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Human embryonic stem cells are a type of pluripotent stem cells (hPSCs) that are used to investigate their differentiation into diverse mature cell types for molecular studies. The mechanisms underlying insulin receptor (IR)-mediated signaling in the maintenance of human pluripotent stem cell (hPSC) identity and cell fate specification are not fully understood. Here, we used two independent shRNAs to stably knock down IRs in two hPSC lines that represent pluripotent stem cells and explored the consequences on expression of key proteins in pathways linked to proliferation and differentiation. We consistently observed lowered pAKT in contrast to increased pERK1/2 and a concordant elevation in pluripotency gene expression. ERK2 chromatin immunoprecipitation, luciferase assays, and ERK1/2 inhibitors established direct causality between ERK1/2 and OCT4 expression. Of importance, RNA sequencing analyses indicated a dysregulation of genes involved in cell differentiation and organismal development. Mass spectrometry-based proteomic analyses further confirmed a global downregulation of extracellular matrix proteins. Subsequent differentiation toward the neural lineage reflected alterations in SOX1 + PAX6 + neuroectoderm and FOXG1 + cortical neuron marker expression and protein localization. Collectively, our data underscore the role of IR-mediated signaling in maintaining pluripotency, the extracellular matrix necessary for the stem cell niche, and regulating cell fate specification including the neural lineage., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The pleiotropic hormone leptin has a pivotal role in regulating energy balance by inhibiting hunger and increasing energy expenditure. Homozygous mutations found in the leptin gene are associated with extreme obesity, marked hyperphagia, and impaired immune function. Although these mutations have been characterized in vivo , a detailed understanding of how they affect leptin structure and function remains elusive. In the current work, we used NMR, differential scanning calorimetry, molecular dynamics simulations, and bioinformatics calculations to characterize the effects of these mutations on leptin structure and function and binding to its cognate receptor. We found that mutations identified in patients with congenital leptin deficiency not only cause leptin misfolding or aggregation, but also cause changes in the dynamics of leptin residues on the receptor-binding interface. Therefore, we infer that mutation-induced leptin deficiency may arise from several distinct mechanisms including (i) blockade of leptin receptor interface II, (ii) decreased affinity in the second step of leptin's interaction with its receptor, (iii) leptin destabilization, and (iv) unsuccessful threading through the covalent loop, leading to leptin misfolding/aggregation. We propose that this expanded framework for understanding the mechanisms underlying leptin deficiency arising from genetic mutations may be useful in designing therapeutics for leptin-associated disorders., (© 2018 Haglund et al.)

WhiB-like (Wbl) proteins are well known for their diverse roles in actinobacterial morphogenesis, cell division, virulence, primary and secondary metabolism, and intrinsic antibiotic resistance. Gene disruption experiments showed that three different Actinobacteria (Mycobacterium smegmatis, Streptomyces lividans, and Rhodococcus jostii) each exhibited a different whiB7-dependent resistance profile. Heterologous expression of whiB7 genes showed these resistance profiles reflected the host's repertoire of endogenous whiB7-dependent genes. Transcriptional activation of two resistance genes in the whiB7 regulon, tap (a multidrug transporter) and erm(37) (a ribosomal methyltransferase), required interaction of WhiB7 with their promoters. Furthermore, heterologous expression of tap genes isolated from Mycobacterium species demonstrated that divergencies in drug specificity of homologous structural proteins contribute to the variation of WhiB7-dependent drug resistance. WhiB7 has a specific tryptophan/glycine-rich region and four conserved cysteine residues; it also has a peptide sequence (AT-hook) at its C terminus that binds AT-rich DNA sequence motifs upstream of the promoters it activates. Targeted mutagenesis showed that these motifs were required to provide antibiotic resistance in vivo. Anaerobically purified WhiB7 from S. lividans was dimeric and contained 2.1 ± 0.3 and 2.2 ± 0.3 mol of iron and sulfur, respectively, per protomer (consistent with the presence of a 2Fe-2S cluster). However, the properties of the dimer's absorption spectrum were most consistent with the presence of an oxygen-labile 4Fe-4S cluster, suggesting 50% occupancy. These data provide the first insights into WhiB7 iron-sulfur clusters as they exist in vivo, a major unresolved issue in studies of Wbl proteins.

The Elongator complex is composed of 6 subunits (Elp1-Elp6) and promotes RNAPII transcript elongation through histone acetylation in the nucleus as well as tRNA modification in the cytoplasm. This acetyltransferase complex directly or indirectly regulates numerous biological processes ranging from exocytosis and resistance to heat shock in yeast to cell migration and neuronal differentiation in higher eukaryotes. The identity of human ELP1 through ELP4 has been reported but human ELP5 and ELP6 have remained uncharacterized. Here, we report that DERP6 (ELP5) and C3ORF75 (ELP6) encode these subunits of human Elongator. We further investigated the importance and function of these two subunits by a combination of biochemical analysis and cellular assays. Our results show that DERP6/ELP5 is required for the integrity of Elongator and directly connects ELP3 to ELP4. Importantly, the migration and tumorigenicity of melanoma-derived cells are significantly decreased upon Elongator depletion through ELP1 or ELP3. Strikingly, DERP6/ELP5 and C3ORF75/ELP6-depleted melanoma cells have similar defects, further supporting the idea that DERP6/ELP5 and C3ORF75/ELP6 are essential for Elongator function. Together, our data identify DERP6/ELP5 and C3ORF75/ELP6 as key players for migration, invasion and tumorigenicity of melanoma cells, as integral subunits of Elongator.

Antifolates, which are among the first antimicrobial agents invented, inhibit cell growth by creating an intracellular state of folate deficiency. Clinical resistance to antifolates has been mainly attributed to mutations that alter structure or expression of enzymes involved in de novo folate synthesis. We identified a Mycobacterium smegmatis mutant, named FUEL (which stands for folate utilization enzyme for leucovorin), that is hypersusceptible to antifolates. Chemical complementation indicated that FUEL is unable to metabolize folinic acid (also known as leucovorin or 5-formyltetrahydrofolate), whose metabolic function remains unknown. Targeted mutagenesis, genetic complementation, and biochemical studies showed that FUEL lacks 5,10-methenyltetrahydrofolate synthase (MTHFS; also called 5-formyltetrahydrofolate cyclo-ligase; EC 6.3.3.2) activity responsible for the only ATP-dependent, irreversible conversion of folinic acid to 5,10-methenyltetrahydrofolate. In trans expression of active MTHFS proteins from bacteria or human restored both antifolate resistance and folinic acid utilization to FUEL. Absence of MTHFS resulted in marked cellular accumulation of polyglutamylated species of folinic acid. Importantly, MTHFS also affected M. smegmatis utilization of monoglutamylated 5-methyltetrahydrofolate exogenously added to the medium. Likewise, Escherichia coli mutants lacking MTHFS became susceptible to antifolates. These results indicate that folinic acid conversion by MTHFS is required for bacterial intrinsic antifolate resistance and folate homeostatic control. This novel mechanism of antimicrobial antifolate resistance might be targeted to sensitize bacterial pathogens to classical antifolates.

We biochemically simulated HIV-1 DNA polymerization in physiological nucleotide pools found in two HIV-1 target cell types: terminally differentiated/non-dividing macrophages and activated/dividing CD4(+) T cells. Quantitative tandem mass spectrometry shows that macrophages harbor 22-320-fold lower dNTP concentrations and a greater disparity between ribonucleoside triphosphate (rNTP) and dNTP concentrations than dividing target cells. A biochemical simulation of HIV-1 reverse transcription revealed that rNTPs are efficiently incorporated into DNA in the macrophage but not in the T cell environment. This implies that HIV-1 incorporates rNTPs during viral replication in macrophages and also predicts that rNTP chain terminators lacking a 3'-OH should inhibit HIV-1 reverse transcription in macrophages. Indeed, 3'-deoxyadenosine inhibits HIV-1 proviral DNA synthesis in human macrophages more efficiently than in CD4(+) T cells. This study reveals that the biochemical landscape of HIV-1 replication in macrophages is unique and that ribonucleoside chain terminators may be a new class of anti-HIV-1 agents specifically targeting viral macrophage infection.

The transcription factor nuclear factor E2-related factor 2 (Nrf2) induces the expression of antioxidant gene products that neutralize reactive oxygen species and restore redox homeostasis. Nrf2 is constitutively degraded by the ubiquitin proteolytic system in unperturbed cells, but this turnover is arrested in response to oxidative stress, thereby leading to Nrf2 accumulation. Yet, a mechanistic understanding of how Nrf2 stabilization and transcriptional activation are coupled remains to be determined. We have discovered that the ubiquitin-conjugating enzyme UbcM2 is a novel regulator of Nrf2. Recombinant Nrf2 and UbcM2 form a complex upon alkylation of a non-catalytic cysteine in UbcM2, Cys-136. Substitution of this cysteine with a phenylalanine (C136F) to mimic cysteine oxidation/alkylation results in constitutive binding of UbcM2 to Nrf2 and an increased half-life of the transcription factor in vivo. We provide evidence that UbcM2 and Nrf2 form a nuclear complex utilizing the DNA binding, Neh1 domain, of Nrf2. Finally, we demonstrate that UbcM2 can enhance the transcriptional activity of endogenous Nrf2 and that Cys-136 and the active-site cysteine, Cys-145, jointly contribute to this regulation. Collectively, these data identify UbcM2 as a novel component of the Nrf2 regulatory circuit and position cysteine 136 as a putative redox sensor in this signaling pathway. This work implicates UbcM2 in the restoration of redox homeostasis following oxidative stress.

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activity is increased in heart failure (HF), a syndrome characterized by markedly increased risk of arrhythmia. Activation of CaMKII increases peak L-type Ca(2+) current (I(Ca)) and slows I(Ca) inactivation. Whether these events are linked mechanistically is unknown. I(Ca) was recorded in acutely dissociated subepicardial and subendocardial murine left ventricular (LV) myocytes using the whole cell patch clamp method. Pressure overload heart failure was induced by surgical constriction of the thoracic aorta. I(Ca) density was significantly larger in subepicardial myocytes than in subendocardial/myocytes. Similar patterns were observed in the cell surface expression of alpha1c, the channel pore-forming subunit. In failing LV, I(Ca) density was increased proportionately in both cell types, and the time course of I(Ca) inactivation was slowed. This typical pattern of changes suggested a role of CaMKII. Consistent with this, measurements of CaMKII activity revealed a 2-3-fold increase (p < 0.05) in failing LV. To test for a causal link, we measured frequency-dependent I(Ca) facilitation. In HF myocytes, this CaMKII-dependent process could not be induced, suggesting already maximal activation. Internal application of active CaMKII in failing myocytes did not elicit changes in I(Ca). Finally, CaMKII inhibition by internal diffusion of a specific peptide inhibitor reduced I(Ca) density and inactivation time course to similar levels in control and HF myocytes. I(Ca) density manifests a significant transmural gradient, and this gradient is preserved in heart failure. Activation of CaMKII, a known pro-arrhythmic molecule, is a major contributor to I(Ca) remodeling in load-induced heart failure.

EDD (or HYD) is an E3 ubiquitin ligase in the family of HECT (homologous to E6-AP C terminus) ligases. EDD contains an N-terminal ubiquitin-associated (UBA) domain, which is present in a variety of proteins involved in ubiquitin-mediated processes. Here, we use isothermal titration calorimetry (ITC), NMR titrations, and pull-down assays to show that the EDD UBA domain binds ubiquitin. The 1.85 A crystal structure of the complex with ubiquitin reveals the structural basis of ubiquitin recognition by UBA helices alpha1 and alpha3. The structure shows a larger number of intermolecular hydrogen bonds than observed in previous UBA/ubiquitin complexes. Two of these involve ordered water molecules. The functional importance of residues at the UBA/ubiquitin interface was confirmed using site-directed mutagenesis. Surface plasmon resonance (SPR) measurements show that the EDD UBA domain does not have a strong preference for polyubiquitin chains over monoubiquitin. This suggests that EDD binds to monoubiquitinated proteins, which is consistent with its involvement in DNA damage repair pathways.

The mechanism of how fluoride causes fluorosis remains unknown. Exposure to fluoride can inhibit protein synthesis, and this may also occur by agents that cause endoplasmic reticulum (ER) stress. When translated proteins fail to fold properly or become misfolded, ER stress response genes are induced that together comprise the unfolded protein response. Because ameloblasts are responsible for dental enamel formation, we used an ameloblast-derived cell line (LS8) to characterize specific responses to fluoride treatment. LS8 cells were growth-inhibited by as little as 1.9-3.8 ppm fluoride, whereas higher doses induced ER stress and caspase-mediated DNA fragmentation. Growth arrest and DNA damage-inducible proteins (GADD153/CHOP, GADD45alpha), binding protein (BiP/glucose-responsive protein 78 (GRP78), the non-secreted form of carbonic anhydrase VI (CA-VI), and active X-box-binding protein-1 (Xbp-1) were all induced significantly after exposure to 38 ppm fluoride. Unexpectedly, DNA fragmentation increased when GADD153 expression was inhibited by short interfering RNA treatment but remained unaffected by transient GADD153 overexpression. Analysis of control and GADD153(-/-) embryonic fibroblasts demonstrated that caspase-3 mediated the increased DNA fragmentation observed in the GADD153 null cells. We also demonstrate that mouse incisor ameloblasts are sensitive to the toxic effects of high dose fluoride in drinking water. Activated Ire1 initiates an ER stress response pathway, and mouse ameloblasts were shown to express activated Ire1. Ire1 levels appeared induced by fluoride treatment, indicating that ER stress may play a role in dental fluorosis. Low dose fluoride, such as that present in fluoridated drinking water, did not induce ER stress.

Synthesis of the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25-(OH)(2)D), by renal epithelial cells is tightly controlled during normal calcium homeostasis. By contrast, macrophage production of 1,25-(OH)(2)D is often dysregulated with potential hypercalcemic complications. We have postulated that this is due to abnormal catabolism of 1,25-(OH)(2)D by the feedback control enzyme, vitamin D-24-hydroxylase (CYP24). Using chick HD-11 and human THP-1 myelomonocytic cell lines, we have shown that macrophage-like cells express a splice variant of the CYP24 gene (CYP24-SV), which encodes a truncated protein. Compared with the holo-CYP24 gene product in chick and human cells (508 and 513 amino acids, respectively), the truncated CYP24-SV versions consisted of 351 and 372 amino acids. These CYP24-SV proteins retained intact substrate-binding domains but lacked mitochondrial targeting sequences and were therefore catalytically inactive. In common with CYP24, expression of the CYP24 variants was induced by 1,25-(OH)(2)D but without a concomitant rise in 24-hydroxylase activity. However, overexpression of CYP24-SV in HD-11 and THP-1 cells reduced synthesis of 1,25-(OH)(2) D (40-50%), whereas antisense CYP24-SV expression increased 1,25-(OH)(2)D production by 2-7-fold. These data suggest that alternative splicing of CYP24 leads to the generation of a dominant negative-acting protein that is catalytically dysfunctional. We theorize that expression of the CYP24-SV may contribute to the extracellular accumulation of 1,25(OH)(2)D in human health and disease.

In Streptomyces, a family of related butyrolactones and their corresponding receptor proteins serve as quorum-sensing systems that can activate morphological development and antibiotic biosynthesis. Streptomyces pristinaespiralis contains a gene cluster encoding enzymes and regulatory proteins for the biosynthesis of pristinamycin, a clinically important streptogramin antibiotic complex. One of these proteins, PapR1, belongs to a well known family of Streptomyces antibiotic regulatory proteins. Gel shift assays using crude cytoplasmic extracts detected SpbR, a developmentally regulated protein that bound to the papR1 promoter. SpbR was purified, and its gene was cloned using reverse genetics. spbR encoded a 25-kDa protein similar to Streptomyces autoregulatory proteins of the butyrolactone receptor family, including scbR from Streptomyces coelicolor. In Escherichia coli, purified SpbR and ScbR produced bound sequences immediately upstream of papR1, spbR, and scbR. SpbR DNA-binding activity was inhibited by an extracellular metabolite with chromatographic properties similar to those of the well known gamma-butyrolactone signaling compounds. DNase I protection assays mapped the SpbR-binding site in the papR1 promoter to a sequence homologous to other known butyrolactone autoregulatory elements. A nucleotide data base search showed that these binding motifs were primarily located upstream of genes encoding Streptomyces antibiotic regulatory proteins and butyrolactone receptors in various Streptomyces species. Disruption of the spbR gene in S. pristinaespiralis resulted in severe defects in growth, morphological differentiation, pristinamycin biosynthesis, and expression of a secreted superoxide dismutase.

Protein prenylation occurs in the protozoan that causes African sleeping sickness (Trypanosoma brucei), and the protein farnesyltransferase appears to be a good target for developing drugs. We have cloned the alpha- and beta-subunits of T. brucei protein farnesyltransferase (TB-PFT) using nucleic acid probes designed from partial amino acid sequences obtained from the enzyme purified from insect stage parasites. TB-PFT is expressed in both bloodstream and insect stage parasites. Enzymatically active TB-PFT was produced by heterologous expression in Escherichia coli. Compared with mammalian protein farnesyltransferases, TB-PFT contains a number of inserts of >25 residues in both subunits that reside on the surface of the enzyme in turns linking adjacent alpha-helices. Substrate specificity studies with a series of 20 peptides SSCALX (where X indicates a naturally occurring amino acid) show that the recombinant enzyme behaves identically to the native enzyme and displays distinct specificity compared with mammalian protein farnesyltransferase. TB-PFT prefers Gln and Met at the X position but not Ser, Thr, or Cys, which are good substrates for mammalian protein farnesyltransferase. A structural homology model of the active site of TB-PFT provides a basis for understanding structure-activity relations among substrates and CAAX mimetic inhibitors.