VOLUME 278 (2003) PAGES 43991–44000This article has been withdrawn by Deniz Ozaslan, Sophie Wang, Arif M. Kocabas, and Fusun Kilic. Despite attempts, Billow A. Ahmed, John C. McCastlain, and Anca Bene could not be reached for conference on this decision. Dr. Kilic contacted the editorial office to report a concern raised in the figures of this article. An investigation by the Journal determined the following. Lane 2 from Fig. 1 was reused in lane 2 of Fig. 6B. Additionally, in Fig. 1, lanes 3 and 6 are duplicates. In Fig. 6A, lanes 2 and 3 are duplicates. Due to the dated materials, the authors could not provide all of the original immunoblots, but they state that the immunoblots were not reused.
Spectrins are cytoskeletal proteins essential for membrane biogenesis and regulation and serve critical roles in protein targeting and cellular signaling. αII spectrin (SPTAN1) is one of two α spectrin genes and αII spectrin dysfunction is linked to alterations in axon initial segment formation, cortical lamination, and neuronal excitability. Furthermore, human αII spectrin loss-of-function variants cause neurological disease. As global αII spectrin knockout mice are embryonic lethal, the in vivo roles of αII spectrin in adult heart are unknown and untested. Here, based on pronounced alterations in αII spectrin regulation in human heart failure we tested the in vivo roles of αII spectrin in the vertebrate heart. We created a mouse model of cardiomyocyte-selective αII spectrin-deficiency (cKO) and used this model to define the roles of αII spectrin in cardiac function. αII spectrin cKO mice displayed significant structural, cellular, and electrical phenotypes that resulted in accelerated structural remodeling, fibrosis, arrhythmia, and mortality in response to stress. At the molecular level, we demonstrate that αII spectrin plays a nodal role for global cardiac spectrin regulation, as αII spectrin cKO hearts exhibited remodeling of αI spectrin and altered β-spectrin expression and localization. At the cellular level, αII spectrin deficiency resulted in altered expression, targeting, and regulation of cardiac ion channels NaV1.5 and KV4.3. In summary, our findings define critical and unexpected roles for the multifunctional αII spectrin protein in the heart. Furthermore, our work provides a new in vivo animal model to study the roles of αII spectrin in the cardiomyocyte.
Small molecules and short peptides that potently and selectively bind RNA are rare, making the molecular structures of these complexes highly exceptional. Accordingly, several recent investigations have provided unprecedented structural insights into how peptides and proteins recognize the HIV-1 transactivation response (TAR) element, a 59-nucleotide-long, noncoding RNA segment in the 5′ long terminal repeat region of viral transcripts. Here, we offer an integrated perspective on these advances by describing earlier progress on TAR binding to small molecules, and by drawing parallels to recent successes in the identification of compounds that target the hepatitis C virus internal ribosome entry site (IRES) and the flavin-mononucleotide riboswitch. We relate this work to recent progress that pinpoints specific determinants of TAR recognition by: (i) viral Tat proteins, (ii) an innovative lab-evolved TAR-binding protein, and (iii) an ultrahigh-affinity cyclic peptide. New structural details are used to model the TAR–Tat–super-elongation complex (SEC) that is essential for efficient viral transcription and represents a focal point for antiviral drug design. A key prediction is that the Tat transactivation domain makes modest contacts with the TAR apical loop, whereas its arginine-rich motif spans the entire length of the TAR major groove. This expansive interface has significant implications for drug discovery and design, and it further suggests that future lab-evolved proteins could be deployed to discover steric restriction points that block Tat-mediated recruitment of the host SEC to HIV-1 TAR.
Vitamin D3 is terminally bioactivated in the kidney to 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) via cytochrome P450 family 27 subfamily B member 1 (CYP27B1), whose gene is regulated by parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), and 1,25(OH)2D3. Our recent genomic studies in the mouse have revealed a complex kidney-specific enhancer module within the introns of adjacent methyltransferase-like 1 (Mettl1) and Mettl21b that mediate basal and PTH-induced expression of Cyp27b1 and FGF23- and 1,25(OH)2D3-mediated repression. Gross deletion of these segments in mice has severe effects on Cyp27b1 regulation and skeletal phenotype but does not affect Cyp27b1 expression in nonrenal target cells (NRTCs). Here, we report a bimodal activity in the Mettl1 intronic enhancer with components responsible for PTH-mediated Cyp27b1 induction and 1,25(OH)2D3-mediated repression and additional activities, including FGF23 repression, within the Mettl21b enhancers. Deletion of both submodules eliminated basal Cyp27b1 expression and regulation in the kidney, leading to systemic and skeletal phenotypes similar to those of Cyp27b1-null mice. However, basal expression and lipopolysaccharide-induced regulation of Cyp27b1 in NRTCs was unperturbed. Importantly, dietary normalization of calcium, phosphate, PTH, and FGF23 rescued the skeletal phenotype of this mutant mouse, creating an ideal in vivo model to study nonrenal 1,25(OH)2D3 production in health and disease. Finally, we confirmed a conserved chromatin landscape in human kidney that is similar to that in mouse. These findings define a finely balanced homeostatic mechanism involving PTH and FGF23 together with protection from 1,25(OH)2D3 toxicity that is responsible for both adaptive vitamin D metabolism and mineral regulation.
Triple-negative breast cancers (TNBCs) lack progesterone and estrogen receptors and do not have amplified human epidermal growth factor receptor 2, the main therapeutic targets for managing breast cancer. TNBCs have an altered metabolism, including an increased Warburg effect and glutamine dependence, making the glutaminase inhibitor CB-839 therapeutically promising for this tumor type. Accordingly, CB-839 is currently in phase I/II clinical trials. However, not all TNBCs respond to CB-839 treatment, and the tumor resistance mechanism is not yet fully understood. Here we classified cell lines as CB-839–sensitive or –resistant according to their growth responses to CB-839. Compared with sensitive cells, resistant cells were less glutaminolytic and, upon CB-839 treatment, exhibited a smaller decrease in ATP content and less mitochondrial fragmentation, an indicator of poor mitochondrial health. Transcriptional analyses revealed that the expression levels of genes linked to lipid metabolism were altered between sensitive and resistant cells and between breast cancer tissues (available from The Cancer Genome Atlas project) with low versus high glutaminase (GLS) gene expression. Of note, CB-839–resistant TNBC cells had increased carnitine palmitoyltransferase 2 (CPT2) protein and CPT1 activity levels. In agreement, CB-839–resistant TNBC cells mobilized more fatty acids into mitochondria for oxidation, which responded to AMP-activated protein kinase and acetyl-CoA carboxylase signaling. Moreover, chemical inhibition of both glutaminase and CPT1 decreased cell proliferation and migration of CB-839–resistant cells compared with single inhibition of each enzyme. We propose that dual targeting of glutaminase and CPT1 activities may have therapeutic relevance for managing CB-839–resistant tumors.
Serine–arginine (SR) proteins are essential splicing factors that promote numerous steps associated with mRNA processing and whose biological function is tightly regulated through multi-site phosphorylation. In the nucleus, the cdc2-like kinases (CLKs) phosphorylate SR proteins on their intrinsically disordered Arg–Ser (RS) domains, mobilizing them from storage speckles to the splicing machinery. The CLKs have disordered N termini that bind tightly to RS domains, enhancing SR protein phosphorylation. The N termini also promote nuclear localization of CLKs, but their transport mechanism is presently unknown. To explore cytoplasmic–nuclear transitions, several classical nuclear localization sequences in the N terminus of the CLK1 isoform were identified, but their mutation had no effect on subcellular localization. Rather, we found that CLK1 amplifies its presence in the nucleus by forming a stable complex with the SR protein substrate and appropriating its NLS for transport. These findings indicate that, along with their well-established roles in mRNA splicing, SR proteins use disordered protein–protein interactions to carry their kinase regulator from the cytoplasm to the nucleus.
Small-molecule inhibitors of insect chitinases have potential applications for controlling insect pests. Insect group II chitinase (ChtII) is the most important chitinase in insects and functions throughout all developmental stages. However, the possibility of inhibiting ChtII by small molecules has not been explored yet. Here, we report the structural characteristics of four molecules that exhibited similar levels of inhibitory activity against OfChtII, a group II chitinase from the agricultural pest Asian corn borer Ostrinia furnacalis. These inhibitors were chitooctaose ((GlcN)8), dipyrido-pyrimidine derivative (DP), piperidine-thienopyridine derivative (PT), and naphthalimide derivative (NI). The crystal structures of the OfChtII catalytic domain complexed with each of the four inhibitors at 1.4–2.0 Å resolutions suggested they all exhibit similar binding modes within the substrate-binding cleft; specifically, two hydrophobic groups of the inhibitor interact with +1/+2 tryptophan and a −1 hydrophobic pocket. The structure of the (GlcN)8 complex surprisingly revealed that the oligosaccharide chain of the inhibitor is orientated in the opposite direction to that previously observed in complexes with other chitinases. Injection of the inhibitors into 4th instar O. furnacalis larvae led to defects in development and pupation. The results of this study provide insights into a general mechanistic principle that confers inhibitory activity against ChtII, which could facilitate rational design of agrochemicals that target ecdysis of insect pests.
Iron–sulfur clusters are essential cofactors of proteins. In eukaryotes, iron–sulfur cluster biogenesis requires a mitochondrial iron–sulfur cluster machinery (ISC) and a cytoplasmic iron–sulfur protein assembly machinery (CIA). Here we used mitochondria and cytoplasm isolated from yeast cells, and [35S]cysteine to detect cytoplasmic Fe–35S cluster assembly on a purified apoprotein substrate. We showed that mitochondria generate an intermediate, called (Fe–S)int, needed for cytoplasmic iron–sulfur cluster assembly. The mitochondrial biosynthesis of (Fe–S)int required ISC components such as Nfs1 cysteine desulfurase, Isu1/2 scaffold, and Ssq1 chaperone. Mitochondria then exported (Fe–S)int via the Atm1 transporter in the inner membrane, and we detected (Fe–S)int in active form. When (Fe–S)int was added to cytoplasm, CIA utilized it for iron–sulfur cluster assembly without any further help from the mitochondria. We found that both iron and sulfur for cytoplasmic iron–sulfur cluster assembly originate from the mitochondria, revealing a surprising and novel mitochondrial role. Mitochondrial (Fe–S)int export was most efficient in the presence of cytoplasm containing an apoprotein substrate, suggesting that mitochondria respond to the cytoplasmic demand for iron–sulfur cluster synthesis. Of note, the (Fe–S)int is distinct from the sulfur intermediate called Sint, which is also made and exported by mitochondria but is instead used for cytoplasmic tRNA thiolation. In summary, our findings establish a direct and vital role of mitochondria in cytoplasmic iron–sulfur cluster assembly in yeast cells.
Dlouhy et al. (1) recently refined how β-amyloid precursor protein (APP)2 participates in iron (Fe) efflux and hinted that APP may be unnecessary for ferroportin-supported Fe efflux. APP and Fe efflux have long been our interest, given recent work on the APP mRNA 5′-UTR and how regulation of APP translation operates through an interleukin-1 acute box, an iron response element (IRE), where iron-responsive protein 1 (IRP1) binds, and a target sequence for microRNA-346 (2). Untangling the roles of IRE, IRP1, interleukin-1, and miR-346 at the APP 5′-UTR is critical in Alzheimer's disease (AD). Fe regulation of APP levels through this site is well-documented (3). For example, that APP knockout alters Fe efflux in model mice (4) argues in favor of APP regulation of Fe efflux. Dlouhy's group (1) reported that cellular APP plays no role in Fe homeostasis, at least with ferroportin, and secreted APP (sAPP) stabilizes ferroportin at the cell membrane (5). In normal cells, APP is processed by α- or β-secretase to generate sAPPα or sAPPβ, respectively. Post-translational secretase modification of APP alters neuronal Fe homeostasis (6). Because the APP they generated used a C-terminal tag that probably prevented secretase processing, it would have eliminated sAPP. They examined the specific form of APP that was not implicated in Fe metabolism in their own previous work (5) and suitably found further lack of implication. Nevertheless, their work of APP on Fe efflux will encourage researchers to examine effects of more AD relevance, such as the sAPPα (nonamyloidogenic pathway) versus...
Malaria remains a major global health issue, affecting millions and killing hundreds of thousands of people annually. Efforts to break the transmission cycle of the causal Plasmodium parasite, and to cure those that are afflicted, rely upon functional characterization of genes essential to the parasite's growth and development. These studies are often based upon manipulations of the parasite genome to disrupt or modify a gene of interest to understand its importance and function. However, these approaches can be limited by the availability of selectable markers and the time required to generate transgenic parasites. Moreover, there also is a risk of disrupting native gene regulatory elements with the introduction of exogenous sequences. To address these limitations, we have developed CRISPR-RGR, a Streptococcus pyogenes (Sp)Cas9-based gene editing system for Plasmodium that utilizes a ribozyme–guide–ribozyme (RGR) single guide RNA (sgRNA) expression strategy with RNA polymerase II promoters. Using rodent-infectious Plasmodium yoelii, we demonstrate that both gene disruptions and coding sequence insertions are efficiently generated, producing marker-free parasites with homology arms as short as 80–100 bp. Additionally, we find that the common practice of using one sgRNA can produce both unintended plasmid integration and desired locus replacement editing events, whereas the use of two sgRNAs results in only locus replacement editing. Lastly, we show that CRISPR-RGR can be used for CRISPR interference (CRISPRi) by binding catalytically dead SpCas9 (dSpCas9) to the region upstream of a gene of interest, resulting in a position-dependent, but strand-independent reduction in gene expression. This robust and flexible system facilitates efficient genetic characterizations of rodent-infectious Plasmodium species.