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The study of the mechanisms by which melatonin protects against cadmium (Cd) toxicity in plants is still in its infancy, particularly at the molecular level. In this study, the gene encoding a novel serotonin N-acetyltransferase 3 (SNAT3) in rice, a pivotal enzyme in the melatonin biosynthetic pathway, was cloned. Rice (Oryza sativa) OsSNAT3 is the first identified plant ortholog of archaeon Thermoplasma volcanium SNAT. The purified recombinant OsSNAT3 catalyzed the conversion of serotonin and 5-methoxytryptamine to N-acetylserotonin and melatonin, respectively. The suppression of OsSNAT3 by RNAi led to a decline in endogenous melatonin levels followed by a reduction in Cd tolerance in transgenic RNAi rice lines. In addition, the expression levels of genes encoding the endoplasmic reticulum (ER) chaperones BiP3, BiP4, and BiP5 were much lower in RNAi lines than in the wild type. In transgenic rice plants overexpressing OsSNAT3 (SNAT3-OE), however, melatonin levels were higher than in wild-type plants. SNAT3-OE plants also tolerated Cd stress, as indicated by seedling growth, malondialdehyde, and chlorophyll levels. BiP4 expression was much higher in the SNAT3-OE lines than in the wild type. These results indicate that melatonin engineering could help crops withstand Cd stress, resulting in high yields in Cd-contaminated fields. [ABSTRACT FROM AUTHOR]

This review systematically investigates the critical role of natural binding proteins (NBPs), encompassing DNA-, RNA-, carbohydrate-, fatty acid-, and chitin-binding proteins, in the realms of oncology and diagnostics. In an era where cancer continues to pose significant challenges to healthcare systems worldwide, the innovative exploration of NBPs offers a promising frontier for advancing both the diagnostic accuracy and therapeutic efficacy of cancer management strategies. This manuscript provides an in-depth examination of the unique mechanisms by which NBPs interact with specific molecular targets, highlighting their potential to revolutionize cancer diagnostics and therapy. Furthermore, it discusses the burgeoning research on aptamers, demonstrating their utility as 'nucleic acid antibodies' for targeted therapy and precision diagnostics. Despite the promising applications of NBPs and aptamers in enhancing early cancer detection and developing personalized treatment protocols, this review identifies a critical knowledge gap: the need for comprehensive studies to understand the diverse functionalities and therapeutic potentials of NBPs across different cancer types and diagnostic scenarios. By bridging this gap, this manuscript underscores the importance of NBPs and aptamers in paving the way for next-generation diagnostics and targeted cancer treatments. [ABSTRACT FROM AUTHOR]

Cryptosporidium parvum is an apicomplexan parasite causing persistent diarrhea in humans and animals. Issuing from target-based drug development, calcium-dependent protein kinase 1 inhibitors, collectively named bumped kinase inhibitors (BKIs), with excellent efficacies in vitro and in vivo have been generated. Some BKIs including BKI-1748 share a core structure with similarities to the first-generation antiprotozoal drug quinine, which is known to exert notorious side effects. Unlike quinine, BKI-1748 rapidly interfered with C. parvum proliferation in the human colon tumor (HCT) cell line HCT-8 cells and caused dramatic effects on the parasite ultrastructure. To identify putative BKI targets in C. parvum and in host cells, we performed differential affinity chromatography with cell-free extracts from non-infected and infected HCT-8 cells using BKI-1748 and quinine epoxy-activated sepharose columns followed by mass spectrometry. C. parvum proteins of interest were identified in eluates from columns coupled to BKI-1748, or in eluates from both BKI-1748 and quinine columns. However, no C. parvum proteins could be identified binding exclusively to BKI-1748. In contrast, 25 BKI-1748-specific binding proteins originating from HCT-8 cells were detected. Moreover, 29 C. parvum and 224 host cell proteins were identified in both BKI-1748 as well as in quinine eluates. In both C. parvum and host cells, the largest subset of binding proteins was involved in RNA binding and modification, with a focus on ribosomal proteins and proteins involved in RNA splicing. These findings extend previous results, showing that BKI-1748 interacts with putative targets involved in common, essential pathways such as translation and RNA processing. [ABSTRACT FROM AUTHOR]

Background: Arginase 1 (Arg1) encodes a key enzyme that catalyzes the metabolism of arginine to ornithine and urea. In our recent study, we found that knockdown of Arg1 in the lungs of fetal mice induces apoptosis of epithelial cells and dramatically delays initiation of labor. As the most abundant internal mRNA modification, N6‐methyladenosine (m6A) has been found to play important roles in lung development and cellular differentiation. However, if the knockdown of Arg1 affects the RNA m6A modification in fetal lungs remains unknown. Methods: In the current study, the RNA m6A levels and the expression of RNA m6A related enzymes were validated in 13.0 dpc fetal lungs that Arg1 was knocked down by adeno‐associated virus carrying Arg1‐shRNA, using western blot, immunofluorescence, and RT‐qPCR. Results: No statistical differences were found in the expression of methyltransferase, demethylases, and binding proteins in the fetal lungs between AAV‐shArg1‐injected mice and AAV‐2/9‐injected mice. Besides, there is no significant change of overall RNA m6A level in fetal lungs from AAV‐shArg1‐injected mice, compared with that from AAV‐2/9‐injected mice. Conclusions: These results indicate that arginase 1 does not affect RNA m6A methylation in mouse fetal lung, and the mechanisms other than RNA m6A modification underlying the effects of Arg1 knockdown on the fetal lung development and their interaction with labor initiation need to be further explored. [ABSTRACT FROM AUTHOR]

Based on insights obtained during the past decade, the classical concept of an activated hypothalamus-pituitary-adrenocortical axis in response to critical illness is in need of revision. After a brief central hypothalamus-pituitary-adrenocortical axis activation, the vital maintenance of increased systemic cortisol availability and action in response to critical illness is predominantly driven by peripheral adaptations rather than by an ongoing centrally activated several-fold increased production and secretion of cortisol. Besides the known reduction of cortisol-binding proteins that increases free cortisol, these peripheral responses comprise suppressed cortisol metabolism in liver and kidney, prolonging cortisol half-life, and local alterations in expression of 11βHSD1, glucocorticoid receptor-α (GRα), and FK506 binding protein 5 (FKBP51) that appear to titrate increased GRα action in vital organs and tissues while reducing GRα action in neutrophils, possibly preventing immune-suppressive off-target effects of increased systemic cortisol availability. Peripherally increased cortisol exerts negative feed-back inhibition at the pituitary level impairing processing of pro-opiomelanocortin into ACTH, thereby reducing ACTH-driven cortisol secretion, whereas ongoing central activation results in increased circulating pro-opiomelanocortin. These alterations seem adaptive and beneficial for the host in the short term. However, as a consequence, patients with prolonged critical illness who require intensive care for weeks or longer may develop a form of central adrenal insufficiency. The new findings supersede earlier concepts such as "relative," as opposed to "absolute," adrenal insufficiency and generalized systemic glucocorticoid resistance in the critically ill. The findings also question the scientific basis for broad implementation of stress dose hydrocortisone treatment of patients suffering from acute septic shock solely based on assumption of cortisol insufficiency. [ABSTRACT FROM AUTHOR]

Malignant tumor is still a major problem worldwide. During tumorigenesis or tumor development, tumor suppressor p53-binding protein 2 (TP53BP2), also known as apoptosis stimulating protein 2 of p53 (ASPP2), plays a critical role in p53 dependent and independent manner. Expression of TP53BP2 is highly correlated with the prognosis and survival rate of malignant tumor patients. TP53BP2 can interact with p53, NF-κB p65, Bcl-2, HCV core protein, PP1, YAP, CagA, RAS, PAR3, and other proteins to regulate cell function. Moreover, TP53BP2 can also regulate the proliferation, apoptosis, autophagy, migration, EMT and drug resistance of tumor cells through downstream signaling pathways, such as NF-κB, RAS/MAPK, mevalonate, TGF-β1, PI3K/AKT, aPKC-ι/GLI1 and autophagy pathways. As a potential therapeutic target, TP53BP2 has been attracted more attention. We review the role of TP53BP2 in tumorigenesis or tumor development and the signal pathway involved in TP53BP2, which may provide more deep insight and strategies for tumor treatment.

The free hormone hypothesis postulates that only the nonbound fraction (the free fraction) of hormones that otherwise circulate in blood bound to their carrier proteins is able to enter cells and exert biologic effects. In this review, I will examine four hormone groups-vitamin D metabolites (especially 25OHD), thyroid hormones (especially thyroxine [T4]), sex steroids (especially testosterone), and glucocorticoids (especially cortisol)-that are bound to various degrees to their respective binding proteins-vitamin D-binding protein (DBP), thyroid-binding globulin (TBG), sex hormone-binding globulin (SHBG), and cortisol-binding globulin (CBG)-for which a strong case can be made that measurement of the free hormone level provides a better assessment of hormonal status than the measurement of total hormonal levels under conditions in which the binding proteins are affected in levels or affinities for the hormones to which they bind. I will discuss the rationale for this argument based on the free hormone hypothesis, discuss potential exceptions to the free hormone hypothesis, and review functions of the binding proteins that may be independent of their transport role. I will then review the complications involved with measuring the free hormone levels and the efforts to calculate those levels based on estimates of binding constants and levels of both total hormone and total binding protein. In this review, the major focus will be on DBP and free 25OHD, but the parallels and differences with the other binding proteins and hormones will be highlighted. Vitamin D and its metabolites, thyroid hormones, sex steroids, and glucocorticoids are transported in blood bound to serum proteins. The tightness of binding varies depending on the hormone and the binding protein such that the percent free varies from 0.03% for T4 and 25OHD to 4% for cortisol with testosterone at 2%. Although the major function of the primary carrier proteins (DBP, TBG, SHBG, and CBG) may be to transport their respective lipophilic hormones within the aqueous media that is plasma, these proteins may have other functions independent of their transport function. For most tissues, these hormones enter the cell as the free hormone presumably by diffusion (the free hormone hypothesis), although a few tissues such as the kidney and reproductive tissues express megalin/cubilin enabling by endocytosis protein-bound hormone to enter the cell. Measuring the free levels of these protein-bound hormones is likely to provide a better measure of the true hormone status than measuring the total levels in situations where the levels and/or affinities of the binding proteins are altered. Methods to measure free hormone levels are problematic as the free levels can be quite low, the methods require separation of bound and free that could disturb the steady state, and the means of separating bound and free are prone to error. Calculation of free levels using existing data for association constants between the hormone and its binding protein are likewise prone to error because of assumptions of linear binding models and invariant association constants, both of which are invalid. © 2020 The Author. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

This review systematically investigates the critical role of natural binding proteins (NBPs), encompassing DNA-, RNA-, carbohydrate-, fatty acid-, and chitin-binding proteins, in the realms of oncology and diagnostics. In an era where cancer continues to pose significant challenges to healthcare systems worldwide, the innovative exploration of NBPs offers a promising frontier for advancing both the diagnostic accuracy and therapeutic efficacy of cancer management strategies. This manuscript provides an in-depth examination of the unique mechanisms by which NBPs interact with specific molecular targets, highlighting their potential to revolutionize cancer diagnostics and therapy. Furthermore, it discusses the burgeoning research on aptamers, demonstrating their utility as ‘nucleic acid antibodies’ for targeted therapy and precision diagnostics. Despite the promising applications of NBPs and aptamers in enhancing early cancer detection and developing personalized treatment protocols, this review identifies a critical knowledge gap: the need for comprehensive studies to understand the diverse functionalities and therapeutic potentials of NBPs across different cancer types and diagnostic scenarios. By bridging this gap, this manuscript underscores the importance of NBPs and aptamers in paving the way for next-generation diagnostics and targeted cancer treatments.

DNA G-quadruplexes (G4s) are non-canonical secondary structures formed in guanine-rich sequences. Within the human genome, G4s are found in regulatory regions such as gene promoters and telomeres to control replication, transcription, and telomere lengthening. In the cellular context, there are several proteins named as G4-binding proteins (G4BPs) that interact with G4s, either anchoring upon, stabilizing, and/or unwinding them. These proteins may play different key roles in the regulation of the endogenous G4 landscape and its associated functions. The present review summarizes the current literature on G4BPs in terms of their targets and functions, providing updated insights into the regulation of G4s in living organisms.

Caldicellulosiruptor species are proficient at solubilizing carbohydrates in lignocellulosic biomass through surface (S)-layer bound and secretomic glycoside hydrolases. Tāpirins, surface-associated, non-catalytic binding proteins in Caldicellulosiruptor species, bind tightly to microcrystalline cellulose, and likely play a key role in natural environments for scavenging scarce carbohydrates in hot springs. However, the question arises: If tāpirin concentration on Caldicellulosiruptor cell walls increased above native levels, would this offer any benefit to lignocellulose carbohydrate hydrolysis and, hence, biomass solubilization? This question was addressed by engineering the genes for tight-binding, non-native tāpirins into C. bescii. The engineered C. bescii strains bound more tightly to microcrystalline cellulose (Avicel) and biomass compared to the parent. However, tāpirin overexpression did not significantly improve solubilization or conversion for wheat straw or sugarcane bagasse. When incubated with poplar, the tāpirin-engineered strains increased solubilization by 10% compared to the parent, and corresponding acetate production, a measure of carbohydrate fermentation intensity, was 28% higher for the Calkr_0826 expression strain and 18.5% higher for the Calhy_0908 expression strain. These results show that enhanced binding to the substrate, beyond the native capability, did not improve C. bescii solubilization of plant biomass, but in some cases may improve conversion of released lignocellulose carbohydrates to fermentation products. [ABSTRACT FROM AUTHOR]

DNA G-quadruplexes (G4s) are non-canonical secondary structures formed in guanine-rich sequences. Within the human genome, G4s are found in regulatory regions such as gene promoters and telomeres to control replication, transcription, and telomere lengthening. In the cellular context, there are several proteins named as G4-binding proteins (G4BPs) that interact with G4s, either anchoring upon, stabilizing, and/or unwinding them. These proteins may play different key roles in the regulation of the endogenous G4 landscape and its associated functions. The present review summarizes the current literature on G4BPs in terms of their targets and functions, providing updated insights into the regulation of G4s in living organisms. [ABSTRACT FROM AUTHOR]

Acyl‐CoA binding protein (ACBP), also known as diazepam binding inhibitor (DBI), has recently emerged as a hypothalamic and brainstem gliopeptide regulating energy balance. Previous work has shown that the ACBP‐derived octadecaneuropeptide exerts strong anorectic action via proopiomelanocortin (POMC) neuron activation and the melanocortin‐4 receptor. Importantly, targeted ACBP loss‐of‐function in astrocytes promotes hyperphagia and diet‐induced obesity while its overexpression in arcuate astrocytes reduces feeding and body weight. Despite this knowledge, the role of astroglial ACBP in adaptive feeding and metabolic responses to acute metabolic challenges has not been investigated. Using different paradigms, we found that ACBP deletion in glial fibrillary acidic protein (GFAP)‐positive astrocytes does not affect weight loss when obese male mice are transitioned from a high fat diet to a chow diet, nor metabolic parameters in mice fed with a normal chow diet (e.g., energy expenditure, body temperature) during fasting, cold exposure and at thermoneutrality. In contrast, astroglial ACBP deletion impairs meal pattern and feeding responses during refeeding after a fast and during cold exposure, thereby showing that ACBP is required to stimulate feeding in states of increased energy demand. These findings challenge the general view that astroglial ACBP exerts anorectic effects and suggest that regulation of feeding by ACBP is dependent on metabolic status. [ABSTRACT FROM AUTHOR]

BACKGROUND: Lack of control agents and development of bacterial resistance are emergent problems in the chemical control of rice bacterial blight, therefore novel bactericides against Xanthomonas oryzae pv. oryzae (Xoo, the causal agent of rice bacterial blight) are urgently needed. We previously found that parthenolide (PTL) is a potential lead against Xoo, and PTL inhibits Xoo growth via oxidative stress. However, the mechanism of action of PTL against Xoo needs further elucidation. RESULTS: In this study, a biotinylated PTL probe was synthesized, and two important subunits in the respiratory chain (NuoF of complex I and SdhB of complex II) of Xoo were captured with the probe and identified with liquid chromatography tandem mass spectrometry (LC–MS/MS). The binding between them was verified with pull‐down and drug affinity responsive target stability technologies. In addition, purified proteins of NuoF and SdhB greatly lowered the antibacterial activity of PTL, and PTL evidently inhibited the enzyme activities of complexes I and II. Moreover, knockout of nuoF and sdhB in Xoo caused elevated reactive oxygen species (ROS) levels and increased sensitivity to PTL. Furthermore, molecular simulations indicated that PTL may form covalent bonds with Cys105 and Cys187 in NuoF and Cys106 in SdhB. CONCLUSION: PTL can directly bind to NuoF and SdhB, which impairs the enzyme functions of complexes I and II in the respiratory chain, leading to ROS accumulation in Xoo. This study will provide deep insight into the mechanism of action of PTL against Xoo. © 2022 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Tumor suppressor WWOX inhibits cancer growth and retards Alzheimer's disease (AD) progression. Supporting evidence shows that the more strongly WWOX binds intracellular protein partners, the weaker is cancer cell growth in vivo. Whether this correlates with retardation of AD progression is unknown. Two functional forms of WWOX exhibit opposite functions. pY33-WWOX is proapoptotic and anticancer, and is essential for maintaining normal physiology. In contrast, pS14-WWOX is accumulated in the lesions of cancers and AD brains, and suppression of WWOX phosphorylation at S14 by a short peptide Zfra abolishes cancer growth and retardation of AD progression. In parallel, synthetic Zfra4-10 or WWOX7-21 peptide strengthens the binding of endogenous WWOX with intracellular protein partners leading to cancer suppression. Indeed, Zfra4-10 is potent in restoring memory loss in triple transgenic mice for AD (3xTg) by blocking the aggregation of amyloid beta 42 (Aβ42), enhancing degradation of aggregated proteins, and inhibiting activation of inflammatory NF-κB. In light of the findings, Zfra4-10-mediated suppression of cancer and AD is due, in part, to an enhanced binding of endogenous WWOX and its binding partners. In this perspective review article, we detail the molecular action of WWOX in the HYAL-2/WWOX/SMAD4 signaling for biological effects, and discuss WWOX phosphorylation forms in interacting with binding partners, leading to suppression of cancer growth and retardation of AD progression. [ABSTRACT FROM AUTHOR]

Objectives: Insulin‐like growth factor‐I and its binding proteins are involved in cancer development, progression, and metastasis. In urothelial carcinoma, the impact of this pathway is still poorly investigated. The present large cohort study aimed to evaluate the association of preoperative circulating levels of insulin‐like growth factor‐I, insulin‐like growth factor‐I binding protein‐2 and ‐3 on outcomes after radical cystectomy. Methods: A retrospective cohort study of the plasma specimens from 1036 consecutive urothelial carcinoma patients who were treated with radical cystectomy. The primary and secondary outcomes were adverse histopathological features and survival outcomes. Binominal logistic regression and multivariable Cox regression analyses were performed to assess the association of plasma levels of insulin‐like growth factor‐I, insulin‐like growth factor‐I binding protein‐2 and ‐3 with outcomes. Results: On multivariable analysis adjusting for the effects of preoperative variables, lower insulin‐like growth factor‐I binding protein‐2 levels were associated with an increased risk of lymph node metastasis and (any non‐organ confined disease) any non‐organ confined disease. Insulin‐like growth factor‐I binding protein‐3 levels were also inversely independently associated with lymph node metastasis. Receiver operating characteristic curve analysis showed that the addition of insulin‐like growth factor‐I binding proteins biomarkers to a reference model significantly improved the discriminating ability for the prediction of lymph node metastasis (+10.0%, P < 0.001). On multivariable Cox regression models, lower levels of both insulin‐like growth factor‐I binding protein‐2 and ‐3 plasma levels were associated with recurrence‐free survival, cancer‐specific survival, and overall survival. insulin‐like growth factor‐I binding protein‐2 and ‐3 levels and improved the discrimination of a standard reference model for the prediction of recurrence‐free survival, cancer‐specific survival, and overall survival (+4.9%, 4.9%, 2.3%, respectively). Conclusions: Preoperative insulin‐like growth factor‐I binding protein‐2 and ‐3 are significantly associated with features of biologically and clinically aggressive urothelial carcinoma. These biomarkers improved prognostic urothelial carcinoma models. [ABSTRACT FROM AUTHOR]

Acromegaly is a rare disorder characterized by the excessive secretion of growth hormone (GH), mostly caused by pituitary adenomas. While in full-blown cases the diagnosis is easy to establish, milder cases are more challenging. Additionally, establishing whether full cure after surgery is reached may be difficult. In this article, we will review the challenges posed by the variability in measurements of GH and its main effector insulin-like growth factor I (IGF-I) due to both biological changes, co-morbidities, and assays variability. Interpretation of GH and IGF-I assays is important in establishing an early diagnosis of acromegaly, in avoiding misdiagnosis, and in establishing if cure is achieved by surgery. Physicians should be familiar with the variables that affect measurements of these 2 hormones, and with the performance of the assays available in their practice. [ABSTRACT FROM AUTHOR]

Retinol is an essential nutrient in animals. Its metabolites, specifically retinoic acid (RA), are crucial for cell differentiation, including adipogenesis. Retinol binding protein 7 (Rbp7) is under the control of PPARγ, the master regulator of adipogenesis. However, the role of RBP7 in adipogenesis is unclear. Our study showed that Rbp7 was abundantly expressed in white and brown mouse adipose tissues and had a higher expression in adipocytes than in stromal vascular fraction. Rbp7 overexpression promoted 3T3-L1 preadipocyte differentiation with increased triglyceride accumulation and up-regulation of Pparγ, Fabp4, C/ebpα, and AdipoQ. Rbp7 deficient adipocytes had opposite effects of the overexpression, which were rescued by RA supplementation. Indirect assessment of relative nuclear RA levels using RAR response element (RARE)-Luc reporter assay demonstrated that Rbp7 overexpression significantly increased RARE-Luc reporter activity. Rbp7 overexpression significantly increased expression of Raldh1, responsible for RA production, and up-regulation of Lrat and Cyp26a1, involved in retinol storage and RA catabolism, respectively, in 3T3-L1 adipocytes. Rbp7 deficient adipocytes had opposite effects of the overexpression of those genes involved in retinol metabolism. These data suggest that RBP7 increases transcriptional activity of RARE that may induce negative feedback responses via regulation of the gene expression for retinol homeostasis. Our data indicate critical RBP7 functions in adipocytes: regulation of transcriptional activity of RARE and adipocytes differentiation, potentially providing a new target for obesity therapy.

ABSTRACT In addition to auxiological, clinical and metabolic features measurements of growth hormone (GH) and insulin-like growth factor I (IGF-I) complement our tools in diagnosis and follow-up of GH-related disorders. While comparably robust during the pre-analytical phase, measurement and interpretation of concentrations of both hormones can be challenging due to analytical issues and biological confounders. Assay methods differ in terms of antibody specificity, interference from binding proteins, reference preparations and sensitivity. GH assays have different specificity towards different GH-isoforms (e.g. 20 kDa GH, placental GH) and interference from the GH antagonist Pegvisomant. The efficacy to prevent binding protein interference is most important in IGF-I assays. Methodological differences between assays require that reference intervals and diagnostic cut-offs are assay-specific. Among biological variables, pubertal development and age are most relevant for IGF-I, making detailed reference intervals mandatory for interpretation. GH has pulsatile secretion and short half-life. Its concentration is modified by acute factors such as stress, exercise and sleep, but also by intake of oral estrogens and anthropometric factors (e.g. BMI). Other GH dependent biomarkers such as free IGF-I, IGF binding protein 3 (IGFBP 3) and acid labile subunit (ALS) have been proposed. Their concentrations largely mirror the information obtained through measurement of IGF-I, but their measurement can be helpful in particular situations. In this review, we describe the evolution of analytical methods to measure biomarkers of GH action, the impact of the methodological changes on laboratory results and the need to include biological variables in their interpretation. Arch Endocrinol Metab. 2019;63(6):618-29

Organic acid metabolites exhibit acidic properties. These metabolites serve as intermediates in major carbon metabolic pathways and are involved in several biochemical pathways, including the tricarboxylic acid (TCA) cycle and glycolysis. They also regulate cellular activity and play crucial roles in epigenetics, tumorigenesis, and cellular signal transduction. Knowledge of the binding proteins of organic acid metabolites is crucial for understanding their biological functions. However, identifying the binding proteins of these metabolites has long been a challenging task owing to the transient and weak nature of their interactions. Moreover, traditional methods are unsuitable for the structural modification of the ligands of organic acid metabolites because these metabolites have simple and similar structures. Even minor structural modifications can significantly affect protein interactions. Thermal proteome profiling (TPP) provides a promising avenue for identifying binding proteins without the need for structural modifications. This approach has been successfully applied to the identification of the binding proteins of several metabolites. In this study, we investigated the binding proteins of two TCA cycle intermediates, i.e., succinate and fumarate, and lactate, an end-product of glycolysis, using the matrix thermal shift assay (mTSA) technique. This technique involves combining single-temperature (52 ℃) TPP and dose-response curve analysis to identify ligand-binding proteins with high levels of confidence and determine the binding affinity between ligands and proteins. To this end, HeLa cells were lysed, followed by protein desalting to remove endogenous metabolites from the cell lysates. The desalted cell lysates were treated with fumarate or succinate at final concentrations of 0.004, 0.04, 0.4, and 2 mmol/L in the experimental groups or 2 mmol/L sodium chloride in the control group. Considering that the cellular concentration of lactate can be as high as 2-30 mmol/L, we then applied lactate at final concentrations of 0.2, 1, 5, 10, and 25 mmol/L in the experimental groups or 25 mmol/L sodium chloride in the control group. Using high-sensitivity mass spectrometry coupled with data-independent acquisition (DIA) quantification, we quantified 5870, 5744, and 5816 proteins in succinate, fumarate, and lactate mTSA experiments, respectively. By setting stringent cut-off values (i.e., significance of changes in protein thermal stability ( p -value)<0.001 and quality of the dose-response curve fitting (square of Pearson's correlation coefficient, R )>0.95), multiple binding proteins for these organic acid metabolites from background proteins were confidently determined. Several known binding proteins were identified, notably fumarate hydratase (FH) as a binding protein for fumarate, and 2 -ketoglutarate-dependent dioxygenase (FTO) as a binding protein for both fumarate and succinate. Additionally, the affinity data for the interactions between these metabolites and their binding proteins were obtained, which closely matched those reported in the literature. Interestingly, ornithine aminotransferase (OAT), which is involved in amino acid biosynthesis, and 3-mercaptopyruvate sulfurtransferase (MPST), which acts as an antioxidant in cells, were identified as lactate-binding proteins. Subsequently, an orthogonal assay technique developed in our laboratory, the solvent-induced precipitation (SIP) technique, was used to validate the mTSA results. SIP identified OAT as the top target candidate, validating the mTSA-based finding that OAT is a novel lactate-binding protein. Although MPST was not identified as a lactate-binding protein by SIP, statistical analysis of MPST in the mTSA experiments with 10 or 25 mmol/L lactate revealed that MPST is a lactate-binding protein with a high level of confidence. Peptide-level empirical Bayes α -tests combined with Fisher's exact test also supported the conclusion that MPST is a lactate-binding protein. Lactate is structurally similar to pyruvate, the known binding protein of MPST. Therefore, assuming that lactate could potentially occupy the binding site of pyruvate on MPST. Overall, the novel binding proteins identified for lactate suggest their potential involvement in amino acid synthesis and redox balance regulation.t -tests combined with Fisher's exact test also supported the conclusion that MPST is a lactate-binding protein. Lactate is structurally similar to pyruvate, the known binding protein of MPST. Therefore, assuming that lactate could potentially occupy the binding site of pyruvate on MPST. Overall, the novel binding proteins identified for lactate suggest their potential involvement in amino acid synthesis and redox balance regulation.

FK506-binding proteins (FKBPs) alter the conformation of proteins via cis--trans isomerization of prolyl-peptide bonds. While this activity can be demonstrated in vitro, the intractability of detecting prolyl isomerization events in cells has limited our understanding of the biological processes regulated by FKBPs. Here we report that FKBP25 is an active participant in the repair of DNA double-strand breaks (DSBs). FKBP25 influences DSB repair pathway choice by promoting homologous recombination (HR) and suppressing single-strand annealing (SSA). Consistent with this observation, cells depleted of FKBP25 form fewer Rad51 repair foci in response to etoposide and ionizing radiation, and they are reliant on the SSA repair factor Rad52 for viability. We find that FKBP25's catalytic activity is required for promoting DNA repair, which is the first description of a biological function for this enzyme activity. Consistent with the importance of the FKBP catalytic site in HR, rapamycin treatment also impairs homologous recombination, and this effect is at least in part independent of mTor. Taken together these results identify FKBP25 as a component of the DNA DSB repair pathway. Key words: DNA repair, prolyl isomerase, homologous recombination, FKBP, olaparib. Resume: Les proteines de liaison du FK506 (FKBP) modifient la conformation des proteines au moyen de l'isomerisation cis--trans des liens peptidiques de la proline. Alors que cette activite peut etre demontree in vitro, la grande difficulte a detecter les manifestations de l'isomerisation de la proline dans les cellules a limite notre comprehension des processus biologiques regules par les FKBP. Les auteurs rapportent ici que la FKBP25 est un participant actif dans la reparation des bris double brin (BDB) d'ADN. La FKBP25 influence le choix de la voie de reparation des BDB en favorisant la recombinaison homologue (RH) et en supprimant la renaturation simple brin. En accord avec cette observation, les cellules depourvues de FKBP25 forment moins de foyers de reparation comportant Rad51 en reponse a l'etoposide et a la radiation ionisante, et elles dependent du facteur de reparationrecombinaison Rad52 pour assurer leur viabilite. Ils ont trouve que l'activite catalytique de FKBP25 est requise pour favoriser la reparation d'ADN, ce qui constitue la premiere description de la fonction biologique de l'activite de cette enzyme. En accord avec l'importance du site catalytique de FKBP dans la RH, le traitement a la rapamycine diminue aussi la recombinaison homologue et cet effet est au moins partiellement independant de mTor. Dans leur ensemble, ces resultats identifient la FKBP25 comme element de la voie de reparation des BDB de l'ADN. [Traduit par la Redaction] Mots-cles : reparation d'ADN, prolyl isomerase, recombinaison homologue, FKBP, olaparib., Introduction FK506-binding proteins (FKBPs) are enzymes that catalyze the cis--trans isomerization of prolyl-peptide bonds to regulate substrate protein structure and function. Fifteen FKBPs are found in the human proteome and [...]

P-loop NTPases comprise one of the major superfamilies of nucleotide binding proteins, which mediate a variety of cellular processes, such as mRNA translation, signal transduction, cell motility, and growth regulation. In this review, we discuss the structure and function of two members of the ancient Obg-related family of P-loop GTPases: human Obg-like ATPase 1 (hOLA1), and its bacterial/plant homolog, YchF. After a brief discussion of nucleotide binding proteins in general and the classification of the Obg-related family in particular, we discuss the sequence and structural features of YchF and hOLA1. We then explore the various functional roles of hOLA1 in mammalian cells during stress response and cancer progression, and of YchF in bacterial cells. Finally, we directly compare and contrast the structure and function of hOLA1 with YchF before summarizing the future perspectives of hOLA1 research. This review is timely, given the variety of recent studies aimed at understanding the roles of hOLA1 and YchF in such critical processes as cellular-stress response, oncogenesis, and protein synthesis. Key words: hOLA1, YchF, cellular stress, cancer progression, structure--function. Les NTPases a boucle P constituent une des principales superfamilles de proteines de liaison des nucleotides qui interviennent dans une variete de processus cellulaires tels que la traduction de l'ARNm, la transduction de signaux, la motilite cellulaire et la regulation de la croissance. Dans cette synthese, les auteurs discutent de la structure et de la fonction de deux membresdelafamille anciennedeGTPasesabouclePrelieesaObg:l'ATPasedetype obg humaine hOLA1etson homologue chez les bacteries et les vegetaux, YchF. Apres une breve presentation des proteines de liaison des nucleotides en general et de la classification de la famille reliee a Obg en particulier, les auteurs discutent des caracteristiques de sequences et structurales de YchF et de hOLA1. Ils explorent ensuite les differents roles fonctionnels de hOLA1 dans les cellules de mammiferes durant la reponse au stress et la progression du cancer, ainsi que ceux de YchF dans les cellules bacteriennes. Finalement, ils comparent et exposent directement les differences entre la structure et la fonction de hOLA1 et celles de YchF, avant de resumer les perspectives de larecherche sur hOLA1. Cette synthese tombe a point nomme, compte tenu dela variete d'etudes recentes visant a comprendre les roles de hOLA1 et de YchF dans des processus importants comme la reponse cellulaire au stress, l'oncogenese et la synthese des proteines. [Traduit par la Redaction] Mots-cles : hOLA1, YchF, stress cellulaire, progression du cancer, structure--fonction., Introduction Nucleotide binding proteins Nucleotide binding proteins have vital roles in cellular processes including mRNA translation, signal transduction, cell motility, cell division, cell growth, chromatin remodeling, and cytoskeleton organization (Milligan [...]

Mutations in genes encoding components of the mitochondrial DNA (mtDNA) replication machinery cause mtDNA depletion syndromes (MDSs), which associate ocular features with severe neurological syndromes. Here, we identified heterozygous missense mutations in single-strand binding protein 1 (SSBP1) in 5 unrelated families, leading to the R38Q and R107Q amino acid changes in the mitochondrial single-stranded DNA-binding protein, a crucial protein involved in mtDNA replication. All affected individuals presented optic atrophy, associated with foveopathy in half of the cases. To uncover the structural features underlying SSBP1 mutations, we determined a revised SSBP1 crystal structure. Structural analysis suggested that both mutations affect dimer interactions and presumably distort the DNA-binding region. Using patient fibroblasts, we validated that the R38Q variant destabilizes SSBP1 dimer/tetramer formation, affects mtDNA replication, and induces mtDNA depletion. Our study showing that mutations in SSBP1 cause a form of dominant optic atrophy frequently accompanied with foveopathy brings insights into mtDNA maintenance disorders., Introduction Mitochondrial diseases are clinically heterogeneous and can be caused by mutations in either nuclear or mitochondrial DNA (mtDNA) (1). Mutations in genes known to be essential for mtDNA maintenance [...]

Tumor suppressor WWOX inhibits cancer growth and retards Alzheimer’s disease (AD) progression. Supporting evidence shows that the more strongly WWOX binds intracellular protein partners, the weaker is cancer cell growth in vivo. Whether this correlates with retardation of AD progression is unknown. Two functional forms of WWOX exhibit opposite functions. pY33-WWOX is proapoptotic and anticancer, and is essential for maintaining normal physiology. In contrast, pS14-WWOX is accumulated in the lesions of cancers and AD brains, and suppression of WWOX phosphorylation at S14 by a short peptide Zfra abolishes cancer growth and retardation of AD progression. In parallel, synthetic Zfra4-10 or WWOX7-21 peptide strengthens the binding of endogenous WWOX with intracellular protein partners leading to cancer suppression. Indeed, Zfra4-10 is potent in restoring memory loss in triple transgenic mice for AD (3xTg) by blocking the aggregation of amyloid beta 42 (Aβ42), enhancing degradation of aggregated proteins, and inhibiting activation of inflammatory NF-κB. In light of the findings, Zfra4-10-mediated suppression of cancer and AD is due, in part, to an enhanced binding of endogenous WWOX and its binding partners. In this perspective review article, we detail the molecular action of WWOX in the HYAL-2/WWOX/SMAD4 signaling for biological effects, and discuss WWOX phosphorylation forms in interacting with binding partners, leading to suppression of cancer growth and retardation of AD progression.

ABSTRACT The free hormone hypothesis postulates that only the nonbound fraction (the free fraction) of hormones that otherwise circulate in blood bound to their carrier proteins is able to enter cells and exert biologic effects. In this review, I will examine four hormone groups—vitamin D metabolites (especially 25OHD), thyroid hormones (especially thyroxine [T4]), sex steroids (especially testosterone), and glucocorticoids (especially cortisol)—that are bound to various degrees to their respective binding proteins—vitamin D‐binding protein (DBP), thyroid‐binding globulin (TBG), sex hormone‐binding globulin (SHBG), and cortisol‐binding globulin (CBG)—for which a strong case can be made that measurement of the free hormone level provides a better assessment of hormonal status than the measurement of total hormonal levels under conditions in which the binding proteins are affected in levels or affinities for the hormones to which they bind. I will discuss the rationale for this argument based on the free hormone hypothesis, discuss potential exceptions to the free hormone hypothesis, and review functions of the binding proteins that may be independent of their transport role. I will then review the complications involved with measuring the free hormone levels and the efforts to calculate those levels based on estimates of binding constants and levels of both total hormone and total binding protein. In this review, the major focus will be on DBP and free 25OHD, but the parallels and differences with the other binding proteins and hormones will be highlighted. Vitamin D and its metabolites, thyroid hormones, sex steroids, and glucocorticoids are transported in blood bound to serum proteins. The tightness of binding varies depending on the hormone and the binding protein such that the percent free varies from 0.03% for T4 and 25OHD to 4% for cortisol with testosterone at 2%. Although the major function of the primary carrier proteins (DBP, TBG, SHBG, and CBG) may be to transport their respective lipophilic hormones within the aqueous media that is plasma, these proteins may have other functions independent of their transport function. For most tissues, these hormones enter the cell as the free hormone presumably by diffusion (the free hormone hypothesis), although a few tissues such as the kidney and reproductive tissues express megalin/cubilin enabling by endocytosis protein‐bound hormone to enter the cell. Measuring the free levels of these protein‐bound hormones is likely to provide a better measure of the true hormone status than measuring the total levels in situations where the levels and/or affinities of the binding proteins are altered. Methods to measure free hormone levels are problematic as the free levels can be quite low, the methods require separation of bound and free that could disturb the steady state, and the means of separating bound and free are prone to error. Calculation of free levels using existing data for association constants between the hormone and its binding protein are likewise prone to error because of assumptions of linear binding models and invariant association constants, both of which are invalid. © 2020 The Author. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Membrane repair is essential to cell survival. In skeletal muscle, injury often associates with plasma membrane disruption. Additionally, muscular dystrophy is linked to mutations in genes that produce fragile membranes or reduce membrane repair. Methods to enhance repair and reduce susceptibility to injury could benefit muscle in both acute and chronic injury settings. Annexins are a family of membrane-associated [Ca.sup.2+]-binding proteins implicated in repair, and annexin A6 was previously identified as a genetic modifier of muscle injury and disease. Annexin A6 forms the repair cap over the site of membrane disruption. To elucidate how annexins facilitate repair, we visualized annexin cap formation during injury. We found that annexin cap size positively correlated with increasing [Ca.sup.2+] concentrations. We also found that annexin overexpression promoted external blebs enriched in [Ca.sup.2+] and correlated with a reduction of intracellular [Ca.sup.2+] at the injury site. Annexin A6 overexpression reduced membrane injury, consistent with enhanced repair. Treatment with recombinant annexin A6 protected against acute muscle injury in vitro and in vivo. Moreover, administration of recombinant annexin A6 in a model of muscular dystrophy reduced serum creatinine kinase, a biomarker of disease. These data identify annexins as mediators of membrane-associated [Ca.sup.2+] release during membrane repair and annexin A6 as a therapeutic target to enhance membrane repair capacity., Introduction Plasma membrane repair occurs after membrane disruption and is a highly conserved process. The active process required for resealing membrane disruptions is thought to rely on [Ca.sup.2+]-dependent vesicle fusion [...]

Nature exploits cage-like proteins for a variety of biological purposes, from molecular packaging and cargo delivery to catalysis. These cage-like proteins are of immense importance in nanomedicine due to their propensity to self-assemble from simple identical building blocks to highly ordered architecture and the design flexibility afforded by protein engineering. However, delivery of protein nanocages to the renal tubules remains a major challenge because of the glomerular filtration barrier, which effectively excludes conventional size nanocages. Here, we show that DNA-binding protein from starved cells (Dps)--the extremely small archaeal antioxidant nanocage--is able to cross the glomerular filtration barrier and is endocytosed by the renal proximal tubules. Using a model of endotoxemia, we present an example of the way in which proximal tubule-selective Dps nanocages can limit the degree of endotoxin- induced kidney injury. This was accomplished by amplifying the endogenous antioxidant property of Dps with addition of a dinuclear manganese cluster. Dps is the first-in-class protein cage nanoparticle that can be targeted to renal proximal tubules through glomerular filtration. In addition to its therapeutic potential, chemical and genetic engineering of Dps will offer a nanoplatform to advance our understanding of the physiology and pathophysiology of glomerular filtration and tubular endocytosis., Introduction One of the major challenges in translational research is the development of therapeutics that are precisely targeted to the desired site of action. Precise targeting is also important for [...]

TAR DNA-binding protein 43 kDa (TDP-43), encoded by TARDBP, is an RNA-binding protein, the nuclear depletion of which is the histopathological hallmark of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder affecting both upper and lower motor neurons. Besides motor symptoms, patients with ALS often develop nonneuronal signs including glucose intolerance, but the underlying pathomechanism is still controversial, i.e., whether it is impaired insulin secretion and/or insulin resistance. Here, we showed that ALS subjects reduced early-phase insulin secretion and that the nuclear localization of TDP-43 was lost in the islets of autopsied ALS pancreas. Loss of TDP-43 inhibited exocytosis by downregulating CaV1.2 calcium channels, thereby reducing early-phase insulin secretion in a cultured [beta] cell line (MIN6) and [beta] cell-specific Tardbp-knockout mice. Overexpression of CaV1.2 restored early-phase insulin secretion in Tardbp- knocked-down MIN6 cells. Our findings suggest that TDP-43 regulates cellular exocytosis mediated by L-type voltage-dependent calcium channels and, thus, plays an important role in the early phase of insulin secretion by pancreatic islets. Thus, nuclear loss of TDP-43 is implicated in not only the selective loss of motor neurons, but also in glucose intolerance due to impaired insulin secretion at an early stage of ALS., Introduction Amyotrophic lateral sclerosis (ALS) is a selective motor neuron disorder leading to death in 3-5 years after the onset, due to rapid progression of muscle atrophy, including atrophy of [...]

Cystic fibrosis (CF) is a multiorgan progressive genetic disease caused by loss of functional cystic fibrosis transmembrane conductance regulator (CFTR) channel. Previously, we identified a significant dysfunction in CF cells and model mice of the transcription factor nuclear factor E2-related factor-2 (Nrf2), a major regulator of redox balance and inflammatory signaling. Here we report that the approved F508del CFTR correctors VX809 and VX661 recover diminished Nrf2 function and colocalization with CFTR in CF human primary bronchial epithelia by proximity ligation assay, immunoprecipitation, and immunofluorescence, concordant with CFTR correction. F508del CFTR correctors induced Nrf2 nuclear translocation, Nrf2-dependent luciferase activity, and transcriptional activation of target genes. Rescue of Nrf2 function by VX809/VX661 was dependent on significant correction of F508del and was blocked by inhibition of corrected channel function, or high-level shRNA knockdown of CFTR or F508del CFTR. Mechanistically, F508del CFTR modulation restored Nrf2 phosphorylation and its interaction with the coactivator CREB-binding protein (CBP). Our findings demonstrate that sufficient modulation of F508del CFTR function corrects Nrf2 dysfunction in CF., Introduction Cystic fibrosis (CF) is an autosomal recessive disease characterized by progressive loss of lung function, pancreatic exocrine dysfunction, and gastrointestinal complications, which culminate in dramatically shortened life expectancy. CF [...]

A purified F-actin-derived actin trimer that interacts with end-binding proteins did not activate or bind the side-binding protein myosin under rigor conditions. Remodeling of the actin trimer by the binding of gelsolin did not rescue myosin binding, nor did the use of different means of inhibiting the polymerization of the trimer. Our results demonstrate that ADP-ribosylation on all actin subunits of an F-actin-derived trimer inhibits myosin binding and that the binding of DNase-I to the pointed end subunits of a crosslinked trimer also remodels the myosin binding site. Taken together, this work highlights the need for a careful balance between modification of actin subunits and maintaining protein-protein interactions to produce a physiologically relevant short F-actin complex. Key words: actomyosin, conformational change, protein crosslinking, polymerization inhibition. Un trimere d'actine forme a partir de F-actine purifiee qui interagit avec les proteines de liaison terminales n'activait ni ne liait la myosine, une proteine de liaison laterale, en condition de rigidite. Le remodelage du trimere d'actine par la liaison de la gelsoline ne retablissait pas la liaison a la myosine, ni l'utilisation de differents moyens pour inhiber la polymerisation du trimere. Les resultats des auteurs demontrent que l'ADP-ribosylation de toutes les sous-unites d'actine d'un trimere forme a partir de F-actine inhibe la liaison de la myosine et que la liaison de la DNase-I aux sous-unites de l'extremite pointue d'un trimere reticule remodele aussi le site de liaison a la myosine. Dans l'ensemble, ce travail met en lumiere le besoin d'un equilibre delicat entre la modification des sous-unites de l'actine et le maintien des interactions proteine-proteine pour produire un complexe court de F-actine d'interet physiologique. [Traduit par la Redaction] Mots-cles : actomyosine, changement conformationnel, reticulation des proteines, inhibition de la polymerisation., Introduction Actin is a highly regulated cytoskeletal protein. Rarely do actin proteins exist on their own in cells, unbound by an actin-binding protein (ABP) modulating where and when actin functions [...]

A protein without natural binding functions was engineered to bind HIV-1 integrase. Phage display selections applied a library of variants based on the C-terminal domain of the eye lens protein human γS-crystallin. Multiple loop regions were altered to encode libraries with ≈3.6 × 10(11) different variants. A crystallin variant, termed integrase binding protein-10 (IBP-10), inhibits integrase catalysis with nanomolar K(i) values. IBP-10 interacts with the integrase C-terminal domain and inhibits integrase substrate affinity. This allosteric mechanism allows IBP-10 to inhibit drug-resistant integrase variants. The results demonstrate the applicability of the crystallin scaffold for the discovery of binding partners and enzyme inhibitors.

The insulin/insulin-like growth factors (IGFs) have crucial tasks in the growth, differentiation, and proliferation of healthy and pernicious cells. They are involved in coordinated complexes, including receptors, ligands, binding proteins, and proteases. However, the systems can become dysregulated in tumorigenesis. Insulin-like growth factor-binding protein 7 (IGFBP7) is a protein belonging to the IGFBP superfamily (also termed GFBP-related proteins). Numerous studies have provided evidence that IGFBP3 and IGFBP7 are involved in a variety of cancers, including hepatocellular carcinoma (HCC), breast cancer, gastroesophageal cancer, colon cancer, prostate cancer, among many others. Still, very few suggest an interaction between these two molecules. In studying several cancer types in our laboratories, we found that both proteins share some crucial signaling pathways. The objective of this review is to present a comprehensive overview of the relationship between IGFBP7 and cancer, as well as highlighting IGFBP3 crosstalk with IGFBP7 reported in recent studies.

Microdialysis allows for a preview into local muscle metabolism and can provide physiological insight that blood measurements cannot.Purpose: To examine the potential differential IGF-I system regulation in interstitial fluid during unilateral stretch shortening cycle exercise.Methods: 10 men (26 ± 7 year) performed unilateral jumping [stretch shortening cycle (SSC) exercise at 50% of optimal jump height] until volitional fatigue on a sled apparatus. Biological sampling took place using a catheter inserted into an antecubital vein (serum), and 100 kDa microdialysis probes inserted into the thigh muscle of each exercise/control leg (dialysate). Serum was drawn before (Pre; −3 h) and after SSC [Post I (+0 h), II (+3 h), or III (+20 h)]; dialysate was sampled for 2 h before (Pre), during/immediately after (Ex), and 3 h into recovery (Rec) following SSC. IGF-I system parameters (free/total IGF-I and IGFBPs 1–6) were measured with immunoassays. Interstitial free IGF-I was estimated from dialysate IGF-I and relative recovery (ethanol) correction. Data were analyzed with repeated measures ANOVA.Results: Serum total IGF-I remained elevated +3 h (Post II: 182.8 ± 37.6 vs. Pre: 168.3 ± 35.0 ng/mL, p < 0.01), but returned to baseline by +20 h (Post III vs. Pre, p = 0.31). No changes in serum free IGF-I were noted. Serum BP-1 and −3 increased over baseline, but not until + 20 h after SSC (Post III vs. Pre: 7.6 ± 4.9 vs. 3.7 ± 2.3 and 1,048.6 ± 269.2 vs. 891.4 ± 171.2 ng/mL, respectively). We observed a decreased serum BP-6 +3 h after SSC (p < 0.01), followed by a return to baseline at +20 h (p = 0.64 vs. Pre). There were no exercise-induced changes in serum BP-2, −4, or −5. Unlike serum, there were no changes in dialysate or interstitial free IGF-I in either leg (p > 0.05). Dialysate BP-1 remained increased in both exercise and control legs through 3 h into recovery (Rec vs. Pre, p < 0.01). Dialysate BP-3 also demonstrated a prolonged elevation over Pre SSC concentrations, but in the exercise leg only (Ex and Rec vs. Pre, p < 0.04). We observed a prolonged decrease in dialysate BP-5 (Ex and Rec vs. Pre, p < 0.03) and an increase in BP-4 IP in the exercise leg only. There were no changes relative to Pre SSC in dialysate BP-2 or −6.Conclusions: Unilateral exercise drives differential regulation of the IGF-I system at both local and systemic levels. More specifically, this is the first study to demonstrate that localized exercise increases IGFBP-3, IGFBP-4 and decreases in IGFBP-5 in muscle interstitial fluid.

Microdialysis allows for a preview into local muscle metabolism and can provide physiological insight that blood measurements cannot. Purpose: To examine the potential differential IGF-I system regulation in interstitial fluid during unilateral stretch shortening cycle exercise. Methods: 10 men (26 ± 7 year) performed unilateral jumping [stretch shortening cycle (SSC) exercise at 50% of optimal jump height] until volitional fatigue on a sled apparatus. Biological sampling took place using a catheter inserted into an antecubital vein (serum), and 100 kDa microdialysis probes inserted into the thigh muscle of each exercise/control leg (dialysate). Serum was drawn before (Pre; −3 h) and after SSC [Post I (+0 h), II (+3 h), or III (+20 h)]; dialysate was sampled for 2 h before (Pre), during/immediately after (Ex), and 3 h into recovery (Rec) following SSC. IGF-I system parameters (free/total IGF-I and IGFBPs 1–6) were measured with immunoassays. Interstitial free IGF-I was estimated from dialysate IGF-I and relative recovery (ethanol) correction. Data were analyzed with repeated measures ANOVA. Results: Serum total IGF-I remained elevated +3 h (Post II: 182.8 ± 37.6 vs. Pre: 168.3 ± 35.0 ng/mL, p < 0.01), but returned to baseline by +20 h (Post III vs. Pre, p = 0.31). No changes in serum free IGF-I were noted. Serum BP-1 and −3 increased over baseline, but not until + 20 h after SSC (Post III vs. Pre: 7.6 ± 4.9 vs. 3.7 ± 2.3 and 1,048.6 ± 269.2 vs. 891.4 ± 171.2 ng/mL, respectively). We observed a decreased serum BP-6 +3 h after SSC (p < 0.01), followed by a return to baseline at +20 h (p = 0.64 vs. Pre). There were no exercise-induced changes in serum BP-2, −4, or −5. Unlike serum, there were no changes in dialysate or interstitial free IGF-I in either leg (p > 0.05). Dialysate BP-1 remained increased in both exercise and control legs through 3 h into recovery (Rec vs. Pre, p < 0.01). Dialysate BP-3 also demonstrated a prolonged elevation over Pre SSC concentrations, but in the exercise leg only (Ex and Rec vs. Pre, p < 0.04). We observed a prolonged decrease in dialysate BP-5 (Ex and Rec vs. Pre, p < 0.03) and an increase in BP-4 IP in the exercise leg only. There were no changes relative to Pre SSC in dialysate BP-2 or −6. Conclusions : Unilateral exercise drives differential regulation of the IGF-I system at both local and systemic levels. More specifically, this is the first study to demonstrate that localized exercise increases IGFBP-3, IGFBP-4 and decreases in IGFBP-5 in muscle interstitial fluid. [ABSTRACT FROM AUTHOR]

The insulin/insulin-like growth factors (IGFs) have crucial tasks in the growth, differentiation, and proliferation of healthy and pernicious cells. They are involved in coordinated complexes, including receptors, ligands, binding proteins, and proteases. However, the systems can become dysregulated in tumorigenesis. Insulin-like growth factor-binding protein 7 (IGFBP7) is a protein belonging to the IGFBP superfamily (also termed GFBP-related proteins). Numerous studies have provided evidence that IGFBP3 and IGFBP7 are involved in a variety of cancers, including hepatocellular carcinoma (HCC), breast cancer, gastroesophageal cancer, colon cancer, prostate cancer, among many others. Still, very few suggest an interaction between these two molecules. In studying several cancer types in our laboratories, we found that both proteins share some crucial signaling pathways. The objective of this review is to present a comprehensive overview of the relationship between IGFBP7 and cancer, as well as highlighting IGFBP3 crosstalk with IGFBP7 reported in recent studies. [ABSTRACT FROM AUTHOR]

• The interaction between combinatorial histone modifications and tandem-domain reader proteins was identified. Four tandem-domain proteins (BPTF-PB, CBP-BP, TRIM24-PB, TAF1-BB) could read the peptides with dual-modifications. • The binding affinities were detected by isothermal titration calorimetry. The interaction between BPTF-PB and peptides with PTMs is the strongest. • The binding proteins to the tandem-domains were quantified. 78 enriched proteins were further characterized. • The molecule network of "histone modification-reader-binding proteins" was analyzed. Histone posttranslational modifications (HPTMs) play important roles in eukaryotic transcriptional regulation. Recently, it has been suggested that combinatorial modification codes that comprise two or more HPTMs can recruit readers of HPTMs, performing complex regulation of gene expression. However, the characterization of the multiplex interactions remains challenging, especially for the molecular network of histone PTMs, readers and binding complexes. Here, we developed an integrated method that combines a peptide library, affinity enrichment, mass spectrometry (MS) and bioinformatics analysis for the identification of the interaction between HPTMs and their binding proteins. Five tandem-domain-reader proteins (BPTF, CBP, TAF1, TRIM24 and TRIM33) were designed and prepared as the enriched probes, and a group of histone peptides with multiple PTMs were synthesized as the target peptide library. First, the domain probes were used to pull down the PTM peptides from the library, and then the resulting product was characterized by MS. The binding interactions between PTM peptides and domains were further validated and measured by isothermal titration calorimetry analysis (ITC). Meanwhile, the binding proteins were enriched by domain probes and identified by HPLC-MS/MS. The interaction network of histone PTMs-readers-binding complexes was finally analyzed via informatics tools. Our results showed that the integrated approach combining MS analysis with ITC assay enables us to understand the interaction between the combinatorial HPTMs and reading domains. The identified network of "HPTMs-reader proteins-binding complexes" provided potential clues to reveal HPTM functions and their regulatory mechanisms. [ABSTRACT FROM AUTHOR]

Bacillus thuringiensis produces insecticidal Cry toxins used in the control of multiple insect pests. Evolution of insect resistance to Bt toxins endangers the use of Cry toxins for pest control. Analysis of the Cry1Ah-binding proteins from brush border membrane vesicles (BBMV) of Ostrinia furnacalis , Asian corn borer (ACB) from the Cry1Ah-resistant (ACB-AhR) and susceptible (ACB-BtS) strains was performed by an improved pull down assay that includes coupling Cry1Ah to NHS-activated Sepharose combined with liquid chromatography–tandem mass spectrometry (LC-MS/MS). Our data show that Cry1Ah bound to alkaline phosphatase (ALP), cadherin-like (CAD), actin, aminopeptidase-N (APN), prophenoloxidase (proPO), serine proteinase inhibitor (SPI), immulectin, and V-ATPase and to other proteins that were not previously characterized as Cry-binding proteins in ACB-BtS strain. Analysis of Cry1Ah-pulled down proteins of the BBMV from ACB-AhR revealed that Cry1Ah toxin did not bind to ALP in ACB-AhR strain, suggesting that this protein may correlate with the resistant phenotype of this strain. Additionally, we analyzed the expression of representative genes coding for Cry1Ah-binding proteins such as ALP, APN, CAD, proPO, SPI , and immulectin by qRT-PCR. ACB-AhR showed increased expression levels of proPO (7.5 fold), ALP (6.2 fold) and APN (1.4 fold) in comparison to ACB-BtS strain. In contrast, the cad gene showed slight decreased expression in ACB-AhR strain (0.7 fold) compared with ACB-BtS strain. Our data suggest that differences in the susceptibility to Cry1Ah toxin in the ACB-AhR strain may be associated with reduced ALP binding sites and with an increased immune response. This study also brings evidence of a possible binding interaction of Cry1Ah toxin to immune related proteins like proPO. Unlabelled Image • Cry1Ah bound to ALP, CAD, actin, APN, prophenoloxidase, SRI, immulectin, and V-ATPase in ACB-BtS. • Cry1Ah toxin in the ACB-AhR strain associated with reduced ALP binding sites and with an increased immune response. • The Cry1Ah-binding proteins showed increased expression levels in ACB-AhR comparison to ACB-BtS strain. • Direct binding of Cry1Ah toxin to immune related proteins like proPO. [ABSTRACT FROM AUTHOR]

Purpose For patients suffering from prolonged critical illness, it is unknown whether and when the hypothalamus-pituitary-adrenal axis alterations recover, and to what extent adrenocortical function parameters relate to sepsis/septic shock, to clinical need for glucocorticoid treatment, and to survival. Methods Patients still in ICU on day 7 (N = 392) and 20 matched healthy subjects were included. Morning blood and 24-h urine were collected daily and cosyntropin tests (250 [micro]g) performed weekly, repeated 1 week after ICU discharge on the regular ward. Results In all patients free of glucocorticoid treatment up until ICU day 28 (N = 347), plasma ACTH always remained low/normal, whereas free cortisol remained high (P [less than or equal to] 0.002) explained by reduced binding proteins (P [less than or equal to] 0.02) and suppressed cortisol breakdown (P [less than or equal to] 0.001). Beyond ICU day 28 (N = 64 long-stayers), plasma (free)cortisol was no longer elevated. One week after ICU discharge, plasma ACTH and (free)cortisol always rose to supra-normal levels (P [less than or equal to] 0.006), most pronounced in long-stayers. Long-stayers always showed low incremental total (P [less than or equal to] 0.001), but normal incremental free cortisol responses to weekly cosyntropin tests, explained by low cortisol plasma binding proteins. Sepsis/septic shock patients were not different from others, patients subsequently receiving glucocorticoids (N = 45) were not different from those who did not, and non-survivors were distinguishable from survivors only by higher (free)cortisol. Conclusions Irrespective of sepsis/septic shock, need for glucocorticoids and survival, low cortisol plasma binding proteins and suppressed cortisol breakdown determine systemic (free)cortisol availability in prolonged critical illness, the latter no longer elevated beyond ICU day 28. The uniform rise in ACTH and cortisol to supra-normal levels 1 week after ICU discharge indicates recovery of a central adrenocortical suppression while in ICU. Low cortisol plasma binding invalidates the cosyntropin test., Author(s): Bram Peeters [sup.1], Philippe Meersseman [sup.1] [sup.2], Sarah Vander Perre [sup.1], Pieter J. Wouters [sup.1], Dimitri Vanmarcke [sup.1], Yves Debaveye [sup.1], Jaak Billen [sup.3], Pieter Vermeersch [sup.3], Lies Langouche [...]

Common genetic contributions to autism spectrum disorder (ASD) reside in risk gene variants that individually have minimal effect sizes. As environmental factors that perturb neurodevelopment also underlie idiopathic ASD, it is crucial to identify altered regulators that can orchestrate multiple ASD risk genes during neurodevelopment. Cytoplasmic polyadenylation element binding proteins 1-4 (CPEB1-4) regulate the translation of specific mRNAs by modulating their poly(A)-tails and thereby participate in embryonic development and synaptic plasticity. Here we find that CPEB4 binds transcripts of most high-confidence ASD risk genes. The brains of individuals with idiopathic ASD show imbalances in CPEB4 transcript isoforms that result from decreased inclusion of a neuron-specific microexon. In addition, 9% of the transcriptome shows reduced poly(A)-tail length. Notably, this percentage is much higher for high-confidence ASD risk genes, correlating with reduced expression of the protein products of ASD risk genes. An equivalent imbalance in CPEB4 transcript isoforms in mice mimics the changes in mRNA polyadenylation and protein expression of ASD risk genes and induces ASD-like neuroanatomical, electrophysiological and behavioural phenotypes. Together, these data identify CPEB4 as a regulator of ASD risk genes.CPEB4 binds the mRNA of genes known to be associated with autism and shows an isoform imbalance in individuals with autism, and an equivalent imbalance in mice induces an autism-like phenotype., Author(s): Alberto Parras [sup.1] [sup.2] , Héctor Anta [sup.3] [sup.4] , María Santos-Galindo [sup.1] [sup.2] , Vivek Swarup [sup.5] , Ainara Elorza [sup.1] [sup.2] , José L. Nieto-González [sup.2] [sup.6] [...]

Over 100 types of cellular RNA modifications have been identified in both coding and a variety of non-coding RNAs. N6-methyladenosine (m6A) is the most prevalent and abundant post-transcriptional RNA modification on eukaryote mRNA, and its biological functions are mediated by special binding proteins (i.e., methyltransferases, demethylases, and effectors) that recognize this modification. The presence of m6A on transcripts contributes to diverse fundamental cellular functions, such as pre-mRNA splicing, nuclear transport, stability, translation, and microRNA biogenesis, implying an association with numerous human diseases. This review principally summarizes recent progress in the study of m6A methylation mechanisms and relevant roles they play in immunoregulation.

Insulin-like growth factor-I (IGF-I) is associated with muscle hypertrophy, and circulating IGF-I levels are correlated with fitness. To test the hypothesis that IGF-I increases with increased physical activity in adolescent males, 38 subjects (16 +/- 0.7 yr old) were randomized to control (n = 18) or increased physical activity groups for 5 wk. Before and after the intervention, we measured thigh muscle volume using magnetic resonance imaging and serum levels of mean growth hormone (GH) by overnight multiple sampling, GH binding protein (GHBP), IGF-I, and IGFBPs 1-5 by standard assays. Energy expenditure was assessed with the doubly labeled water technique toward the end of the study. In the training subjects there was 1) a significant increase in thigh muscle volume (+3.6 +/- 1%), 2) 15.5 +/- 3.3% greater energy expenditure than in controls, and 3) no evidence of weight loss (+1.44 +/- 0.4%). In contrast to our hypothesis, but similar to our recent observations in adolescent females, training decreased IGF-I (-12 +/- 4%, P < 0. 005). Moreover, training substantially reduced GHBP (-21 +/- 4%, P < 0.00002) and increased IGFBP-2 (+40 +/- 16%, P < 0.008). Brief training increased muscle volume in weight-stable adolescent males and, surprisingly, influenced not only IGF-I but GHBP and IGFBP-2 as well in a manner typically found in energy-deficient states.

PurposeThe growth effects of exercise appear to be mediated in part by central neuroendocrine control reflected in circulating levels of growth hormone (GH), insulin-like growth factor-I (IGF-I), and their binding proteins (BP). In previous studies positive correlations between peak VO2 and circulating IGF-I have been demonstrated. The relationship between peak oxygen uptake and these potential regulating factors has not been examined in adolescent males where patterns of GH pulsatility and levels of IGF-I are rapidly changing.MethodsForty-three healthy adolescent males (age 16 +/- 0.7 yr, 70% at Tanner V) performed cycle ergometry to determine p oxygen uptake (peak VO2), and magnetic resonance images to determine the thigh muscle volume. Baseline blood samples were collected for GHBP, the extracellular portion of the GH tissue receptor (by ligand mediated immunofunctional assay), IGF-I (by RIA), and IGFBPs 1-5 (by RIA). Mean GH was determined from samples obtained every 20 min overnight.ResultsPeak VO2/kg was positively correlated with mean overnight GH levels (r = 0.41, P < 0.005). Both peak VO2/kg and thigh muscle volume/kg were negatively correlated with GHBP (r = -0.33, P < 0.02) and IGFBP-4 (r = -0.52, P < 0.005). There were no correlations between peak VO2/kg and IGF-I or IGFBPs 1-3, and 5.ConclusionsGH pulsatility is increased adolescent males who have higher peak VO2, but this did not translate into increases in IGF-I. We speculate that in the fitter males, lower GHBP levels may reduce hepatic sensitivity to GH. Thus, circulating IGF-I was unchanged despite higher mean GH in subjects with higher peak VO2. IGFBP-4 which is known to inhibit IGF-I was negatively correlated with peak VO2 leading, possibly, to increased IGF-I bioactivity. Fitness (as assessed by muscle mass and peak VO2) does modulate the GH-IGF-I axis, but not solely through circulating IGF-I; both GHBP and IGFBPs play important roles.

PurposeThe growth effects of exercise appear to be mediated in part by central neuroendocrine control reflected in circulating levels of growth hormone (GH), insulin-like growth factor-I (IGF-I), and their binding proteins (BP). In previous studies positive correlations between peak VO2 and circulating IGF-I have been demonstrated. The relationship between peak oxygen uptake and these potential regulating factors has not been examined in adolescent males where patterns of GH pulsatility and levels of IGF-I are rapidly changing.MethodsForty-three healthy adolescent males (age 16 +/- 0.7 yr, 70% at Tanner V) performed cycle ergometry to determine p oxygen uptake (peak VO2), and magnetic resonance images to determine the thigh muscle volume. Baseline blood samples were collected for GHBP, the extracellular portion of the GH tissue receptor (by ligand mediated immunofunctional assay), IGF-I (by RIA), and IGFBPs 1-5 (by RIA). Mean GH was determined from samples obtained every 20 min overnight.ResultsPeak VO2/kg was positively correlated with mean overnight GH levels (r = 0.41, P < 0.005). Both peak VO2/kg and thigh muscle volume/kg were negatively correlated with GHBP (r = -0.33, P < 0.02) and IGFBP-4 (r = -0.52, P < 0.005). There were no correlations between peak VO2/kg and IGF-I or IGFBPs 1-3, and 5.ConclusionsGH pulsatility is increased adolescent males who have higher peak VO2, but this did not translate into increases in IGF-I. We speculate that in the fitter males, lower GHBP levels may reduce hepatic sensitivity to GH. Thus, circulating IGF-I was unchanged despite higher mean GH in subjects with higher peak VO2. IGFBP-4 which is known to inhibit IGF-I was negatively correlated with peak VO2 leading, possibly, to increased IGF-I bioactivity. Fitness (as assessed by muscle mass and peak VO2) does modulate the GH-IGF-I axis, but not solely through circulating IGF-I; both GHBP and IGFBPs play important roles.

Cisplatin and its analogues are widely used as chemotherapeutic agents in clinical practice. After being intravenously administrated, a substantial amount of platinum will bind with proteins in the blood. This binding is vital for the transport, distribution, and metabolism of drugs; however, toxicity can also occur from the irreversible binding between biologically active proteins and platinum drugs. Therefore, it is very important to study the protein-binding behavior of platinum drugs in blood. This review summarizes mass spectrometry-based strategies to identify and quantitate the proteins binding with platinum anticancer drugs in blood, such as offline high-performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC–ICP-MS) combined with electrospray ionization mass spectrometry (ESI-MS/MS) and multidimensional LC–ESI-MS/MS. The identification of in vivo targets in blood cannot be accomplished without first studying the protein-binding behavior of platinum drugs in vitro; therefore, relevant studies are also summarized. This knowledge will further our understanding of the pharmacokinetics and toxicity of platinum anticancer drugs, and it will be beneficial for the rational design of metal-based anticancer drugs.

Highlights • This study focused on hepatic igfbp responses to EDCs in Atlantic salmon. • In fry and smolts, hepatic ghr, igf1 and -2 were diminished by EDC exposure. • In fry, EDCs diminished igfbp1b1, -2a, -2b1, -4, -5b2 and -6b1. • In smolts, EDCs diminished igfbp1b1, -4 and -6b1. • igfbp5a was stimulated by EDCs in both fry and smolts. Abstract Feminizing endocrine disrupting compounds (EDCs) affect the growth and development of teleost fishes. The major regulator of growth performance, the growth hormone (Gh)/insulin-like growth-factor (Igf) system, is sensitive to estrogenic compounds and mediates certain physiological and potentially behavioral consequences of EDC exposure. Igf binding proteins (Igfbps) are key modulators of Igf activity, but their alteration by EDCs has not been examined. We investigated two life-stages (fry and smolts) of Atlantic salmon (Salmo salar), and characterized how the Gh/Igf/Igfbp system responded to waterborne 17α-ethinylestradiol (EE 2), 17β-estradiol (E 2) and 4-nonylphenol (NP). Fry exposed to EE 2 and NP for 21 days had increased hepatic vitellogenin (vtg) mRNA levels while hepatic estrogen receptor α (erα), gh receptor (ghr) , igf1 and igf2 mRNA levels were decreased. NP-exposed fry had reduced body mass and total length compared to controls. EE 2 and NP reduced hepatic igfbp1b1 , -2a , -2b1 , -4 , -5b2 and -6b1 , and stimulated igfbp5a. In smolts, hepatic vtg mRNA levels were induced following 4-day exposures to all three EDCs, while erα only responded to EE 2 and E 2. EDC exposures did not affect body mass or fork length; however, EE 2 diminished plasma Gh and Igf1 levels in parallel with reductions in hepatic ghr and igf1. In smolts, EE 2 and E 2 diminished hepatic igfbp1b1 , -4 and -6b1 , and stimulated igfbp5a. There were no signs of compromised ionoregulation in smolts, as indicated by unchanged branchial ion pump/transporter mRNA levels. We conclude that hepatic igfbps respond (directly and/or indirectly) to environmental estrogens during two key life-stages of Atlantic salmon, and thus may modulate the growth and development of exposed individuals. [ABSTRACT FROM AUTHOR]

Poxviruses are large, DNA viruses whose protein capsid is surrounded by one or more lipid envelopes. Embedded into these lipid envelopes are three conserved viral proteins which are thought to mediate binding of virions to target cells. While the function of these proteins has been studied in vitro, their specific roles during the pathogenesis of poxviral disease remain largely unclear. Here we present data demonstrating that the putative chondroitin binding protein M083 from the leporipoxvirus myxoma virus is a significant virulence factor during infection of susceptible Oryctolagus rabbits. Removal of M083 results in a reduced capacity of virus to spread beyond the regional lymph nodes and completely eliminates infectionmediated mortality. In vitro, removal of M083 results in only minor intracellular replication defects but causes a significant reduction in the ability of myxoma virus to spread from infected epithelial cells onto primary lymphocytes. We hypothesize that the physiological role of M083 is therefore to mediate the spread of myxoma virus onto rabbit lymphocytes, allowing these cells to disseminate virus throughout infected rabbits. IMPORTANCE Poxviruses represent both a class of human pathogens and potential therapeutic agents for the treatment of human malignancy. Understanding the basic biology of these agents is therefore significant to human health in a variety of ways. While the mechanisms mediating poxviral binding have been well studied in vitro, how these mechanisms impact poxviral pathogenesis in vivo remains unclear. The current study advances our understanding of how poxviral binding impacts viral pathogenesis by demonstrating that the putative chondroitin binding protein M083 plays a critical role during the pathogenesis of myxoma virus in susceptible Oryctolagus rabbits by impacting viral dissemination through changes in the transfer of virions onto primary splenocytes. [ABSTRACT FROM AUTHOR]

Transgenic rice expressing Bacillus thuringiensis (Bt) Cry1Ac and Cry2Aa toxins have proven resistant to lepidopteran pests in China, including the striped stem borer, Chilo suppressalis . Identifying the molecular mechanisms through which Cry toxins affect insect pests is essential, both for understanding the nature of their toxicity, and for assessing the potential for the development of resistance to such toxins. In this study, both one-dimensional gel electrophoresis (1-DE), combined ligand blots and mass spectrometry, were used to identify proteins binding to the Cry1Ac and Cry2Aa toxins in brush border membrane vesicles (BBMVs) of the C. suppressalis larvae midgut. Aminopeptidase-N (APN), alkaline phosphatase (ALP), cadherin-like (CAD), ATP-binding cassette transporter subfamily C1 protein (ABCC1), actin, ATPase, alpha amylase, and heat shock protein cognate (HSPC), were detected in Cry1Ac binding bands, and APN, actin, ATPase and HSPC were identified as Cry2Aa potential binding proteins. The novel potential binding protein, delta-aminolevulinic acid dehydratase, was also detected in both Cry1Ac and Cry2Aa binding bands. This is the first study to comprehensively identify the proteins that bind to Cry1Ac and Cry2Aa toxins in C. suppressalis . [ABSTRACT FROM AUTHOR]

A technician job can become a satisfying career -- or act as a solid stepping stone.A technician job can become a satisfying career -- or act as a solid stepping stone., Author(s): Charlotte SchubertAuthor Affiliations:How working as a research technician can bolster your scientific career From technician to programme manager: Jenniffer Mabuka-Maroa (right) during a postdoc with her mentor Thumbi Ndung'u. [...]