The ATP synthase complexes in mitochondria make the ATP required to sustain life by a rotary mechanism. Their membrane domains are embedded in the inner membranes of the organelle, and they dimerize ...via interactions between their membrane domains. The dimers form extensive chains along the tips of the cristae with the two rows of monomeric catalytic domains extending into the mitochondrial matrix at an angle to each other. Disruption of the interface between dimers by mutation affects the morphology of the cristae severely. By analysis of particles of purified dimeric bovine ATP synthase by cryo-electron microscopy, we have shown that the angle between the central rotatory axes of the monomeric complexes varies between ca. 76 and 95°. These particles represent active dimeric ATP synthase. Some angular variations arise directly from the catalytic mechanism of the enzyme, and others are independent of catalysis. The monomer-monomer interaction is mediated mainly by j subunits attached to the surface of wedge-shaped protein-lipid structures in the membrane domain of the complex, and the angular variation arises from rotational and translational changes in this interaction, and combinations of both. The structures also suggest how the dimeric ATP synthases might be interacting with each other to form the characteristic rows along the tips of the cristae via other interwedge contacts, molding themselves to the range of oligomeric arrangements observed by tomography of mitochondrial membranes, and at the same time allowing the ATP synthase to operate under the range of physiological conditions that influence the structure of the cristae.
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Researchers and managers need effective tools for monitoring the use of forages by large herbivores. Since 2000, the number of herbivore diet studies has nearly doubled. In this review, we determine ...trends in the field; assess the utility of key techniques against five criteria (cost, accuracy and precision, resolution, utility for long-term monitoring programs, and appropriateness for browsers and grazers); and make recommendations to give managers appropriate tools. Three techniques stand out: microhistology, near infrared reflectance spectroscopy, and deoxyribonucleic acid (DNA) barcoding. Microhistology has a long history of use in rangelands and is often considered the gold standard for understanding diet composition, albeit at a high cost of labor. Near infrared reflectance spectroscopy can resolve the presence of target groups or species more quickly than microhistology, especially for grazers. DNA barcoding provides the greatest resolution of dietary items with less quantitative certainty than microhistology. The costs associated with DNA barcoding come primarily from technology and sequencing, while in microhistology they are associated with labor. Therefore, an improved, streamlined microhistology method could provide rangeland managers a rapid and cost-effective method for diet monitoring. Ultimately, the complex challenges facing rangeland managers today may require the use of more than one method to achieve acceptable resolution within actionable time frames.
The structure of the intact ATP synthase from the α-proteobacterium Paracoccus denitrificans, inhibited by its natural regulatory ζ-protein, has been solved by X-ray crystallography at 4.0 Å ...resolution. The ζ-protein is bound via its N-terminal α-helix in a catalytic interface in the F1 domain. The bacterial F1 domain is attached to the membrane domain by peripheral and central stalks. The δ-subunit component of the peripheral stalk binds to the N-terminal regions of two α-subunits. The stalk extends via two parallel long α-helices, one in each of the related b and b' subunits, down a noncatalytic interface of the F1 domain and interacts in an unspecified way with the a-subunit in the membrane domain. The a-subunit lies close to a ring of 12 c-subunits attached to the central stalk in the F1 domain, and, together, the central stalk and c-ring form the enzyme's rotor. Rotation is driven by the transmembrane proton-motive force, by a mechanism where protons pass through the interface between the a-subunit and c-ring via two half-channels in the a-subunit. These half-channels are probably located in a bundle of four α-helices in the a-subunit that are tilted at ∼30° to the plane of the membrane. Conserved polar residues in the two α-helices closest to the c-ring probably line the proton inlet path to an essential carboxyl group in the c-subunit in the proton uptake site and a proton exit path from the proton release site. The structure has provided deep insights into the workings of this extraordinary molecular machine.
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Mitochondrial respiratory complex I is a product of both the nuclear and mitochondrial genomes. The integration of seven subunits encoded in mitochondrial DNA into the inner membrane, their ...association with 14 nuclear-encoded membrane subunits, the construction of the extrinsic arm from 23 additional nuclear-encoded proteins, iron–sulfur clusters, and flavin mononucleotide cofactor require the participation of assembly factors. Some are intrinsic to the complex, whereas others participate transiently. The suppression of the expression of the NDUFA11 subunit of complex I disrupted the assembly of the complex, and subcomplexes with masses of 550 and 815 kDa accumulated. Eight of the known extrinsic assembly factors plus a hydrophobic protein, C3orf1, were associated with the subcomplexes. The characteristics of C3orf1, of another assembly factor, TMEM126B, and of NDUFA11 suggest that they all participate in constructing the membrane arm of complex I.
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HECT-family E3 ligases ubiquitinate protein substrates to control virtually every eukaryotic process and are misregulated in numerous diseases. Nonetheless, understanding of HECT E3s is limited ...by a paucity of selective and potent modulators. To overcome this challenge, we systematically developed ubiquitin variants (UbVs) that inhibit or activate HECT E3s. Structural analysis of 6 HECT-UbV complexes revealed UbV inhibitors hijacking the E2-binding site and activators occupying a ubiquitin-binding exosite. Furthermore, UbVs unearthed distinct regulation mechanisms among NEDD4 subfamily HECTs and proved useful for modulating therapeutically relevant targets of HECT E3s in cells and intestinal organoids, and in a genetic screen that identified a role for NEDD4L in regulating cell migration. Our work demonstrates versatility of UbVs for modulating activity across an E3 family, defines mechanisms and provides a toolkit for probing functions of HECT E3s, and establishes a general strategy for systematic development of modulators targeting families of signaling proteins.
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•High-affinity and selective UbV modulators for 20 HECT E3 ligases•UbV inhibitors hijack the E2 binding site•N-lobe exosite bound UbVs activate HECT E3 ligases•UbVs function in cells and intestinal organoids to reveal new roles of HECT E3 ligases
Zhang, Wu et al. generated ubiquitin variants (UbVs) that inhibit or activate the catalytic activities of HECT E3 ligases. These variants can be used to delve into E3 mechanisms and to probe new biological functions of HECT E3s.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Although practiced clinically for more than 40 years, the use of hematopoietic stem cell (HSC) transplants remains limited by the ability to expand these cells ex vivo. An unbiased screen with ...primary human HSCs identified a purine derivative, StemRegenin 1 (SR1), that promotes the ex vivo expansion of CD34⁺ cells. Culture of HSCs with SR1 led to a 50-fold increase in cells expressing CD34 and a 17-fold increase in cells that retain the ability to engraft immunodeficient mice. Mechanistic studies show that SR1 acts by antagonizing the aryl hydrocarbon receptor (AHR). The identification of SR1 and AHR modulation as a means to induce ex vivo HSC expansion should facilitate the clinical use of HSC therapy.
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New therapeutic strategies are needed to combat the tuberculosis pandemic and the spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) forms of the disease, which remain a serious ...public health challenge worldwide. The most urgent clinical need is to discover potent agents capable of reducing the duration of MDR and XDR tuberculosis therapy with a success rate comparable to that of current therapies for drug-susceptible tuberculosis. The last decade has seen the discovery of new agent classes for the management of tuberculosis, several of which are currently in clinical trials. However, given the high attrition rate of drug candidates during clinical development and the emergence of drug resistance, the discovery of additional clinical candidates is clearly needed. Here, we report on a promising class of imidazopyridine amide (IPA) compounds that block Mycobacterium tuberculosis growth by targeting the respiratory cytochrome bc1 complex. The optimized IPA compound Q203 inhibited the growth of MDR and XDR M. tuberculosis clinical isolates in culture broth medium in the low nanomolar range and was efficacious in a mouse model of tuberculosis at a dose less than 1 mg per kg body weight, which highlights the potency of this compound. In addition, Q203 displays pharmacokinetic and safety profiles compatible with once-daily dosing. Together, our data indicate that Q203 is a promising new clinical candidate for the treatment of tuberculosis.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Abscission is a mechanism by which plants shed entire organs in response to both developmental and environmental signals. Arabidopsis thaliana, in which only the floral organs abscise, has been used ...extensively to study the genetic, molecular and cellular processes controlling abscission. Abscission in Arabidopsis requires two genes that encode functionally redundant receptor-like protein kinases, HAESA (HAE) and HAESA-LIKE 2 (HSL2). Double hae hsl2 mutant plants fail to abscise their floral organs at any stage of floral development and maturation.
Using RNA-Seq, we compare the transcriptomes of wild-type and hae hsl2 stage 15 flowers, using the floral receptacle which is enriched for abscission zone cells. 2034 genes were differentially expressed with a False Discovery Rate adjusted p < 0.05, of which 349 had two fold or greater change in expression. Differentially expressed genes were enriched for hydrolytic, cell wall modifying, and defense related genes. Testing several of the differentially expressed genes in INFLORESCENCE DEFICIENT IN ABSCISSION (ida) mutants shows that many of the same genes are co-regulated by IDA and HAE HSL2 and support the role of IDA in the HAE and HSL2 signaling pathway. Comparison to microarray data from stamen abscission zones show distinct patterns of expression of genes that are dependent on HAE HSL2 and reveal HAE HSL2- independent pathways.
HAE HSL2-dependent and HAE HSL2-independent changes in genes expression are required for abscission. HAE and HSL2 affect the expression of cell wall modifying and defense related genes necessary for abscission. The HAE HSL2-independent genes also appear to have roles in abscission and additionally are involved in processes such as hormonal signaling, senescence and callose deposition.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
During tissue healing, the dynamic and temporal alterations required for effective repair occur in the structure and composition of the extracellular matrix (ECM). Matricellular proteins (MPs) are a ...group of diverse non-structural ECM components that bind cell surface receptors mediating interactions between the cell and its microenviroment, effectively regulating adhesion, migration, proliferation, signaling, and cell phenotype. Periostin (Postn), a pro-fibrogenic secreted glycoprotein, is defined as an MP based on its expression pattern and regulatory roles during development and healing and in disease processes. Postn consists of a typical signal sequence, an EMI domain responsible for binding to fibronectin, four tandem fasciclin-like domains that are responsible for integrin binding, and a C-terminal region in which multiple splice variants originate. This review focuses specifically on the role of Postn in wound healing and remodeling, an area of intense research during the last 10 years, particularly as related to skin healing and myocardium post-infarction. Postn interacts with cells through various integrin pairs and is an essential downstream effector of transforming growth factor-β superfamily signaling. Across various tissues, Postn is associated with the pro-fibrogenic process: specifically, the transition of fibroblasts to myofibroblasts, collagen fibrillogenesis, and ECM synthesis. Although the complexity of Postn as a modulator of cell behavior in tissue healing is only beginning to be elucidated, its expression is clearly a defining event in moving wound healing through the proliferative and remodeling phases.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
SQ109, a 1,2-diamine related to ethambutol, is currently in clinical trials for the treatment of tuberculosis, but its mode of action remains unclear. Here, we demonstrate that SQ109 disrupts cell ...wall assembly, as evidenced by macromolecular incorporation assays and ultrastructural analyses. SQ109 interferes with the assembly of mycolic acids into the cell wall core of Mycobacterium tuberculosis, as bacilli exposed to SQ109 show immediate inhibition of trehalose dimycolate (TDM) production and fail to attach mycolates to the cell wall arabinogalactan. These effects were not due to inhibition of mycolate synthesis, since total mycolate levels were unaffected, but instead resulted in the accumulation of trehalose monomycolate (TMM), the precursor of TDM and cell wall mycolates. In vitro assays using purified enzymes showed that this was not due to inhibition of the secreted Ag85 mycolyltransferases. We were unable to achieve spontaneous generation of SQ109-resistant mutants; however, analogs of this compound that resulted in similar shutdown of TDM synthesis with concomitant TMM accumulation were used to spontaneously generate resistant mutants that were also cross-resistant to SQ109. Whole-genome sequencing of these mutants showed that these all had mutations in the essential mmpL3 gene, which encodes a transmembrane transporter. Our results suggest that MmpL3 is the target of SQ109 and that MmpL3 is a transporter of mycobacterial TMM.
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