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A novel series of hydantoins incorporating phthalimides has been synthesised by condensation of activated phthalimides with 1-aminohydantoin and investigated for their inhibitory activity against a panel of human (h) carbonic anhydrase (CA, EC 4.2.1.1): the cytosolic isoforms hCA I, hCA II, and hCA VII, secreted isoform hCA VI, and the transmembrane hCA IX, by a stopped-flow CO 2 hydrase assay. Although all newly developed compounds were totally inactive on hCA I and mainly ineffective towards hCA II, they generally exhibited moderate repressing effects on hCA VI, VII, and IX with K I s values in the submicromolar to micromolar ranges. The salts 3a and 3b , followed by derivative 5 , displayed the best inhibitory activity of all the evaluated compounds and their binding mode was proposed in silico . These compounds can also be considered interesting starting points for the development of novel pharmacophores for this class of enzyme inhibitors.

Posttranslational protein arginylation has been shown as a key regulator of cellular processes in eukaryotes by affecting protein stability, function, and interaction with macromolecules. Thus, the enzyme Arginyltransferase and its targets, are of immense interest to modulate cellular processes in the normal and diseased state. While the study on the effect of this posttranslational modification in mammalian systems gained momentum in the recent times, the detail structures of human ATE1 ( h ATE1) enzymes has not been investigated so far. Thus, the purpose of this study was to predict the overall structure and the structure function relationship of h ATE1 enzyme and its four isoforms. The structure of four ATE1 isoforms were modelled and were docked with 3'end of the Arg-tRNA Arg which acts as arginine donor in the arginylation reaction, followed by MD simulation. All the isoforms showed two distinct domains. A compact domain and a somewhat flexible domain as observed in the RMSF plot. A distinct similarity in the overall structure and interacting residues were observed between h ATE1-1 and X4 compared to h ATE1-2 and 5. While the putative active sites of all the h ATE1 isoforms were located at the same pocket, differences were observed in the active site residues across h ATE1 isoforms suggesting different substrate specificity. Mining of nsSNPs showed several nsSNPs including cancer associated SNPs with deleterious consequences on h ATE1 structure and function. Thus, the current study for the first time shows the structural differences in the mammalian ATE1 isoforms and their possible implications in the function of these proteins.Communicated by Ramaswamy H. Sarma.

Vitamin D (VD) is produced by the skin upon exposure to sunlight or is obtained from dietary sources. Several risk factors are associated with VD deficiency including mutations and post-translational modifications in its transport protein known as vitamin D binding protein (VDBP) or GC-globulin. The two common single nucleotide polymorphisms rs7041 and rs4588 create three major isoforms of VDBP, including GC-1F also called wild type, GC1S, and GC-2. The 3D models for both GC-1F and GC-2 were constructed in their glycosylated states to decipher the effect of these mutations on the overall conformational changes and VD-binding affinity. The binding affinities were estimated using the Molecular Mechanics Poison-Boltzmann surface area (MM-PBSA) method and conformational changes were investigated after free energy landscapes estimations. Total free energies suggest that GC-1F exhibits stronger affinity (ΔE = -116.09 kJ/mol) than GC-2 (ΔE = -95 kJ/mol) variant with VD. The GC-1F isoforms had more streamlined motion compared to GC-2 isoforms, predicting a trade-off between cross-talk residues that significantly impacts protein structural stability. The data suggest that glycation at Thr418 plays a vital role in the overall VDBP-VD affinity by stabilizing the N-T loop that holds the domain I (VD-pocket) and domain III intact. The loss of glycation in GC-2 has a pivotal role in the inter-domain conformational stability of VDBP, which may ultimately affect VD transportation and maturation. These findings describe a novel mechanism in how mutations distant from the VD-active site change the overall conformational of the VDBP and abrogate the VDBP-VD interaction.Communicated by Ramaswamy H. Sarma.

The study investigates molecular changes in the lumbosacral (L/S) spine's yellow ligamentum flavum during degenerative stenosis, focusing on the role of transforming growth factor beta 1-3 (TGF-β-1-3). Sixty patients with degenerative stenosis and sixty control participants underwent molecular analysis using real-time quantitative reverse transcription reaction technique (RTqPCR), enzyme-linked immunosorbent assay (ELISA), Western blot, and immunohistochemical analysis (IHC). At the mRNA level, study samples showed reduced expression of TGF-β-1 and TGF-β-3 , while TGF-β-2 increased by only 4%. Conversely, at the protein level, the study group exhibited significantly higher concentrations of TGF-β-1, TGF-β-2, and TGF-β-3 compared to controls. On the other hand, at the protein level, a statistically significant higher concentration of TGF-β-1 was observed (2139.33 pg/mL ± 2593.72 pg/mL vs. 252.45 pg/mL ± 83.89 pg/mL; p  < 0.0001), TGF-β-2 (3104.34 pg/mL ± 1192.74 pg/mL vs. 258.86 pg/mL ± 82.98 pg/mL; p  < 0.0001), TGF-β-3 (512.75 pg/mL ± 107.36 pg/mL vs. 55.06 pg/mL ± 9.83 pg/mL, p  < 0.0001) in yellow ligaments obtained from patients of the study group compared to control samples. The study did not establish a significant correlation between TGF-β-1-3 concentrations and pain severity. The findings suggest that molecular therapy aimed at restoring the normal expression pattern of TGF-β-1-3 could be a promising strategy for treating degenerative stenosis of the L/S spine. The study underscores the potential therapeutic significance of addressing molecular changes at the TGF-β isoforms level for better understanding and managing degenerative spinal conditions.

Calcium signaling has been identified as an important phenomenon in a plethora of cellular processes. Inositol 1,4,5-trisphosphate receptors (IP 3 Rs) are ER-residing intracellular calcium (Ca 2+ ) release channels responsible for cell bioenergetics by transferring calcium from the ER to the mitochondria. The recent availability of full-length IP 3 R channel structure has enabled the researchers to design the IP 3 competitive ligands and reveal the channel gating mechanism by elucidating the conformational changes induced by ligands. However, limited knowledge is available for IP 3 R antagonists and the exact mechanism of action of these antagonists within a tumorigenic environment of a cell. Here in this review a summarized information about the role of IP 3 R in cell proliferation and apoptosis has been discussed. Moreover, structure and gating mechanism of IP 3 R in the presence of antagonists have been provided in this review. Additionally, compelling information about ligand-based studies (both agonists and antagonists) has been discussed. The shortcomings of these studies and the challenges toward the design of potent IP 3 R modulators have also been provided in this review. However, the conformational changes induced by antagonists for channel gating mechanism still display some major drawbacks that need to be addressed. However, the design, synthesis and availability of isoform-specific antagonists is a rather challenging one due to intra-structural similarity within the binding domain of each isoform. HighlightsThe intricate complexity of IP 3 R's in cellular processes declares them an important target whereby, the recently solved structure depicts the receptor's potential involvement in a complex network of processes spanning from cell proliferation to cell death.Pharmacological inhibition of IP 3 R attenuates the proliferation or invasiveness of cancers, thus inducing necrotic cell death.Despite significant advancements, there is a tremendous need to design new potential hits to target IP 3 R, based upon 3D structural features and pharmacophoric patterns.Communicated by Ramaswamy H. Sarma.

Transcripts from the human WDR33 gene, which encodes a central component of the mRNA polyadenylation (PA) machinery, are subject to alternative polyadenylation (APA) within promoter-proximal introns/exons. This APA, which itself involves usage of multiple PA sites, results in the production of two non-canonical protein isoforms, V2 and V3, that are functionally completely unrelated to the full-length protein, with roles in innate immunity. The mechanism and regulation of WDR33 APA are unclear. Here, we report that levels of the PA factor CFIm25 modulate V2 and V3 expression, and that PA site usage of both V2 and V3 varies in distinct immune responses. Using newly developed assays to measure splicing and PA site strength, we show that splicing of V2-associated intron 6 is inefficient, allowing V2 to be produced using weak PA sites. Usage of V3's strong PA sites, on the other hand, is relatively low, reflecting the high efficiency of intron 7 splicing coupled with dependency on usage of an alternative 3' splice site within the intron. Overall, our findings demonstrate that usage of WDR33 alternative PA sites is stochastic, dependent on a complex interplay between splicing and PA, and thus provide new insights into mechanisms underlying APA.

Smyd1, a member of the Smyd lysine methyltransferase family, plays an important role in myofibrillogenesis of skeletal and cardiac muscles. Loss of Smyd1b (a Smyd1 ortholog) function in zebrafish results in embryonic death from heart malfunction. smyd1b encodes two isoforms, Smyd1b&#95;tv1 and Smyd1b&#95;tv2, differing by 13 amino acids due to alternative splicing. While smyd1 alternative splicing is evolutionarily conserved, the isoform-specific expression and function of Smyd1b&#95;tv1 and Smyd1b&#95;tv2 remained unknown. Here we analyzed their expression and function in skeletal and cardiac muscles. Our analysis revealed expression of smyd1b&#95;tv1 predominately in cardiac and smyd1b&#95;tv2 in skeletal muscles. Using zebrafish models expressing only one isoform, we demonstrated that Smyd1b&#95;tv1 is essential for cardiomyocyte differentiation and fish viability, whereas Smyd1b&#95;tv2 is dispensable for heart development and fish survival. Cellular and biochemical analyses revealed that Smyd1b&#95;tv1 differs from Smyd1b&#95;tv2 in protein localization and binding with myosin chaperones. While Smyd1b&#95;tv2 diffused in the cytosol of muscle cells, Smyd1b&#95;tv1 was localized to M-lines and essential for sarcomere organization in cardiomyocytes. Co-IP analysis revealed a stronger binding of Smyd1b&#95;tv1 with chaperones and cochaperones compared with Smyd1b&#95;tv2. Collectively, these findings highlight the nonequivalence of Smyd1b isoforms in cardiomyocyte differentiation, emphasizing the critical role of Smyd1b&#95;tv1 in cardiac function.

The histone variant H2A.Z plays important functions in the regulation of gene expression. In mammals, it is encoded by two genes, giving rise to two highly related isoforms named H2A.Z.1 and H2A.Z.2, which can have similar or antagonistic functions depending on the promoter. Knowledge of the physiopathological consequences of such functions emerges, but how the balance between these isoforms regulates tissue homeostasis is not fully understood. Here, we investigated the relative role of H2A.Z isoforms in intestinal epithelial homeostasis. Through genome-wide analysis of H2A.Z genomic localization in differentiating Caco-2 cells, we uncovered an enrichment of H2A.Z isoforms on the bodies of genes which are induced during enterocyte differentiation, stressing the potential importance of H2A.Z isoforms dynamics in this process. Through a combination of in vitro and in vivo experiments, we further demonstrated the two isoforms cooperate for stem and progenitor cells proliferation, as well as for secretory lineage differentiation. However, we found that they antagonistically regulate enterocyte differentiation, with H2A.Z.1 preventing terminal differentiation and H2A.Z.2 favoring it. Altogether, these data indicate that H2A.Z isoforms are critical regulators of intestine homeostasis and may provide a paradigm of how the balance between two isoforms of the same chromatin structural protein can control physiopathological processes.

CD44 protein and its variant isoforms are expressed in cancer stem cells (CSCs), and various CD44 isoforms can have different functional roles in cells. Our goal was to investigate how different CD44 isoforms contribute to the emergence of stem cell (SC) overpopulation that drives colorectal cancer (CRC) development. Specific CD44 variant isoforms are selectively expressed in normal colonic SCs and become overexpressed in CRCs during tumor development. We created a unique panel of anti-CD44 rabbit genomic antibodies to 16 specific epitopes that span the entire length of the CD44 molecule. Our panel was used to comprehensively investigate the expression of different CD44 isoforms in matched pairs ( n  = 10) of malignant colonic tissue and adjacent normal mucosa, using two (IHC & IF) immunostaining approaches. We found that: i ) CD44v8-10 is selectively expressed in the normal human colonic SC niche; ii ) CD44v8-10 is co-expressed with the SC markers ALDH1 and LGR5 in normal and malignant colon tissues; iii ) colon carcinoma tissues frequently (80%) stain for CD44v8-10 while staining for CD44v6 was less frequent (40%). Given that CD44v8-10 expression is restricted to cells in the normal human colonic SC niche and CD44v8-10 expression progressively increases during CRC development, CD44v8-10 expression likely contributes to the SC overpopulation that drives the development and growth of colon cancers. Since the CD44 variant v8-10 epitope is located on CD44's extracellular region, it offers great promise for targeted anti-CSC treatment approaches.

A one-pot two-step protocol was developed for the synthesis of a series of novel 4-cyanamidobenzenesulfonamides from easily accessible methyl (4-sulfamoylphenyl)-carbamimidothioate. The new sulphonamides were investigated as inhibitors of the enzyme carbonic anhydrase (CA, EC 4.2.1.1), the human (h) cytosolic isoforms hCA I, II, VII, and XIII, as well as three bacterial enzymes belonging to the β-CA class, MscCA from Mammaliicoccus (Staphylococcus) sciuri and StCA1 and StCA2, from Salmonella enterica (serovar Typhimurium ). The human isoforms were generally effectively inhibited by these compounds, with a clear structure-activity relationship privileging long aliphatic chains (C6, C7 and C18) as substituents at the cyanamide functionality. The bacterial CAs were also inhibited by these compounds, but not as effective as the hCAs. The most sensitive enzyme to these inhibitors was StCA1 (K I s of 50.7 - 91.1 nM) whereas SscCA was inhibited in the micromolar range (K I s of 0.86-9.59 µM).

A small library of substituted cyclic guanidine incorporated benzothiazole-6-sulphonamides was synthesized. All obtained compounds were investigated for their inhibitory activity against the key brain-associated human carbonic anhydrase isoform hCA VII (a promising target for the treatment of neuropathic pain) and three isoforms expressed in brain and other tissues, hCA I, II, and IV. Sulphaguanidine derivatives 9a-d were inactive on the all investigated isoforms while the primary sulphonamide containing guanidines 6a-c and 7a-c were inactive towards hCA IV but displayed inhibiting properties on hCA I, II, and VII with K Is values in the low nanomolar to micromolar ranges. The results indicated that isoforms hCA II and VII were potently and selectively inhibited by these compounds, whereas the cytosolic hCA I was less sensitive to inhibition. The derivatives reported in this study might be useful for design of more potent and selective inhibitors of hCA II and VII.

Carbonic anhydrases (CAs) are important regulators of pH homeostasis and participate in many physiological and pathological processes. CA activators (CAAs) are becoming increasingly important in the biomedical field since enhancing CA activity may have beneficial effects at neurological level. Here, we investigate selected antihistamines, phenothiazine-based antipsychotics, and tricyclic antidepressants (TCAs) as potential activators of human CAs I, II, IV, and VII. Our findings indicate that these compounds are more effective at activating hCA II and VII compared to hCA I and IV. Overall, hCA VII was the most efficiently activated isoform, particularly by phenothiazines and TCAs. This is especially relevant since hCA VII is the most abundant isoform in the central nervous system (CNS) and is implicated in neuronal signalling and bicarbonate balance regulation. This study offers additional insights into the pharmacological profiles of clinically employed drugs and sets the ground for the development of novel optimised CAAs.

Pan-histone deacetylase (HDAC) inhibitors often have some toxic side effects. In this study, three series of novel polysubstituted N -alkyl acridone analogous were designed and synthesised as HDAC isoform-selective inhibitors. Among them, 11b and 11c exhibited selective inhibition of HDAC1, HDAC3, and HDAC10, with IC 50 values ranging from 87 nM to 418 nM. However, these compounds showed no inhibitory effect against HDAC6 and HDAC8. Moreover, 11b and 11c displayed potent antiproliferative activity against leukaemia HL-60 cells and colon cancer HCT-116 cells, with IC 50 values ranging from 0.56 μM to 4.21 μM. Molecular docking and energy scoring functions further analysed the differences in the binding modes of 11c with HDAC1/6. In vitro anticancer studies revealed that the hit compounds 11b and 11c effectively induced histone H3 acetylation, S-phase cell cycle arrest, and apoptosis in HL-60 cells in a concentration-dependent manner.

In the relentless search for new cancer treatments, organoselenium compounds, and carbonic anhydrase (CA) inhibitors have emerged as promising drug candidates. CA isoforms IX and XII are overexpressed in many types of cancer, and their inhibition is associated with potent antitumor/antimetastatic effects. Selenium-containing compounds, particularly selenols, have been shown to inhibit tumour-associated CA isoforms in the nanomolar range since the properties of the selenium atom favour binding to the active site of the enzyme. In this work, two series of selenoesters (1a-19a and 1b-19b), which gathered NSAIDs, carbo/heterocycles, and fragments from natural products, were evaluated against hCA I, II, IX, and XII. Indomethacin (17b) and flufenamic acid (19b) analogs exhibited selectivity for tumour-associated isoform IX in the low micromolar range. In summary, selenoesters that combine NSAIDs with fragments derived from natural sources have been developed as promising nonclassical inhibitors of the tumour-associated CA isoforms.

High aldehyde dehydrogenase (ALDH) activity is a metabolic feature of adult stem cells and various cancer stem cells (CSCs). The ALDEFLUOR system is currently the most commonly used method for evaluating ALDH enzyme activity in viable cells. This system is applied extensively in the isolation of normal stem cells and CSCs from heterogeneous cell populations. For many years, ALDH1A1 has been considered the most important subtype among the 19 ALDH family members in determining ALDEFLUOR activity. However, in recent years, studies of many types of normal and tumour tissues have demonstrated that other ALDH subtypes can also significantly influence ALDEFLUOR activity. In this article, we briefly review the relationships between various members of the ALDH family and ALDEFLUOR activity. The clinical significance of these ALDH isoforms in different cancers and possible directions for future studies are also summarised.

LAMP2 (lysosomal associated membrane protein 2) is one of the major protein components of the lysosomal membrane. There currently exist three LAMP2 isoforms, LAMP2A, LAMP2B and LAMP2C, and they vary in distribution and function. LAMP2A serves as a receptor and channel for transporting cytosolic proteins in a process called chaperone-mediated autophagy (CMA). LAMP2B is required for autophagosome-lysosome fusion in cardiomyocytes and is one of the components of exosome membranes. LAMP2C is primarily implicated in a novel type of autophagy in which nucleic acids are taken up into lysosomes for degradation. In this review, the current evidence for the function of each LAMP2 isoform in various pathophysiological processes and human diseases, as well as their possible mechanisms, are comprehensively summarized. We discuss the evolutionary patterns of the three isoforms in vertebrates and provide technical guidance on investigating these isoforms. We are also concerned with the newly arising questions in this particular research area that remain unanswered. Advances in the functions of the three LAMP2 isoforms will uncover new links between lysosomal dysfunction, autophagy and human diseases. Abbreviation: ACSL4: acyl-CoA synthetase long-chain family member 4; AD: Alzheimer disease; Ag: antigens; APP: amyloid beta precursor protein; ATG14: autophagy related 14; AVSF: autophagic vacuoles with unique sarcolemmal features; BBC3/PUMA: BCL2 binding component 3; CCD: C-terminal coiled coil domain; CMA: chaperone-mediated autophagy; CVDs: cardiovascular diseases; DDIT4/REDD1: DNA damage inducible transcript 4; ECs: endothelial cells; ER: endoplasmic reticulum; ESCs: embryonic stem cells; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GBA/β-glucocerebrosidase: glucosylceramidase beta; GSCs: glioblastoma stem cells; HCC: hepatocellular carcinoma; HD: Huntington disease; HSCs: hematopoietic stem cells; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; IL3: interleukin 3; IR: ischemia-reperfusion; LAMP2: lysosomal associated membrane protein 2; LDs: lipid droplets; LRRK2: leucine rich repeat kinase 2; MA: macroautophagy; MHC: major histocompatibility complex; MST1: macrophage stimulating 1; NAFLD: nonalcoholic fatty liver disease; NFE2L2/NRF2: NFE2 like bZIP transcription factor 2; NLRP3: NLR family pyrin domain containing 3; PARK7: Parkinsonism associated deglycase; PD: Parkinson disease; PEA15/PED: proliferation and apoptosis adaptor protein 15; PKM/PKM2: pyruvate kinase M1/2; RA: rheumatoid arthritis; RARA: retinoic acid receptor alpha; RCAN1: regulator of calcineurin 1; RCC: renal cell carcinoma; RDA: RNautophagy and DNautophagy; RNAi: RNA interference; RND3: Rho Family GTPase 3; SG-NOS3/eNOS: deleterious glutathionylated NOS3; SLE: systemic lupus erythematosus; TAMs: tumor-associated macrophages; TME: tumor microenvironment; UCHL1: ubiquitin C-terminal hydrolase L1; VAMP8: vesicle associated membrane protein 8.

FBXW7 is the substrate recognition component of the E3 ubiquitin ligase SCF FBW7 complex which controls the levels of CYCLINE, c-MYC and HIF1α proteins crucial for cell growth and differentiation. Mutations in FBXW7 are frequently associated with tumourigenesis. While examining FBXW7 regulation we were compelled to reevaluate a commonly used anti-FBXW7 antibody. Retinal microvascular endothelial cells (RMEC) were exposed to normoxia (21% oxygen) or hypoxia (1% oxygen) for 24 h or treated with MG132 and protein extracted for western blotting. Flag-tagged FBXW7-α, β or γ isoforms were transfected into HEK293A cells and processed using denaturing and native extraction protocols for western blotting or immunoprecipitation analysis. Two anti-FBXW7 antibodies were used, one raised to the unique FBXW7α N-terminus and the other to the C-terminus region common to all isoforms. Initial studies showed that the pan-isoform C-terminus antibody detected a single 64kDa band in RMEC rather than any of the predicted sizes for FBXW7. In contrast, expression of the isoform-specific constructs, detected with an anti-Flag antibody, confirmed the expected migratory distance of 110kDa, 68kDa and 65kDa for α, β and γ respectfully. Similarly, the N-terminus FBXW7α antibody also detected the 110kDa product. Notably, the C-terminus antibody did not recognize any of the isoforms but continued to detect a 64kDa band in all samples, including the non-transfected controls. Immunoprecipitation confirmed this lack of specificity and the inability to detect overexpressed or endogenous FBXW7α in HEK293A cells and RMEC. A commonly used C-terminus FBXW7 antibody does not detect FBXW7 under standard western blotting conditions.

The profiling of gene expression patterns to glean biological insights from single cells has become commonplace over the last few years. However, this approach overlooks the transcript contents that can differ between individual cells and cell populations. In this review, we describe early work in the field of single-cell short-read sequencing as well as full-length isoforms from single cells. We then describe recent work in single-cell long-read sequencing wherein some transcript elements have been observed to work in tandem. Based on earlier work in bulk tissue, we motivate the study of combination patterns of other RNA variables. Given that we are still blind to some aspects of isoform biology, we suggest possible future avenues such as CRISPR screens which can further illuminate the function of RNA variables in distinct cell populations.

Breast Cancer Gene 1 (BRCA1) is a tumour suppressor protein that modulates multiple biological processes including genomic stability and DNA damage repair. Although the main BRCA1 protein is well characterized, further proteomics studies have already identified additional BRCA1 isoforms with lower molecular weights. However, the accurate nucleotide sequence determination of their corresponding mRNAs is still a barrier, mainly due to the increased mRNA length of BRCA1 (~5.5 kb) and the limitations of the already implemented sequencing approaches. In the present study, we designed and employed a multiplexed hybrid sequencing approach (Hybrid-seq), based on nanopore and semi-conductor sequencing, aiming to detect BRCA1 alternative transcripts in a panel of human cancer and non-cancerous cell lines. The implementation of the described Hybrid-seq approach led to the generation of highly accurate long sequencing reads that enabled the identification of a wide spectrum of BRCA1 splice variants ( BRCA1 sv.7 - sv.52), thus deciphering the transcriptional landscape of the human BRCA1 gene. In addition, demultiplexing of the sequencing data unveiled the expression profile and abundance of the described BRCA1 mRNAs in breast, ovarian, prostate, colorectal, lung and brain cancer as well as in non-cancerous human cell lines. Finally, in silico analysis supports that multiple detected mRNAs harbour open reading frames, being highly expected to encode putative protein isoforms with conserved domains, thus providing new insights into the complex roles of BRCA1 in genomic stability and DNA damage repair.

In the present study, we report that a GFP fusion tag facilitated the soluble expression of a pea actin isoform (PEAc1) in E. coli . To investigate the influence of a GFP fusion tag on PEAc1 structure and activity, PEAc1, His-tagged PEAc1 (His-PEAc1), His-tagged GFP (His-GFP), and His-tagged PEAc1 fusion with GFP (His-PEAc1-GFP) were expressed in E. coli . SDS-PAGE and western blot analyses reveal that the solubility of His-PEAc1-GFP was higher than that of PEAc1 and His-PEAc1. The His-PEAc1-GFP and His-GFP fusion proteins were purified from the supernatant of cell homogenate on a Ni-affinity column, and PEAc1 and His-PEAc1 were purified from inclusion bodies. CD spectrum analysis of the four purified proteins indicated that the proportion of α-helix and β-sheet in PEAc1 was closest to the predicted data in His-PEAc1-GFP (compared with His-PEAc1 or PEAc1). In addition, the actin-associated activities of His-PEAc1-GFP, including polymerization to microfilaments under specific ionic conditions and DNase I inhibition by monomers, were more similar to those of muscle actin (compared with PEAc1 and His-PEAc1). These improvements in PEAc1 solubility and activity are likely the result of correct PEAc1 folding mediated by GFP fusion.

All the previously reported phenylpyrazoles as carbonic anhydrase inhibitors (CAIs) were found to have small sizes and high levels of flexibility, and hence showed low selectivity profiles toward a particular isoform of CA. Herein, we report the development of a more rigid ring system bearing a sulfonamide hydrophilic head and a lipophilic tail to develop novel molecules that are suggested to have a better selectivity toward a special CA isoform. Accordingly, three novel sets of pyrano[2,3- c ]pyrazoles attached with sulfonamide head and aryl hydrophobic tail were synthesized to enhance the selectivity toward a specific isoform of human carbonic anhydrases ( h CAs). The impact of both attachments on the potency and selectivity has been extensively discussed in terms of in vitro cytotoxicity evaluation under hypoxic conditions, structure-activity relationship and carbonic anhydrase enzyme assay. All of the new candidates displayed good cytotoxic activities against breast and colorectal carcinomas. Results of the carbonic anhydrase enzyme assay demonstrated the preferential of compounds 22 , 24 and 27 to inhibit the isoform IX of h CAs selectively. Wound-healing assay has also been performed and revealed the potential of 27 to decrease the wound closure percentage in MCF-7 cells. Molecular docking and molecular orbital analysis have finally been conducted. Results indicate the potential binding interactions of 24 and 27 with several crucial amino acids of the h CA IX.Communicated by Ramaswamy H. Sarma.

In this work, different series of benzothiazole-based sulphonamides 8a-c, 10, 12, 16a-b and carboxylic acids 14a-c were developed as novel SLC-0111 analogues with the goal of generating potent carbonic anhydrase (CA) inhibitors. The adopted strategy involved replacing the 4-fluorophenyl tail in SLC-0111 with a benzothiazole motif that attached to the ureido linker to produce compounds 8c and its regioisomers 8a-b . In addition, the ureido spacer was elongated by methylene or ethylene groups to afford the counterparts 10 and 12 . In turn, the primary sulfamoyl zinc binding group (ZBG) was either substituted or replaced by carboxylic acid functionality in order to provide the secondary sulphonamide-based SLC-0111 analogues 16a-b , and the carboxylic acid derivatives 14a-c , respectively. All compounds ( 8a-c, 10, 12, 14a-c and 16a-b ) were tested for their ability to inhibit CA isoforms CA I, II, IX and XII. Additionally, the in vitro anticancer properties of the developed CAIs were evaluated.

Cytochrome P4501B1 is a ubiquitous family protein that is majorly overexpressed in tumors and is responsible for biotransformation-based inactivation of anti-cancer drugs. This inactivation marks the cause of resistance to chemotherapeutics. In the present study, integrated in-silico approaches were utilized to identify selective CYP1B1 inhibitors. To achieve this objective, we initially developed different machine learning models corresponding to two isoforms of the CYP1 family i.e. CYP1A1 and CYP1B1. Subsequently, small molecule databases including ChemBridge, Maybridge, and natural compound library were screened from the selected models of CYP1B1 and CYP1A1. The obtained CYP1B1 inhibitors were further subjected to molecular docking and ADMET analysis. The selectivity of the obtained hits for CYP1B1 over the other isoforms was also judged with molecular docking analysis. Finally, two hits were found to be the most stable which retained key interactions within the active site of CYP1B1 after the molecular dynamics simulations. Novel compound with CYP-D9 and CYP-14 IDs were found to be the most selective CYP1B1 inhibitors which may address the issue of resistance. Moreover, these compounds can be considered as safe agents for further cell-based and animal model studies. Communicated by Ramaswamy H. Sarma.

Sirtuins are NAD + -dependent protein lysine deacylase and mono-ADP ribosylases present in both prokaryotes and eukaryotes. The sirtuin family comprises seven isoforms in mammals, each possessing different subcellular localization and biological functions. Sirtuins have received increasing attention in the past two decades given their pivotal functions in a variety of biological contexts, including cytodifferentiation, transcriptional regulation, cell cycle progression, apoptosis, inflammation, metabolism, neurological and cardiovascular physiology and cancer. Consequently, modulation of sirtuin activity has been regarded as a promising therapeutic option for many pathologies. In this review, we provide an up-to-date overview of sirtuin biology and pharmacology. We examine the main features of the most relevant inhibitors and activators, analyzing their structure-activity relationships, applications in biology, and therapeutic potential.

Aberrant alternative splicing (AS) of pre-mRNAs promotes the development and proliferation of cancerous cells. Accordingly, we had previously observed higher levels of the aryl hydrocarbon receptor nuclear translocator (ARNT) spliced variant isoform 1 in human lymphoid malignancies compared to that in normal lymphoid cells, which is a consequence of increased inclusion of alternative exon 5. ARNT is a transcription factor that has been implicated in the survival of various cancers. Notably, we found that ARNT isoform 1 promoted the growth and survival of lymphoid malignancies, but the regulatory mechanism controlling ARNT AS is unclear. Here, we report cis- and trans -regulatory elements which are important for the inclusion of ARNT exon 5. Specifically, we identified recognition motifs for the RNA-binding protein RBFOX2, which are required for RBFOX2-mediated exon 5 inclusion. RBFOX2 upregulation was observed in lymphoid malignancies, correlating with the observed increase in ARNT exon 5 inclusion. Moreover, suppression of RBFOX2 significantly reduced ARNT isoform 1 levels and cell growth. These observations reveal RBFOX2 as a critical regulator of ARNT AS in lymphoid malignancies and suggest that blocking the ARNT -specific RBFOX2 motifs to decrease ARNT isoform 1 levels is a viable option for targeting the growth of lymphoid malignancies.

There are two main types of growth hormone (GH) in the circulatory system. One is 22 kD-GH, which is the predominant isoform in the circulating system, 90% GH is present as a 22 kD protein, and 10% of GH is present as a 20 kD protein. Amino acid sequences are identical between 20 kD-GH and 22 kD-GH protein, except that 20 kD-GH lacks 15 amino acid residues 32 to 46. Studies have shown that GH has many important biological effects on mesenchymal stem cells (MSCs). However, so far, the cellular characteristics of the two types of GH have not been studied in BM-MSCs. Furthermore, the biological activity of 20 kD-GH has not been explored in BM-MSCs. For this, in the current work, BM-MSCs were used as in vitro cell model. We have carried out the current research using a series of experimental techniques (such as Western-blot and indirect immunofluorescence). Firstly, we explored the cell behavior of two types of GH in the Bm-MSC model and found that they showed different biological characteristics; Secondly, we investigated the biological characteristics of 20 kD-GH and 22 kD-GH, and results showed that 22 kD-GH and 20 kD-GH exhibited different signaling profiles; Thirdly, we found that the 20 kD-GH and 22 kD-GH Gexhibited different regulatory effects on the osteogenic differentiation of BM-MSCs. The current research lays a solid foundation for further studies on the regulatory effects of GH on MSCs.

Smoking could predispose individuals to a more severe COVID-19 by upregulating a particular gene known as mdig , which is mediated through a number of well-known histone modifications. Smoking might regulate the transcription-activating H3K4me3 mark, along with the transcription-repressing H3K9me3 and H3K27me3 marks, in a way to favor SARS-CoV-2 entry by enhancing the expression of ACE2, NRP1 and NRP2, AT1R, CTSD and CTSL, PGE2 receptors 2-4, SLC6A20 and IL-6, all of which interact either directly or indirectly with important receptors, facilitating viral entry in COVID-19.

The with-no-lysine (WNK) kinase causes pseudohypoaldosteronism type II, a genetic form of hypertension. Due to ∼80% similarity among four isoforms (WNK1/2/3/4) of the WNK protein family, the discovery of an ATP-competitive inhibitor renders a significant challenge. Here, we combined molecular modeling and molecular dynamics simulations to study the structural and conformational properties of the WNK kinase isoforms bound to an ATP competitive inhibitor (WNK463). We have also investigated the effect of phosphorylation on the conformational properties of each isoform. The largest deviation of C α atoms is observed for the unphosphorylated uWNK4 complex, while the least deviation is obtained for uWNK3. The G-loop and αC-helix regions are also more flexible in uWNK4 compared to the other three unphosphorylated isoforms. However, in uWNK1, the A-loop region is the most flexible compared to other complexes. In all cases, phosphorylation stabilizes different regions of the protein-inhibitor complexes. In the case of uWNK4, relatively higher anti-correlated motions are observed compared to the other three unphosphorylated complexes. Furthermore, in the case of uWNK4, the distance between N- and C-lobes is found to be slightly higher than other complexes. This distance is reduced in all four complexes after the phosphorylation. Principal component analyses suggest that the phosphorylation leads to structural stabilization in WNK1 and WNK4, while it causes more flexibility in WNK2 and WNK3. Overall, our study provides comprehensive and comparative information on the structural dynamics of the WNK isoform family with the known competitive inhibitor that would aid in the development of a new inhibitor.Communicated by Ramaswamy H. Sarma.

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein that is characteristic for astrocytes and neural stem cells, and their malignant analogues in glioma. Since the discovery of the protein 50 years ago, multiple alternative splice variants of the GFAP gene have been discovered, leading to different GFAP isoforms. In this review, we will describe GFAP isoform expression from gene to protein to network, taking the canonical isoforms GFAPα and the main alternative variant GFAPδ as the starting point. We will discuss the relevance of studying GFAP and its isoforms in disease, with a specific focus on diffuse gliomas.

Thymidylate kinase (TMPK) phosphorylates deoxythymidine monophosphate (dTMP) and plays an important role in genome stability. Deficiency in TMPK activity due to genetic alterations of DTYMK , i.e., the gene coding for TMPK, causes severe microcephaly in humans. However, no defects were observed in other tissues, suggesting the existence of a compensatory enzyme for dTTP synthesis. In search for this compensatory enzyme we analyzed 6 isoforms of TMPK mRNA deposited in the GenBank. Of these, only isoform 1 has been characterized and represents the known human TMPK. Our results reveal that isoform 2, 3, 4 and 5 lack essential structural elements for substrate binding and, thus, they are considered as nonfunctional isoforms. Isoform 6, however, has intact catalytic centers, i.e., dTMP-binding, DRX motif, ATP-binding p-loop and lid region, which are the key structural elements of an active TMPK, suggesting that isoform 6 may function as TMPK. When isoform 6 was expressed and purified, it showed only minimal activity (<0.1%) as compared with isoform 1. A putative isoform 6 was detected in a cancer cell line, in addition to the dominant isoform 1. However, because of its low activity, isoform 6 is unlikely be able to compensate for the loss of TMPK activity caused by deletions and/or point mutations of the DTYMK gene. Thereby, future studies to identify and characterize the compensatory TMPK enzyme found in patients with DTYMK mutations may contribute to the understanding of dTTP synthesis and of the pathophysiological role of DTYMK mutations in neurodegenerative disorders.

4IgB7-H3 ( 4Ig ) and 2IgB7-H3 ( 2Ig ) expression characteristics in acute myeloid leukemia (AML) remain unknown. This study investigated mRNA and membrane protein expression of two B7-H3 isoforms in AML cell lines and de novo patients by using RT-PCR and flow cytometry, and analyzed the B7-H3 promoter methylation state by utilizing RQ-MSP. 4Ig was the dominant isoform. 2Ig mRNA expression rate and abundance were elevated in AML in comparison with controls ( P = 0.000 and 0.000), while no significant difference in 4Ig ( P = 0.802, P = 0.398). Membrane protein levels of B7-H3 isoforms in AML was higher than controls, detected by total B7-H3 expression rate ( P = 0.002), total B7-H3 mean fluorescence intensity (MFI) ratio of blast cells and lymphocytes (MFI ratio) ( P = 0.000), and 4Ig B7-H3 MFI ratio ( P = 0.005). Compared with 2Ig low group, 2Ig high patients had older age, lower NPM1 mutation, higher FLT3-ITD mutation, and declining complete remission (CR) rates ( P = 0.026, 0.012, 0.047, and 0.028). In B7-H3 high group, there was a trend toward older age, M4 and M5, worse karyotype, and lower CR rates, although with no marked difference ( P > 0.05). The overall survival (OS) of 2Ig high and B7-H3 high groups were shorter than that of 2Ig low and B7-H3 low groups in the whole and non-M3 AML cohorts ( P = 0.006 and 0.046; P = 0.003 and 0.032). Besides, an unmethylated B7-H3 promoter was identified. In conclusion, 2Ig mRNA and total B7-H3 membrane protein tended to have potential diagnostic value for AML. Specifically, high 2Ig mRNA and total B7-H3 membrane protein expression indicate worse OS. 4Ig and 2Ig expression are methylation-independent.

As the main exchanger of electroneutral NaCl absorption, sodium-hydrogen exchanger isoform 3 (NHE3) circulates in the epithelial brush border (BB) and intracellular compartments in a multi-protein complex. The size of the NHE3 complex changes during rapid regulation events. Recycling regulation of NHE3 in epithelial cells can be roughly divided into three stages. First, when stimulated by Ca 2+ , cGMP, and cAMP-dependent signaling pathways, NHE3 is converted from an immobile complex found at the apical microvilli (MV) into an easily internalized and mobile form that relocates to a compartment near the base of the MV. Second, NHE3 is internalized by clathrin and albumin-dependent pathways into cytoplasmic endosomal compartments, where the complex is reprocessed and reassembled. Finally, NHE3 is translocated from the recycling endosomes (REs) to the apex of epithelial cells, a process that can be stimulated by an increase in sodium-glucose cotransporter 1 (SGLT1) activity, epidermal growth factor receptor (EGFR) signaling, Ca 2+ signaling, and binding to βPix and SH3 and multiple ankyrin repeat domains 2 (Shank2) proteins. This review describes the molecular steps and protein interactions involved in the recycling movement of NHE3 from the apex of epithelial cells, into vesicles, where it is reprocessed and reassembled, and returned to its original location on the plasma membrane, where it exerts its physiological function.

Aberrant chaperone-mediated autophagy (CMA) activation has been suggested as a tumorigenesis-promoting event in various cancers, although its roles in prostate cancer (PCa) remain elusive. Emerging evidence indicates that TPD52 isoform 1 , a prostate-specific and androgen-responsive gene, contributes to the malignant progression of PCa. Here, we demonstrate that TPD52 enhances CMA activation by interacting with HSPA8/HSC70 and enhancing substrate degradation in PCa. Elevation of TPD52 is essential for CMA-induced PCa cell proliferation and stress resistance in vitro and in vivo . Furthermore, TPD52 is acetylated by KAT2B at K163, which is a process that can be antagonized by HDAC2. Inactivation of HDAC2 results in elevated TPD52 acetylation, which compromises the interaction between TPD52 and HSPA8, leading to impaired CMA function and tumor growth in vivo . Taken together, our findings reveal that acetylation-dependent regulation of TPD52 modulates CMA oncogenic function in PCa, thereby suggesting the possibility of targeting the TPD52-mediated CMA pathway to control the progression of PCa. Abbreviations : CMA: chaperone-mediated autophagy; HDAC2: histone deacetylase 2; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; KAT2B: lysine acetyltransferase 2B; LAMP2A: lysosomal associated membrane protein 2A; PCa: prostate cancer; TPD52: tumor protein D52.

Background: Circulating levels of soluble receptor for advanced glycation end products (sRAGE) and advanced glycation end products (AGEs) correlate with aging/cardiovascular risk, which is delayed in long-living individuals (LLIs). AGEs/sRAGE isoforms (cleaved RAGE [cRAGE] and secretory RAGE [esRAGE]) ratio is a valuable marker for disease risk. Results: We evaluated circulating sRAGE isoforms, and AGEs in LLIs (n = 95; 90-105 years) and controls (n = 94; 11-89 years). cRAGE decreased with age in controls and further declined in LLIs. esRAGE increased in LLIs. AGEs rose with age in controls and decreased in LLIs that were characterized by a lower AGEs/sRAGE ratio. Notably, cRAGE and AGE/esRAGE ratio better discriminated controls from LLIs. Conclusion: circulating cRAGE could be considered a reliable marker of chronological age while esRAGE a protective factor for longevity.

The GLUT is a key regulator of glucose metabolism and is widely expressed on the surface of most cells of the body. GLUT provides a variety of nutrients for the growth, proliferation and differentiation of cells. In recent years, the development of drugs affecting the energy intake of tumor cells has become a research hotspot. GLUT inhibitors are gaining increased attention because they can block the energy supply of malignant tumors. Herein, we elaborate on the structure and function of GLUT1, the structural and functional differences among GLUT1-4 transporters and the relationship between GLUT1 and tumor development, as well as GLUT1 transporter inhibitors, to provide a reference for the development of new GLUT1 inhibitors.

Proper base-pairing of a miRNA with its target mRNA is a key step in miRNA-mediated mRNA repression. RNA remodelling by RNA-binding proteins (RBPs) can improve access of miRNAs to their target mRNAs. The largest isoform p45 of the RBP AUF1 has previously been shown to remodel viral or AU-rich RNA elements. Here, we show that AUF1 is capable of directly promoting the binding of the miRNA let-7b to its target site within the 3'UTR of the POLR2D mRNA. Our data suggest this occurs in two ways. First, the helix-destabilizing RNA chaperone activity of AUF1 disrupts a stem-loop structure of the target mRNA and thus exposes the miRNA target site. Second, the RNA annealing activity of AUF1 drives hybridization of the miRNA and its target site within the mRNA. Interestingly, the RNA remodelling activities of AUF1 were found to be isoform-specific. AUF1 isoforms containing a YGG motif are competent RNA chaperones, whereas isoforms lacking the YGG motif are not. Overall, our study demonstrates that AUF1 has the ability to modulate a miRNA-target site interaction, thus revealing a new regulatory function for AUF1 proteins during post-transcriptional control of gene expression. Moreover, tests with other RBPs suggest the YGG motif acts as a key element of RNA chaperone activity.

Casein kinase 1 (CK1) is an extensively expressed serine/threonine kinase family, with six highly conserved isoforms of human CK1. Due to its involvement in many biological processes, CK1 is a promising target for several pathological states, including circadian sleep disorder, neurodegenerative diseases, cancer and inflammation. However, due to the structural similarities between the six CK1 members, the design of CK1 inhibitors is intricate. So far, no effective CK1 inhibitors are reported to reach clinical trials; thus, approaches to obtaining both selective and effective CK1 inhibitors are in great demand. Here we analyze several CK1 inhibitors that provide successful experience for structure-based drug design and rational structure modification, which could provide references for further drug design.

We have earlier shown that p53-FL and its translational isoform ∆40p53 are differentially regulated. In this study, we have investigated the cellular effect of ∆40p53 regulation on downstream gene expression, specifically miRNAs. Interestingly, ∆40p53 showed antagonistic regulation of miR-186-5p as compared to either p53 alone or a combination of both the isoforms. We have elucidated the miR-186-5p mediated effect of ∆40p53 in cell proliferation. Upon expression of ∆40p53, we observed a significant decrease in YY1 levels, an established target of miR-186-5p, which is involved in cell proliferation. Further assays with anti-miR-186 established the interdependence of ∆40p53- miR-186-5p-YY1- cell proliferation. The results unravel a new dimension toward the understanding of ∆40p53 functions, which seems to regulate cellular fate independent of p53FL.

LARP1 is an oncogenic RNA-binding protein required for ribosome biogenesis and cancer cell survival. From published in vitro studies, there is disparity over which of two different LARP1 protein isoforms (termed the long LI-LARP1 and short SI-LARP1) is the canonical. Here, after conducting a series of biochemical and cellular assays, we conclude that LI-LARP1 (NM&#95;033551.3 > NP&#95;056130.2) is the dominantly expressed form. We observe that SI-LARP1 (NM&#95;015315.5> NP&#95;056130.2) is epigenetically repressed and that this repression is evolutionarily conserved in all but a small subclade of mammalian species. As with other LARP family members, there are multiple potential LARP1 mRNA isoforms that appear to be censored within the nucleus. The capacity of the cell to modulate splicing and expression of these apparently 'redundant' mRNAs hints at contextually specific mechanisms of LARP1 expression.

Gap-junction channels formed by two connexin hemichannels play diverse and pivotal roles in intercellular communication and regulation. Normally hemichannels at the plasma membrane participate in autocrine and paracrine signaling, but abnormal increase in their activity can lead or contribute to various diseases. Selective inhibitors toward connexin hemichannels are of great interest. Among more than 20 identified isoforms of connexins, connexin 43 (Cx43) attracts the most interest due to its prevalence and link to cell damage in many disorders or diseases. Traditional antibacterial kanamycin decorated with hydrophobic groups yields amphiphilic kanamycins that show low cytotoxicity and prominent inhibitory effect against Cx43. This review focuses on the development of amphiphilic kanamycins as connexin hemichannel inhibitors and their future perspective.

Our body responds to environmental stress by changing the expression levels of a series of cytoprotective enzymes/proteins through multilayered regulatory mechanisms, including the KEAP1-NRF2 system. While NRF2 upregulates the expression of many cytoprotective genes, there are fundamental limitations in short-read RNA sequencing (RNA-Seq), resulting in confusion regarding interpreting the effectiveness of cytoprotective gene induction at the transcript level. To precisely delineate isoform usage in the stress response, we conducted independent full-length transcriptome profiling (isoform sequencing; Iso-Seq) analyses of lymphoblastoid cells from three volunteers under normal and electrophilic stress-induced conditions. We first determined the first exon usage in KEAP1 and NFE2L2 (encoding NRF2) and found the presence of transcript diversity. We then examined changes in isoform usage of NRF2 target genes under stress conditions and identified a few isoforms dominantly expressed in the majority of NRF2 target genes. The expression levels of isoforms determined by Iso-Seq analyses showed striking differences from those determined by short-read RNA-Seq; the latter could be misleading concerning the abundance of transcripts. These results support that transcript usage is tightly regulated to produce functional proteins under electrophilic stress. Our present study strongly argues that there are important benefits that can be achieved by long-read transcriptome sequencing., (Copyright © 2021 American Society for Microbiology.)

Growth hormone-activated STAT5b is an essential regulator of sex-differential gene expression in mouse liver; however, its impact on hepatic gene expression and epigenetic responses is poorly understood. Here, we found a substantial, albeit incomplete loss of liver sex bias in hepatocyte-specific STAT5a/STAT5b (collectively, STAT5)-deficient mouse liver. In male liver, many male-biased genes were downregulated in direct association with the loss of STAT5 binding; many female-biased genes, which show low STAT5 binding, were derepressed, indicating an indirect mechanism for repression by STAT5. Extensive changes in CpG methylation were seen in STAT5-deficient liver, where sex differences were abolished at 88% of ∼1,500 sex-differentially methylated regions, largely due to increased DNA methylation upon STAT5 loss. STAT5-dependent CpG hypomethylation was rarely found at proximal promoters of STAT5-dependent genes. Rather, STAT5 primarily regulated the methylation of distal enhancers, where STAT5 deficiency induced widespread hypermethylation at genomic regions enriched for accessible chromatin, enhancer histone marks (histone H3 lysine 4 monomethylation [H3K4me1] and histone H3 lysine 27 acetylation [H3K27ac]), STAT5 binding, and DNA motifs for STAT5 and other transcription factors implicated in liver sex differences. Thus, the sex-dependent binding of STAT5 to liver chromatin is closely linked to the sex-dependent demethylation of distal regulatory elements linked to STAT5-dependent genes important for liver sex bias., (Copyright © 2021 American Society for Microbiology.)

Tropomyosin receptor kinase A (Trk A) is a receptor tyrosine kinase activated by nerve growth factor (NGF). TrkA plays an important role in pain sensation, which leads to significant interest in the development of small molecule inhibitors of TrkA. However, TrkA and the other two highly homologous isoforms, TrkB and TrkC, are highly conserved in the ATP binding site, which suggests that achieving TrkA subtype selectivity over TrkB and TrkC in this site may be extremely challenging. Allosteric inhibitors without making any interactions with the conserved ATP binding site may present a more promising approach. Recently, selective TrkA inhibitors 1 and 2 were reported to be allosteric inhibitors targeting the DFG-out allosteric pocket. In the present study, molecular dynamics simulations and free energy calculations were carried out on TrkA in complex with ligands 1 and 2 , which was expected to provide a basis for the rational drug design of TrkA allosteric inhibitors.

A large library of saccharin and acesulfame derivatives has been synthesised and evaluated against four isoforms of human carbonic anhydrase, the two off-targets hCA I/II and the tumour related isoforms hCA IX/XII. Different strategies of scaffold modification have been attempted on both saccharin as well as acesulfame core leading to the obtainment of 60 compounds. Some of them exhibited inhibitory activity in the nanomolar range, albeit some of the performed changes led to either micromolar activity or to its absence, against hCA IX/XII. Molecular modelling studies focused the attention on the binding mode of these compounds to the enzyme. The proposed inhibition mechanism is the anchoring to zinc-bound water molecule. Docking studies along with molecular dynamics also underlined the importance of the compounds flexibility (e.g. achieved through the insertion of methylene group) which favoured potent and selective hCA inhibition.

Alterations of cell adhesion are involved in cancer progression, but the mechanisms underlying the progression and cell adhesion have remained poorly understood. Focusing on the complex between EpCAM, claudins and tetraspanins, we described a sequence of events by which of the molecules associate each other in ovarian cancer. The interactions between molecules were evaluated by immunoprecipitations and then immunoblotting. To identify the effects of complex formation on the ovarian cancer progression, the different types of ovarian cancer cell lines were compared. In this study, we report the identification of the EpCAM-claudin-4 or -7-CD82 complex in the ovarian cancer progression and metastasis in vitro. Additionally, we demonstrated palmitoylation and intra- or extra-cellular regions are critically required for the complex formation. These results represent the first direct evidence for the link between the dynamism of cell adhesion molecules and ovarian cancer progression.

IFNL2 is a potent antiviral interferon, but the regulation of its gene expression is not fully clear. Here, we report the regulation of ATG10S for IFNL2 transcription. Through sequential deletion of the IFNL2 promoter sequence, we found LP1-1 , a fragment of the promoter responding to ATG10S activity. Subcellular localization and DNA immunoprecipitation assays showed ATG10S translocating into the nucleus and binding to LP1-1 . Online prediction for transcription factor binding sites showed an IRF1 targeting locus in LP1-1 . Luciferase assays, RT-PCR, and western blot analysis revealed a core motif (CAAGAC) existing in LP1-1 , which determined ATG10S and IRF1 activity; individual nucleotide substitution showed that the functional nucleotides of ATG10S targeting were C1, A3, and C6, and the ones associated with IRF1 were A3 and G4 within the core motif. Co-immunoprecipitation assays revealed ATG10S combination with KPNA1/importin α, KPNB1/importin β, and IRF1. The knockdown of endogenous IRF1 increased ATG10S activity on IFNL2 transcription. These results indicate that ATG10S as a transcription factor competes with IRF1 for the same binding site to promote IFNL2 gene transcription. Abbreviations: ATG10: autophagy related 10; ATG10S: the shorter isoform of autophagy related 10; BD: binding domain; CM: core motif; co-IP: co-immunoprecipitation; GFP: green fluorescent protein; HCV: hepatitis C virus; IF: immunofluorescence; IFN: interferon; IRF: interferon regulatory factor; LP: lambda promoter; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; RLU: relative light unit; SQSTM1: sequestosome 1.

The p53 family transcriptional factor p73 plays a pivotal role in development. Ablation of p73 results in severe neurodevelopmental defects, chronic infections, inflammation and infertility. In addition to this, Trp73 -\- mice display severe alteration in the ciliated epithelial lining and the full-length N -terminal isoform TAp73 has been implicated in the control of multiciliogenesis transcriptional program. With our recently generated Trp73 Δ13/Δ13 mouse model, we interrogate the physiological role of p73 C-terminal isoforms in vivo . Trp73 Δ13/Δ13 mice lack exon 13 in Trp73 gene, producing an ectopic switch from the C-terminal isoforms p73α to p73β. Trp73 Δ13/Δ13 mice show a pattern of expression of TAp73 comparable to the wild-type littermates, indicating that the α to β switch does not significantly alter the expression of the gene in this cell type. Moreover, Trp73 Δ13/Δ13 do not display any significant alteration in the airway ciliated epithelium, suggesting that in this context p73β can fully substitute the function of the longer isoform p73α. Similarly, Trp73 Δ13/Δ13 ciliated epithelium of the brain ependyma also does appear defective. In this district however expression of TAp73 is not detectable, indicating that expression of the gene might be compensated by alternative mechanisms. Overall our work indicates that C-terminus p73 is dispensable for the multiciliogenesis program and suggests a possible tissue-specific effect of p73 alternative splicing.

Vascular endothelial growth factor (VEGF)-A is differentially spliced to give two functionally different isoform families; pro-angiogenic, pro-permeability VEGF-A xxx and anti-angiogenic, anti-permeability VEGF-A xxx b. VEGF-A splicing is dysregulated in several pathologies, including cancer, diabetes, and peripheral arterial disease. The bichromatic VEGF-A splicing-sensitive fluorescent reporter harboured in a transgenic mouse is a novel approach to investigate the splicing patterns of VEGF-A in vivo . We generated a transgenic mouse harbouring a splicing-sensitive fluorescent reporter designed to mimic VEGF-A terminal exon splicing (VEGF8ab) by insertion into the ROSA26 genomic locus. dsRED expression denotes proximal splice site selection (VEGF-A xxx ) and eGFP expression denotes distal splice site selection (VEGF-A xxx b). We investigated the tissue-specific expression patterns in the eye, skeletal muscle, cardiac muscle, kidney, and pancreas, and determined whether the splicing pattern could be manipulated in the same manner as endogenous VEGF-A by treatment with the SRPK1 inhibitor SPHINX 31. We confirmed expression of both dsRED and eGFP in the eye, skeletal muscle, cardiac muscle, kidney, and pancreas, with the highest expression of both fluorescent proteins observed in the exocrine pancreas. The ratio of dsRED and eGFP matched that of endogenous VEGF-A xxx and VEGF-A xxx b. Treatment of the VEGF8ab mice with SPHINX 31 increased the mRNA and protein eGFP/dsRED ratio in the exocrine pancreas, mimicking endogenous VEGF-A splicing. The VEGF-A exon 8 splicing-sensitive fluorescent reporter mouse is a novel tool to assess splicing regulation in the individual cell-types and tissues, which provides a useful screening process for potentially therapeutic splicing regulatory compounds in vivo .

The extra 5' guanine nucleotide (G-1) on tRNA His is a nearly universal feature that specifies tRNA His identity. The G-1 residue is either genome encoded or post-transcriptionally added by tRNA His guanylyltransferase (Thg1). Despite Caenorhabditis elegans being a Thg1-independent organism, its cytoplasmic tRNA His (CetRNA n His ) retains a genome-encoded G-1. Our study showed that this eukaryote possesses a histidyl-tRNA synthetase (CeHisRS) gene encoding two distinct HisRS isoforms that differ only at their N-termini. Most interestingly, its mitochondrial tRNA His (CetRNA m His ) lacks G-1, a scenario never observed in any organelle. This tRNA, while lacking the canonical identity element, can still be efficiently aminoacylated in vivo. Even so, addition of G-1 to CetRNA m His strongly enhanced its aminoacylation efficiency in vitro. Overexpression of CeHisRS successfully bypassed the requirement for yeast THG1 in the presence of CetRNA n His without G-1. Mutagenesis assays showed that the anticodon takes a primary role in CetRNA His identity recognition, being comparable to the universal identity element. Consequently, simultaneous introduction of both G-1 and the anticodon of tRNA His effectively converted a non-cognate tRNA to a tRNA His -like substrate. Our study suggests that a new balance between identity elements of tRNA His relieves HisRS from the absolute requirement for G-1.

Introduction : Progressive skeletal muscle wasting is the manifesting symptom of Duchenne muscular dystrophy, an X-linked inherited disorder triggered by primary abnormalities in the DMD gene. The almost complete loss of dystrophin isoform Dp427 causes a multi-system pathology that features in addition to skeletal muscle weakness also late-onset cardio-respiratory deficiencies, impaired metabolism and abnormalities in the central nervous system. Areas covered : This review focuses on the mass spectrometry-based proteomic characterization of X-linked muscular dystrophy with special emphasis on the identification of novel biomarker candidates in skeletal muscle tissues, as well as non-muscle tissues and various biofluids. Individual sections focus on molecular and cellular aspects of the pathogenic changes in dystrophinopathy, proteomic workflows used in biomarker research, the proteomics of the dystrophin-glycoprotein complex and the potential usefulness of newly identified protein markers involved in fibre degeneration, fibrosis and inflammation. Expert opinion : The systematic application of large-scale proteomic surveys has identified a distinct cohort of both tissue- and biofluid-associated protein species with considerable potential for improving diagnostic, prognostic and therapy-monitoring procedures. Novel proteomic markers include components involved in fibre contraction, cellular signalling, ion homeostasis, cellular stress response, energy metabolism and the immune response, as well as maintenance of the cytoskeletal and extracellular matrix.