to the same degree for both the homogenizers; however, the membrane fractions obtained using either homogenizer were not enriched in Na+/K+-ATPase and transporters localized in the basolateral membrane. These results indicate that fractionation, using the bead homogenizer, yielded transporter-enriched plasma membrane fractions from mouse liver and kidney tissues; however, no enrichment of basolateral transporters was observed in plasma membrane fractions prepared from kidney tissues.

Leucine-rich α-2 glycoprotein 1 (LRG1) is a diagnostic marker candidate for glioblastoma. Although LRG1 has been associated with angiogenesis, it has been suggested that its biomarker role differs depending on the type of tumor. In this study, a clinicopathological examination of LRG1’s role as a biomarker for glioblastoma was performed. We used tumor tissues of 155 cases with diffuse gliomas (27 astrocytomas, 14 oligodendrogliomas, 114 glioblastomas). The immunohistochemical LRG1 intensity scoring was classified into 2 groups: low expression and high expression. Mutations of IDH1, IDH2, and TERT promoter were analyzed through the Sanger method. We examined the relationship between LRG1 expression level in glioblastoma and clinical parameters, such as age, preoperative Karnofsky performance status, tumor location, extent of resection, O6-methylguanine DNA methyltransferase promoter, and prognosis. LRG1 high expression rate was 41.2% in glioblastoma, 3.7% in astrocytoma, and 21.4% in oligodendroglioma. Glioblastoma showed a significantly higher LRG1 expression than lower-grade glioma (p = 0.0003). High expression of LRG1 was an independent favorable prognostic factor (p = 0.019) in IDH-wildtype glioblastoma and correlated with gross total resection (p = 0.002) and the tumor location on nonsubventricular zone (p = 0.00007). LRG1 demonstrated multiple potential as a diagnostic, prognostic, and regional biomarker for glioblastoma.

Brain delivery of nanoparticles and macromolecular drugs depends on blood-brain barrier (BBB)-permeable carriers. In this study, we searched for cyclic heptapeptides facilitating BBB permeation of M13 phages by phage library screening using a transcellular permeability assay with hCMEC/D3 cell monolayers, a human BBB model. The M13 phage, which is larger than macromolecular drugs and nanoparticles, served as a model macromolecule. The screen identified cyclic heptapeptide SLSHSPQ (SLS) as a human BBB-permeable peptide. The SLS-displaying phage (SLS-phage) exhibited improved permeation across the cell monolayer of monkey and rat BBB co-culture models. The SLS-phage internalized into hCMEC/D3 cells via macropinocytosis and externalized via the exosome excretion pathway. SLS-phage distribution into brain parenchyma was observed in mice after intravenous administration. Moreover, liposome permeated across the BBB as cyclic SLS peptide conjugates. In conclusion, the cyclic SLS heptapeptide is a novel carrier candidate for brain delivery of macromolecular drugs and nanoparticles.

The extracellular matrix (ECM) initiates mechanical cues that activate intracellular signaling through matrix–cell interactions. In blood vessels, additional mechanical cues derived from the pulsatile blood flow and pressure play a pivotal role in homeostasis and disease development. Currently, the nature of the cues from the ECM and their interaction with the mechanical microenvironment in large blood vessels to maintain the integrity of the vessel wall are not fully understood. Here, we identified the matricellular protein thrombospondin-1 (Thbs1) as an extracellular mediator of matrix mechanotransduction that acts via integrin αvβ1 to establish focal adhesions and promotes nuclear shuttling of Yes-associated protein (YAP) in response to high strain of cyclic stretch. Thbs1-mediated YAP activation depends on the small GTPase Rap2 and Hippo pathway and is not influenced by alteration of actin fibers. Deletion of Thbs1 in mice inhibited Thbs1/integrin β1/YAP signaling, leading to maladaptive remodeling of the aorta in response to pressure overload and inhibition of neointima formation upon carotid artery ligation, exerting context-dependent effects on the vessel wall. We thus propose a mechanism of matrix mechanotransduction centered on Thbs1, connecting mechanical stimuli to YAP signaling during vascular remodeling in vivo.

Single-chain Fv (scFv) is a recombinant antibody in which the variable regions of the heavy chain (VH) and light chain (VL) are connected by a short flexible polypeptide linker. Compared with monoclonal antibodies, scFvs have the advantages of low-cost production using Escherichia coli and easy genetic manipulation. ScFvs are, therefore, regarded as useful modules for producing next-generation medical antibodies. The practical use of scFvs has been limited due to their aggregation propensity mediated by interchain VH–VL interactions. To overcome this problem, we recently reported a cyclic scFv whose N-terminus and C-terminus were connected by sortase A-mediated ligation. Preparation of cyclic scFv is, however, a time-consuming process. To accelerate the application study of cyclic scFv, we developed a method to produce cyclic scFv by the combined use of a protein ligation technique based on protein trans-splicing reaction (PTS) by split intein and a chaperone co-expression system. This method allows for the preparation of active cyclic scFv from the cytoplasm of E. coli. The present method was applied to the production of cyclic 73MuL9-scFv, a GA-pyridine antibody, as a kind of advanced glycation end-product. These findings are expected to evoke further application study of cyclic scFv.

OBJECTIVE An extracellular matrix such as collagen is an essential component of the tumor microenvironment. Collagen alpha-2(I) chain (COL1A2) is a chain of type I collagen whose triple helix comprises two alpha-1 chains and one alpha-2 chain. The authors’ proteomics data showed that COL1A2 is significantly higher in the blood of patients with glioblastoma (GBM) compared with healthy controls. COL1A2 has many different functions in various types of cancers. However, the functions of COL1A2 in GBM are poorly understood. In this study, the authors analyzed the functions of COL1A2 and its signaling pathways in GBM. METHODS Surgical specimens and GBM cell lines (T98, U87, and U251) were used. The expression level of COL1A2 was examined using GBM tissues and normal brain tissues by quantitative real-time polymerase chain reaction. The clinical significance of these levels was evaluated using Kaplan-Meier analysis. Small interfering RNA (siRNA) and small hairpin RNA of COL1A2 were transfected into GBM cell lines to investigate the function of COL1A2 in vitro and in vivo. Flow cytometry was introduced to analyze the alteration of cell cycles. Western blot and immunohistochemistry were performed to analyze the underlying mechanisms. RESULTS The expression level of COL1A2 was upregulated in GBM compared with normal brain tissues. A higher expression of COL1A2 was correlated with poor progression-free survival and overall survival. COL1A2 inhibition significantly suppressed cell proliferation in vitro and in vivo, likely due to G1 arrest. The invasion ability was notably deteriorated by inhibiting COL1A2. Cyclin D1, cyclin-dependent kinase 1, and cyclin-dependent kinase 4, which are involved in the cell cycle, were all downregulated after blockade of COL1A2 in vitro and in vivo. Phosphoinositide 3-kinase inhibitor reduced the expression of COL1A2. Although downregulation of COL1A2 decreased the protein kinase B (Akt) phosphorylation, Akt activator can phosphorylate Akt in siRNA-treated cells. This finding suggests that Akt phosphorylation is partially dependent on COL1A2. CONCLUSIONS COL1A2 plays an important role in driving GBM progression. COL1A2 inhibition attenuated GBM proliferation by promoting cell cycle arrest, indicating that COL1A2 could be a promising therapeutic target for GBM treatment.

Podocalyxin (PODXL) is a highly sialylated transmembrane protein that is expressed on the luminal membrane of brain microvascular endothelial cells. To clarify the role of PODXL in the blood-brain barrier (BBB), the present study aimed to investigate the effect of PODXL-knockdown on protein expression, especially the expression of ABCB1/MDR1, in human microvascular endothelial cells (hCMEC/D3). By quantitative proteomics, gene ontology enrichment with differentially expressed proteins showed that PODXL-knockdown influenced the immune response and intracellular trafficking. Among transporters, the protein expression of ABCB1/MDR1 and ABCG2/BCRP was significantly elevated by approximately 2-fold in the PODXL-knockdown cells. In the knockdown cells, the efflux activity of ABCB1/MDR1 was significantly increased, while its mRNA expression was not significantly different from that of the control cells. As receptors and tight junction proteins, levels of low-density lipoprotein receptor-related protein 1 and occludin were significantly increased, while those of transferrin receptor and claudin-11 were significantly decreased in the knockdown cells. The present results suggest that PODXL functions as a modulator of BBB function, including transport, tight junctions, and immune responses. Furthermore, PODXL post-transcriptionally regulates the protein expression and efflux activity of ABCB1/MDR1 at the BBB, which may affect drug distribution in the brain.

Recent advances in single-cell proteomics highlight the promise of sensitive analyses in limited cell populations. However, technical challenges remain for sample recovery, throughput, and versatility. Here, we first report a water droplet-in-oil digestion (WinO) method based on carboxyl-coated beads and phase transfer surfactants for proteomic analysis using limited sample amounts. This method was developed to minimize the contact area between the sample solution and the container to reduce the loss of proteins and peptides by adsorption. This method increased protein and peptide recovery 10-fold as well as the number of quantified transmembrane proteins compared to an in-solution digestion (ISD) method. The proteome profiles obtained from 100 cells using the WinO method highly correlated with those from 10000 cells using the ISD method. We successfully applied the WinO method to single-cell proteomics and quantified 462 proteins. Using the WinO method, samples can be easily prepared in a multi-well plate, making it a widely applicable and suitable method for single-cell proteomics.

Metastasis of cancer cells to lymph nodes (LN) is a common modality of metastasis in clinical settings, but the mechanisms involved in lymphatic metastasis remain unclear compared to hematogenous metastasis to bones and the brain. To elucidate the molecular mechanisms responsible for melanoma LN metastasis, we first generated LN metastasis-prone melanoma cells (C8161F2) by the sequential in vivo transplantation of parental melanoma cells (C8161F0). Although the in vitro/in vivo proliferative potential of these melanoma cells were similar, the metastatic potential of the C8161F2 for LNs was significantly enhanced. We then conducted a proteomics analysis to identify the proteins and pathways that contribute to LN metastasis. We identified six proteins (three: up-regulated and three: down-regulated) whose expressions were statistically significantly different by more than 2-fold in the two cell groups. Some of these genes are responsible for the activation of the transforming growth factor-β (TGF-β)-related pathway, a well-known inducer of epithelial-mesenchymal transition (EMT). In addition, a gene ontology analysis revealed that the enhanced cell-cell adhesion appears to be involved in lymphatic metastasis. In conclusion, we established highly lymphatic metastatic melanoma cells, which would be valuable for studies of the molecular mechanisms responsible for lymphatic metastasis.

Histone methyltransferase SETD1A is critical for acute myeloid leukemia (AML) cell survival, but the molecular mechanism driving SETD1A gene regulation remains elusive. To delineate the role of SETD1A, we utilize a protein degrader technology to induce rapid SETD1A degradation in AML cell lines. SETD1A degradation results in immediate downregulation of transcripts associated with DNA repair and heme biosynthesis pathways. CRISPR-based functional analyses and metabolomics reveal an essential role of SETD1A to maintain mitochondrial respiration in AML cells. These SETD1A targets are enriched in head-to-head (H2H) genes. SETD1A degradation disrupts a non-enzymatic SETD1A domain-dependent cyclin K function, increases the Ser5P RNA polymerase II (RNAPII) at the transcriptional start site (TSS), and induces the promoter-proximal pausing of RNAPII in a strand-specific manner. This study reveals a non-enzymatic role for SETD1A in transcriptional pause release and provides insight into the mechanism of RNAPII pausing and its function in cancer.

Uncontrolled proliferation of intestinal epithelial cells caused by mutations in genes of the WNT/β-catenin pathway is associated with development of intestinal cancers. We previously reported that intestinal stromal cell-derived angiopoietin-like protein 2 (ANGPTL2) controls epithelial regeneration and intestinal immune responses. However, the role of tumor cell-derived ANGPTL2 in intestinal tumorigenesis remained unclear. Here, we show that tumor cell-derived ANGPTL2 promotes β-catenin-driven intestinal tumorigenesis. ANGPTL2 deficiency suppressed intestinal tumor development in an experimental mouse model of sporadic colon cancer. We also found that increased ANGPTL2 expression in colorectal cancer (CRC) cells augments β-catenin pathway signaling and promotes tumor cell proliferation. Relevant to mechanism, our findings suggest that tumor cell-derived ANGPTL2 upregulates expression of OB-cadherin, which then interacts with β-catenin, blocking destruction complex-independent proteasomal degradation of β-catenin proteins. Moreover, our observations support a model whereby ANGPTL2-induced OB-cadherin expression in CRC cells is accompanied by decreased cell surface integrin α5β1 expression. These findings overall provide novel insight into mechanisms of β-catenin-driven intestinal tumorigenesis.

Lysine demethylase 5A (KDM5A) is a negative regulator of histone H3 lysine 4 trimethy­lation (H3K4me3), a histone mark associated with activate gene transcription. We identify that KDM5A interacts with the P-TEFb complex and cooperates with MYC to control MYC-targeted genes in multiple myeloma cells. We develop a cell-permeable and selective KDM5 inhibitor, JQKD82, that increases H3K4me3 but paradoxically inhibits downstream MYC-driven transcriptional output in vitro and in vivo. Using genetic ablation together with our inhibitor, we establish that KDM5A supports MYC target gene transcription independent of MYC itself by supporting TFIIH (CDK7)- and P-TEFb (CDK9)–mediated phosphorylation of RNAPII. These data identify KDM5A as a unique vulnerability in multiple myeloma functioning through regulation of MYC target gene transcription and establish JQKD82 as a tool compound to block KDM5A function as a potential therapeutic strategy for multiple myeloma. Significance: We delineate the function of KDM5A in activating the MYC-driven transcriptional landscape. We develop a cell-permeable KDM5 inhibitor to define the activating role of KDM5A on MYC target gene expression and implicate the therapeutic potential of this compound in mouse models and multiple myeloma patient samples. See related video from the AACR Annual Meeting 2021: https://vimeo.com/554896826

The purpose of the present study was to identify a biomarker that can predict the response to preoperative chemoradiotherapy (PCRT) in esophageal cancer patients. Twenty-five serum samples collected from patents with esophageal cancer before PCRT (responder = 13, non-responder = 12) were analyzed by quantitative proteomics, and 248 proteins were identified. Among them, the serum levels of leucine-rich alpha-2-glycoprotein 1 (LRG1) were significantly different (p 0.01) and well discriminated (area under the curve (AUC) of the receiver operating characteristic (ROC) curve0.8) between responder and non-responder groups. The combination of LRG1 with C-reactive protein (CRP) and soluble interleukin-6 receptor (sIL-6R), which were previously reported as biomarkers predicting PCRT response, further improved the predictive performance, providing an AUC of greater than 0.9. The present results suggest that LRG1 and its combination with CRP and sIL-6R are promising biomarker candidates to predict response to PCRT in esophageal cancer patients.

Visualizing tissue distribution of steroid hormones is a promising application of MALDI mass spectrometry imaging (MSI). On-tissue chemical derivatization using Girard's T reagent has enhanced the ionization efficiency of steroids. However, discriminating between structural isomers with distinct bioactivities remains a challenge. Herein, we used ion trap MS/tandem MS (MS3) to distinguish a mineralcorticoid aldosterone (Aldo) and a glucocorticoid cortisol (F), from their structural isomers. Our method is also useful to detect hybrid steroids (18-hydroxycortisol [18-OHF] and 18-oxocortisol) with sufficient signal-to-noise ratio. The clinical applicability of the tandem MS method was evaluated by analyzing F, Aldo, and 18-OHF distributions in human adrenal glands. In such clinical specimens, small Aldo-producing cell clusters (APCCs) were identified and were first found to produce a high level of Aldo and not to contain F. Moreover, a part of APCCs produced 18-OHF, presumably converted from F by APCC-specific CYP11B2 activity. Catecholamine species were also visualized with another derivatization reagent (TAHS), and those profiling successfully discriminated pheochromocytoma species. These tandem MSI-methods, coupled with on-tissue chemical derivatization has proven to be useful for detecting low-abundance steroids, including Aldo and hybrid steroids and thus identifying steroid hormone-producing lesions.