Podcasts about atpases

Become a supporter of this podcast: https://www.spreaker.com/podcast/viced-rhino-the-podcast--4623273/support.Today, Creation Ministries International Canada (CMI Canada) tries to explain why DNA proves creation...and they seem to have gotten in over their heads.Cards:Critiquing Deflate's Critique of a Religious Critique!:https://youtu.be/cTCYOX4RnIcOriginal Video: http://tinyurl.com/25cc8hqwSources:DNA is life's blueprint? No, there's far more to it than that: http://tinyurl.com/29sytd7pRNA Polymerase and Transcription Mechanisms: The Forefront of Physicochemical Studies of Chemical Reactions: http://tinyurl.com/2824baycLife as a manifestation of the second law of thermodynamics: http://tinyurl.com/2876sudu The Origin of Life: A case is made for the descent of electrons: http://tinyurl.com/y4qa5esaPew Research Center's 2014 Survey of AAAS Members: http://tinyurl.com/24zzsyud On the Origin of Species: http://tinyurl.com/2a95jcwpInventing the dynamo machine: the evolution of the F-type and V-type ATPases: http://tinyurl.com/23lk6px5 The Ancestral Shape of the Access Proton Path of Mitochondrial ATP Synthases Revealed by a Split Subunit-a: http://tinyurl.com/297h7od3ATP synthases from archaea: The beauty of a molecular motor: http://tinyurl.com/299cxoceThe evolution of A-, F-, and V-type ATP synthases and ATPases: reversals in function and changes in the H+/ATP coupling ratio: http://tinyurl.com/299cxoceATP synthase: Evolution, energetics, and membrane interactions: http://tinyurl.com/2dhkofl2ATP synthases: structure, function and evolution of unique energy converters: http://tinyurl.com/23ujjohxThe progenitor of ATP synthases was closely related to the current vacuolar H+-ATPase: http://tinyurl.com/2cd4ptgdStepwise formation of the bacterial flagellar system: http://tinyurl.com/26j7wyejA reducibly complex mousetrap: http://tinyurl.com/2b8oz478Mitochondrial ATP synthase: architecture, function and pathology: http://tinyurl.com/2d3729ug Droplets: Unconventional Protocell Model with Life-Like Dynamics and Room to Grow: http://tinyurl.com/24f4tmoe Reading and writing single-atom magnets: http://tinyurl.com/24tk7qceSpontaneous formation and base pairing of plausible prebiotic nucleotides in water: http://tinyurl.com/2c2n655rIdentifying the wide diversity of extraterrestrial purine and pyrimidine nucleobases in carbonaceous meteorites: http://tinyurl.com/yyqbrajwNucleobase synthesis in interstellar ices: http://tinyurl.com/25pththmSynthetic Life with Alternative Nucleic Acids as Genetic Materials: http://tinyurl.com/27lj75h4 Origin and evolution of the genetic code: the universal enigma: http://tinyurl.com/y2e3e25xA Production of Amino Acids Under Possible Primitive Earth Conditions: http://tinyurl.com/28lz85c8A search for tryptophan in the gas of the IC 348 star cluster of the Perseus molecular cloud: http://tinyurl.com/2xmg67ysAmino acids in meteorites: http://tinyurl.com/2bvbjppmMiller–Urey experiment (Wikipedia): http://tinyurl.com/7hngnybAsymmetric autocatalysis. Chiral symmetry breaking and the origins of homochirality of organic molecules: http://tinyurl.com/2ypr22myIntelligent Design Is Not Creationism: http://tinyurl.com/28rwc67kThe Cost Of Natural Selection: https://tinyurl.com/2xr7pgy4Evolutionary Rate at the Molecular Level: https://tinyurl.com/2caxvttyAn Upper Limit on the Functional Fraction of the Human Genome: https://tinyurl.com/2cp73d77Low Fertility in Evolutionary Perspective: https://tinyurl.com/2c32ox8vWhy do humans have an Appendix?: https://tinyurl.com/2gcyaxhqConceptual and Empirical Challenges of Ascribing Functions to Transposable Elements: https://tinyurl.com/2cbvs8f7On the Immortality of Television Sets: "Function" in the Human Genome According to the Evolution-Free Gospel of ENCODE: https://tinyurl.com/y7wvwdvpComparing genomes to computer operating systems in terms of the topology and evolution of their regulatory control networks: https://tinyurl.com/27fpj2okAll my various links can be found here:http://links.vicedrhino.com

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.04.552066v1?rss=1 Authors: Charles-Orszag, A., van Wolferen, M., Lord, S. J., Albers, S.-V., Mullins, D. Abstract: Type IV pili are ancient and widespread filamentous organelles found in most bacterial and archaeal phyla where they support a wide range of functions, including substrate adhesion, DNA uptake, self aggregation, and cell motility. In most bacteria, PilT-family ATPases disassemble adhesion pili, causing them to rapidly retract and produce twitching motility, important for surface colonization. As archaea do not possess homologs of PilT, it was thought that archaeal pili cannot retract. Here, we employ live-cell imaging under native conditions (75{degrees}C and pH 2), together with automated single-cell tracking, high-temperature fluorescence imaging, and genetic manipulation to demonstrate that S. acidocaldarius exhibits bona fide twitching motility, and that this behavior depends specifically on retractable adhesion pili. Our results demonstrate that archaeal adhesion pili are capable of retraction in the absence of a PilT retraction ATPase and suggests that the ancestral type IV pilus machinery in the last universal common ancestor (LUCA) relied on such a bifunctional ATPase for both extension and retraction. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.19.537613v1?rss=1 Authors: Gupta, A., Ruturaj,, Mishra, M., Saha, S., Maji, S., Rodriguez-Boulan, E., Schreiner, R. Abstract: We suggest a model of apico-basolateral sorting in polarized epithelia using homologous Cu-ATPases as membrane cargoes. In polarized epithelia, upon copper treatment, homologous copper-ATPases ATP7A and ATP7B traffic from trans-Golgi network (TGN) to basolateral and apical membranes respectively. We characterized sorting pathways of Cu-ATPases between TGN and plasma-membrane and identified the machinery involved. ATP7A and ATP7B reside on distinct domains of TGN and in high copper, ATP7A traffics directly to basolateral membrane, whereas ATP7B traverses common-recycling, apical-sorting and apical-recycling endosomes en-route to apical membrane. Mass-spectrometry identified regulatory partners of ATP7A and ATP7B that include Adaptor Protein-1 complex. Upon knocking-out pan-AP-1, sorting of both copper-ATPases are disrupted. ATP7A loses polarity and localizes on both apical and basolateral surfaces in high copper. Contrastingly, ATP7B loses TGN-retention but retains apical polarity that becomes copper-independent. Using isoform-specific knockouts, we found that AP-1A provides directionality and TGN-retention for both Cu-ATPases, whereas, AP-1B governs polarized trafficking of ATP7B solely. Trafficking phenotypes of Wilson disease-causing ATP7B mutants that disrupts putative ATP7B-AP1 interaction further substantiates the role of AP-1 in apical sorting of ATP7B. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Title: When ketones will kill you & when they'll protect youIt's important to know when ketones are good for you and when they are bad for you. What do we know about heart function on chronic ketone body exposure? What do ketones have to do with mTORC1 and v-ATPases? How did amino acids sneak into this episode??? Check out Episode 30 of vlmd rounds. For more info, visit www.VyvyaneLohMD.com

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.08.515201v1?rss=1 Authors: Seneviratne, P. B., Lidagoster, S., Valbuena-Castor, S., Lashley, K., Saha, S., Kreitzer, G. Abstract: Kinesin family motors are microtubule (MT)-stimulated ATPases known best as transporters of cellular cargoes through the cytoplasm, regulators of MT dynamics, organizers of the mitotic spindle, and for insuring equal division of DNA during mitosis. Several kinesins have also been shown to regulate transcription by interacting with transcriptional cofactors and regulators, nuclear receptors, or with specific promotor elements on DNA. We previously showed that an LxxLL nuclear receptor box motif in the kinesin-2 family motor KIF17 mediates binding to the orphan nuclear receptor estrogen related receptor alpha (ERR1) and is responsible for the suppression of ERR1-dependent transcription by KIF17. Analysis of all kinesin family proteins revealed that multiple kinesins contain this LxxLL motif, raising the question as to whether additional kinesins motors contribute to regulation of ERR1. In this study, we interrogated the effects of multiple kinesins with LxxLL motifs on ERR1-mediated transcription. We demonstrate that the kinesin-3 motor KIF1B contains two LxxLL motifs, one of which binds to ERR1. In addition, we show that expression of a KIF1B fragment containing this LxxLL motif inhibits ERR1-dependent transcription by regulating nuclear entry of ERR1. We also provide evidence that the effects of expressing the KIF1B-LxxLL fragment on ERR1 activity are mediated by a mechanism distinct from that of KIF17. Because LxxLL domains are found in many kinesins, our data suggest an expanded role for kinesins in nuclear receptor mediated transcriptional regulation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Antihypertensive and antioxidant activity in black beans National Polytechnic Institute (Mexico)   16 July 2022    Beans are one of the most important crops for the Mexican population due to its nutritional qualities. In fact, the country is one of the top 10 producers of this legume in the world, and several studies have reflected the correlation between consumption and decreased chronic degenerative diseases, cardiovascular diseases, obesity and diabetes.   Therefore, the National School of Biological Sciences of the National Polytechnic Institute (IPN-ENCB), conducted an investigation to evaluate the antioxidant and antihypertensive activity in black beans, and found that in addition to these qualities, proteins in the bean can remove heavy metals from the body.    The polytechnic research identified bioactive peptides in the legume that have a beneficial effect as antihypertensive and antioxidant, which could favor the prevention and treatment of cardiovascular diseases, with a specific effect on blood pressure and oxidative stress.    It was determined that fasolina and lectin hydrolysates (main proteins in the Jamapa black bean) had chelating activity (removal of heavy metals in the body) and, when hydrolyzed with pepsin-pancratin, they release peptides (amino acids) with antihypertensive and antioxidant effects.    Could Eating Fruit More Often Keep Depression At Bay? Aston University (UK), July 15, 2022 People who frequently eat fruit are more likely to report greater positive mental well-being and are less likely to report symptoms of depression than those who do not, according to new research from the College of Health and Life Sciences, Aston University The study found frequent fruit eaters had greater positive mental wellbeing The study surveyed 428 adults and looked at the relationship between their consumption of fruit, vegetables, sweet and savoury food snacks and their psychological health The more often people ate fruit, the lower they scored for depression and the higher for mental well-being. Published in the British Journal of Nutrition, the study surveyed 428 adults from across the UK and looked at the relationship between their consumption of fruit, vegetables, sweet and savoury food snacks, and their psychological health. People who frequently snacked on nutrient-poor savoury foods (such as crisps) were more likely to experience 'everyday mental lapses' (known as subjective cognitive failures) and report lower mental wellbeing. A greater number of lapses, was associated with higher reported symptoms of anxiety, stress and depression, and lower mental wellbeing scores.       Effect of Short-term Quercetin, Caloric Restriction in Late Life Effective to Counter Age-Related Oxidative Macromolecular Damage. Pondicherry University (India), July 13, 2022   According to news from Pondicherry, India, research stated, "Aging is characterized by gradual accumulation of macromolecular damage leading to progressive loss of physiological function and increased susceptibility to diverse diseases. Effective anti-aging strategies involving caloric restriction or antioxidant supplementation are receiving growing attention to attenuate macromolecular damage in age associated pathology." Research from Pondicherry University, "In the present study, we for the first time investigated the effect of quercetin, caloric restriction and combined treatment (caloric restriction with quercetin) on oxidative stress parameters, acetylcholinesterase and ATPases enzyme activities in the cerebral cortex of aged male Wistar rats.  Our results demonstrate that combined treatment of caloric restriction and quercetin significantly improved the age associated decline in the activities of endogenous antioxidant enzymes [such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)] and glutathione (GSH), and nitric oxide (NO).  According to the news reporters, the research concluded: "Finally, we conclude that combined treatment of caloric restriction and quercetin in late life is an effective anti-aging therapy to counteract the age related accumulation of oxidative macromolecular damage."       How Drinking Soda on a Hot Day Can Damage Kidneys, Leading to Diabetes, Heart Disease University of Buffalo, July 18, 2022 Research demonstrates the acute deleterious effects soda can have on your kidney function when used to quench your thirst during exertion on a hot day. Drinking soda causes dehydration and raises markers for kidney disease when consumed after performing manual labor or exercise in 95-degree Fahrenheit weather. When you exert yourself in a hot environment, your body regulates blood pressure and conserves water by reducing blood flow to your kidneys. A sudden and steep drop in blood flow through your kidneys can cause acute kidney injury due to the fact that it reduces the amount of oxygen being delivered to your kidneys. Your diet has an overriding influence over the health of your kidneys, with sugar and excess protein topping the list of food components known to cause problems when consumed regularly. The study, published in the American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, found soda caused dehydration and raised markers for kidney disease when consumed after performing manual labor in 95-degree F. weather. “The volunteers completed … a 30-minute treadmill workout followed by three different five-minute lifting, dexterity and sledgehammer swinging activities. After 45 minutes of exercise, the volunteers rested for 15 minutes … while drinking 16 ounces of either a high-fructose, caffeinated soft drink or water. After the break, they repeated the cycle three more times for a total of four hours. Before leaving the laboratory, the volunteers were given more of their assigned beverages to drink before consuming any further fluids. The volume was either 1 liter or a volume equal to 115% of their body weight lost through sweating if that amount was greater.” When volunteers drank soda, 75% of them had elevated levels of creatinine in their blood, a marker for kidney injury. Only 8% of participants in the water trial had elevated creatinine. When drinking soda, volunteers also had: A lower glomerular filtration rate, another marker for kidney injury Higher uric acid levels Mild dehydration Higher levels of vasopressin, an antidiuretic hormone that raises blood pressure       Black Seed Oil Extract Causes Oral Cancer Cells To Self-Destruct Institute of Biochemistry and Biotechnology (Taiwan), July 20, 2022      A range of chemotherapeutic options for treating cancer are available, however many of the treatments are themselves associated with significant morbidity and mortality. Today many cancer sufferers search for alternatives to conventional chemotherapy. Increasingly natural alternative options are becoming available, often with little or no side-effects and concrete science is proving the effects of many natural substances against cancer.   One substance which is increasingly making the headlines is thymoquinone, an active component of Nigella sativa or black seed oil. Scientists from the Institute of Biochemistry and Biotechnology, Taiwan, have found that this remarkable compound elicits cytotoxic effects on various squamous cancer cell lines through various mechanisms.  The study examined a highly malignant strain of squamous cell carcinoma, which was taken from various patients with oral cancer; this particular cancer type also causes many other cancers of the head and neck.   The cell lines were grown in a lab and treated with different concentrations of thymoquinone. The results showed that after just 24 hours of treatment there was a significant concentration-dependent cytotoxic effect on these cells. The study further examined why this was happening. Their findings are quite remarkable as they were able to demonstrate that thymoquinone was a potent inhibitor of oral cancer cell viability via two distinct anti-neoplastic mechanisms.     No bones about it: Cannabis may be used to treat fractures Tel Aviv University researcher finds non-psychotropic compound in marijuana can help heal bone fissures Tel Aviv University (Israel), July 19, 2022 A study published in the Journal of Bone and Mineral Research by Tel Aviv University and Hebrew University researchers explores another promising new medical application for marijuana. According to the research, the administration of the non-psychotropic component cannabinoid cannabidiol (CBD) significantly helps heal bone fractures. The study, conducted on rats with mid-femoral fractures, found that CBD -- even when isolated from tetrahydrocannabinol (THC), the major psychoactive component of cannabis -- markedly enhanced the healing process of the femora after just eight weeks. Undeniable clinical potential The same team, in earlier research, discovered that cannabinoid receptors within our bodies stimulated bone formation and inhibited bone loss. This paves the way for the future use of cannabinoid drugs to combat osteoporosis and other bone-related diseases. "We found that CBD alone makes bones stronger during healing, enhancing the maturation of the collagenous matrix, which provides the basis for new mineralization of bone tissue," said Dr. Gabet. "After being treated with CBD, the healed bone will be harder to break in the future." The researchers injected one group of rats with CBD alone and another with a combination of CBD and THC. After evaluating the administration of THC and CBD together in the rats, they found CBD alone provided the necessary therapeutic stimulus.   OCCUPY PEACE & FREEDOM RALLY INFO Saturday, July 23 -- 2:00 pm Kingston, NY (at the historical 4 corners -- Crown and John Streets) Speakers: Gerald Celente Judge Andrew Napolitano Gary Null Scott Ritter Phil Giraldi (former CIA official) Live music, food and drink

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.17.383745v1?rss=1 Authors: Reyes-Aldrete, E., Dill, E., Bussetta, C., Szymanski, M. R., Diemer, G. S., Maindola, P., White, M., Choi, K., Morais, M. Abstract: Double-stranded DNA viruses package their genomes into pre-assembled protein procapsids. This process is driven by macromolecular motors that transiently assemble at a unique vertex of the procapsid and utilize homomeric ring ATPases to couple genome encapsidation to ATP hydrolysis. Here we describe biochemical and biophysical characterization of the packaging ATPase from Lactococcus lactis phage ascc{varphi}28. Size-exclusion chromatography, analytical ultracentrifugation, small angle x-ray scattering, and negative stain TEM indicate that the ~45 kDa protein formed a 443 kDa cylindrical assembly with a maximum dimension of ~155 Angstroms and radius of gyration of ~54 Angstroms. Together with the dimensions of the crystallographic asymmetric unit from preliminary X-ray diffraction experiments, these results indicate that gp11 forms a decameric D5-symmetric complex consisting of two pentameric rings related by 2-fold symmetry. Additional kinetic analysis shows that recombinantly expressed gp11 has ATPase activity comparable to that of functional ATPase rings assembled on procapsids in other genome packaging systems. Hence, gp11 forms rings in solution that likely reflect the fully assembled ATPases in active virus-bound motor complexes. Whereas ATPase functionality in other dsDNA phage packaging systems requires assembly on viral capsids, the ability to form functional rings in solution imparts gp11 with significant advantages for high resolution structural studies and rigorous biophysical/biochemical analysis. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.13.381962v1?rss=1 Authors: Cheng, C. L., Wong, M. K., Li, Y., Hochstrasser, M. Abstract: The proteasome is a large protease complex that degrades both misfolded and regulatory proteins. In eukaryotes, the 26S proteasome contains six different AAA+ ATPase subunits, Rpt1-Rpt6, which form a hexameric ring as part of the base subcomplex that drives unfolding and translocation of substrates into the proteasome core. Archaeal proteasomes contain only a single type of ATPase subunit, the proteasome-activating nucleotidase (PAN), which forms a trimer-of-dimers and is homologous to the eukaryotic Rpt subunits. A key PAN proline residue (P91) forms cis and trans peptide bonds in successive subunits around the ring, allowing efficient dimerization through upstream coiled coils. The importance of the equivalent Rpt prolines in eukaryotic proteasome assembly was unknown. We show an equivalent proline is strictly conserved in Rpt3 (in S. cerevisiae, P93) and Rpt5 (P76), well conserved in Rpt2 (P103), and loosely conserved in Rpt1 (P96) in deeply divergent eukaryotes, but in no case is its mutation strongly deleterious to yeast growth. However, the rpt2-P103A, rpt3-P93A, and rpt5-P76A mutations all cause synthetic defects with specific base assembly chaperone deletions. The Rpt5-P76A mutation decreases the levels of the protein and induces a mild proteasome assembly defect. The yeast rpt2-P103A rpt5-P76A double mutant has strong growth defects attributable to defects in proteasome base formation. Several Rpt subunits in this mutant form aggregates that are cleared, at least in part, by the Hsp42-mediated protein quality control (PQC) machinery. We propose that the conserved Rpt linker prolines promote efficient 26S proteasome base assembly by facilitating specific ATPase heterodimerization. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.28.317487v1?rss=1 Authors: Llorca, O., Lopez-Perrote, A., Hug, N., Gonzalez-Corpas, A., Rodriguez, C. F., Serna, M., Garcia-Martin, C., Boskovic, J., Fernandez-Leiro, R., Caceres, J. F. Abstract: Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that degrades aberrant mRNAs and also regulates the expression of a wide range of physiological transcripts. RUVBL1 and RUVBL2 AAA-ATPases form an hetero-hexameric ring that is part of several macromolecular complexes such as INO80, SWR1 and R2TP. Interestingly, RUVBL1-RUVBL2 ATPase activity is required for NMD activation by an unknown mechanism. Here, we show that DHX34, an RNA helicase regulating NMD initiation, directly interacts with RUVBL1-RUVBL2 in vitro and in cells. Cryo-EM reveals that DHX34 induces extensive changes in the N-termini of every RUVBL2 subunit in the complex, stabilizing a conformation that does not bind nucleotide and thereby down-regulates ATP hydrolysis of the complex. Using ATPase-deficient mutants, we find that DHX34 acts exclusively on the RUVBL2 subunits. We propose a model, where DHX34 acts to couple RUVBL1-RUVBL2 ATPase activity to the assembly of factors required to initiate the NMD response. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.20.213124v1?rss=1 Authors: Saeed, A. F. U. H., Chan, C., Guo, H., Gong, B., Guo, P., Cheng, X., Ouyang, S. Abstract: Biological motors, ubiquitous in living systems, convert chemical energy into different kinds of mechanical motions critical to cellular functions. Most of these biomotors belong to a group of enzymes known as ATPases, which adopt a multi-subunit ring-shaped structure and hydrolyze adenosine triphosphate (ATP) to generate forces. The gene product 16 (gp16), an ATPase in bacteriophage {square}29, is among the most powerful biomotors known. It can overcome substantial resisting forces from entropic, electrostatic, and DNA bending sources to package double-stranded DNA (dsDNA) into a preformed protein shell (procapsid). Despite numerous studies of the {square}29 packaging mechanism, a structure of the full-length gp16 is still lacking, let alone that of the packaging motor complex that includes two additional molecular components: a connector gp10 protein and a prohead RNA (pRNA). Here we report the crystal structure of the C-terminal domain of gp16 (gp16-CTD). Structure-based alignment of gp16-CTD with related RNase H-like nuclease domains revealed a nucleic acid binding surface in gp16-CTD, whereas no nuclease activity has been detected for gp16. Subsequent molecular dynamics (MD) simulations showed that this nucleic acid binding surface is likely essential for pRNA binding. Furthermore, our simulations of a full-length gp16 structural model highlighted a dynamic interplay between the N-terminal domain (NTD) and CTD of gp16, which may play a role in driving DNA movement into the procapsid, providing structural support to the previously proposed inchworm model. Lastly, we assembled an atomic structural model of the complete {square}29 dsDNA packaging motor complex by integrating structural and experimental data from multiple sources. Collectively, our findings provided a refined inchworm-revolution model for dsDNA translocation in bacteriophage {square}29 and suggested how the individual domains of gp16 work together to power such translocation. ABSTRACT (SHORT)Biological motors, ubiquitous in living systems, convert chemical energy into different kinds of mechanical motions critical to cellular functions. The gene product 16 (gp16) in bacteriophage {square}29 is among the most powerful biomotors known, which adopts a multi-subunit ring-shaped structure and hydrolyzes ATP to package double-stranded DNA (dsDNA) into a preformed procapsid. Here we report the crystal structure of the C-terminal domain of gp16 (gp16-CTD). Structure-based alignment and molecular dynamics (MD) simulations revealed an essential binding surface of gp16-CTD for prohead RNA (pRNA), a unique component of the motor complex. Furthermore, our simulations highlighted a dynamic interplay between the N-terminal domain (NTD) and CTD of gp16, which may play a role in driving DNA movement into the procapsid. Lastly, we assembled an atomic structural model of the complete {square}29 dsDNA packaging motor complex by integrating structural and experimental data from multiple sources. Collectively, our findings provided a refined inchworm-revolution model for dsDNA translocation in bacteriophage {square}29 and suggested how the individual domains of gp16 work together to power such translocation. Copy rights belong to original authors. Visit the link for more info

Peroxisomes are highly dynamic organelles of eukaryotic cells, carrying out essential oxidative metabolic processes. These organelles scavenge reactive oxygen species such as hydrogen peroxide (H2O2) and catabolise fatty acids, which are particular hallmarks and highly conserved features of peroxisomes among different species. Peroxisomal proteins and enzymes are encoded by nuclear DNA and therefore, targeted post-translationally into the peroxisomal matrix. A special class of proteins, collectively called peroxins, perform certain cellular tasks, such as peroxisomal matrix protein import or membrane development in order to maintain peroxisome biogenesis as well as a constant flux of matrix proteins into peroxisomes. The type II AAA+ peroxins Pex1/Pex6 are a core component of the peroxisomal matrix protein import system. ATPases of the AAA+ family of proteins generally assemble into large, macromolecular machines, structurally remodelling their substrate protein, which is driven by the hydrolysis of ATP. The main function of Pex1/6 complexes is to release the receptor Pex5 from peroxisomal membranes after matrix protein import. This relocation of Pex5 into the cytosol ensures a constant pool of available receptor molecules for subsequent cycles of protein import into peroxisomes. Accordingly, certain mutations in mammalian Pex1/Pex6 proteins compromise peroxisome biogenesis and thus, lipid metabolism, causing severe genetic Zellweger diseases in humans. In collaboration with Professor Ralf Erdmann and colleagues at the Ruhr-Universität Bochum, we characterize the structure and function of the AAA+ Pex1/6 complex from yeast Saccharomyces cerevisiae. Single particle electron microscopy (EM) in combination with biochemical assays allows us to analyze how ATP turnover is related to the biological function of the Pex1/6 complex. This study presents EM structures of Pex1/6 complexes assembled in the presence of ADP, ATP, ADP-AlFx and ATPγS, providing a comprehensive structural characterization of the heterohexameric type II AAA+ complex in different nucleotide states. Our EM reconstructions reveal an unexpected triangular overall shape, different than observed for the closely related and well-characterized homohexameric AAA+ protein p97. We show that the heterohexameric Pex1/6 complex is composed of a trimer of heterodimers with alternating subunit arrangement of Pex1 and Pex6 moieties. Furthermore, our results suggest that conserved aromatic residues, lining the central pore of the Pex1/6 D2 ring mediate substrate interactions. These residues correspond to substrate interaction regions in related AAA+ proteins. Comparing Pex1/6 EM reconstructions in different nucleotide states implicates that the mechanical function of Pex1/6 involves an N- to C-terminal protein translocation mechanism along the central pore. The Pex1/6 EM structures resolve symmetric and asymmetric large-scale domain motions, which likely create a power stroke during cycles of ATP binding and hydrolysis. We conclude that Pex5 is probably partially or completely unfolded while it is threaded through the central pore of Pex1/6 complexes. In addition, ATP hydrolysis assays of Pex1/Pex6 complexes containing single amino acid exchanges in individual Walker B motifs reveal that not all active sites are functionally equivalent. In isolated complexes, ATP turnover mainly occurs in Pex6 D2 domains, while Pex1 subunits sustain the structural integrity of the complex. We further resolve the structures of Pex1/6 Walker B variants and observe mutually exclusive protomer-protomer communication. In the Pex1/6 complex, a Walker B mutation induces ATP hydrolysis in the adjacent D2 domain, presenting a structural framework of protomer-protomer communication in the AAA+ heterohexamer.

Dr Erika Mancini explains the role of chromatin in the regulation of gene transcription. Chromatin, the physical packaging of DNA within the nucleus, plays an important role in the regulation of gene expression. All our cells contain the same set of genes, but only some of them are transcribed at any point in a particular tissue. The movement of nucleosomes, packing and unpacking DNA, is governed by chromatin remodelling ATPases. Malfunctions in chromatin structure regulation often leads to complex multi-system diseases and cancer, notably leukemia. Dr Erika Mancini is interested in the role of chromatin in the regulation of gene transcription.

Dr Erika Mancini explains the role of chromatin in the regulation of gene transcription. Chromatin, the physical packaging of DNA within the nucleus, plays an important role in the regulation of gene expression. All our cells contain the same set of genes, but only some of them are transcribed at any point in a particular tissue. The movement of nucleosomes, packing and unpacking DNA, is governed by chromatin remodelling ATPases. Malfunctions in chromatin structure regulation often leads to complex multi-system diseases and cancer, notably leukemia. Dr Erika Mancini is interested in the role of chromatin in the regulation of gene transcription.

Dr Erika Mancini explains how malfunctions in the regulation of chromatin structure often leads to complex multi-system diseases and cancer, notably leukemia. Dr Erika Mancini is interested in the role of chromatin in the regulation of gene transcription. All our cells contain the same set of genes, but only some of them are transcribed at any point in a particular tissue. Regulation of gene transcription is strongly linked to chromatin, physical packaging of the DNA within the nucleus. Molecular Mechanisms influencing DNA packaging Chromatin plays an important role in the regulation of gene expression. The movement of nucleosomes, packing and unpacking DNA, is governed by chromatin remodelling ATPases. Malfunctions in the regulation of chromatin structure often leads to complex multi-system diseases and cancer, notably leukemia.

Dr Erika Mancini explains how malfunctions in the regulation of chromatin structure often leads to complex multi-system diseases and cancer, notably leukemia. Dr Erika Mancini is interested in the role of chromatin in the regulation of gene transcription. All our cells contain the same set of genes, but only some of them are transcribed at any point in a particular tissue. Regulation of gene transcription is strongly linked to chromatin, physical packaging of the DNA within the nucleus. Molecular Mechanisms influencing DNA packaging Chromatin plays an important role in the regulation of gene expression. The movement of nucleosomes, packing and unpacking DNA, is governed by chromatin remodelling ATPases. Malfunctions in the regulation of chromatin structure often leads to complex multi-system diseases and cancer, notably leukemia.

The aerobic, haloalkaliphilic archaeon Natronomonas pharaonis is able to survive in salt-saturated lakes of pH 11. With genome-wide shotgun proteomics, 886 soluble proteins (929 proteins in total) of the theoretical Natronomonas pharaonis soluble proteome consisting of 2187 proteins have been confidentially identified by MS/MS. By comparing the identified proteins of Natronomonas pharaonis with homologues of other organisms, both extreme diversity between halophiles and occasional extraordinary sequence conservation to proteins from unrelated species were observed, substantiating genetic exchange between organisms that are evolutionary nearly unrelated to cope with several extreme conditions. Alternative and largely overlapping open reading frames (called overprinting) could not be identified in the genome of neither Natronomonas pharaonis nor Halobacterium salinarum, leading to the conclusion that in halophiles, not more than one protein can be produced from the same genomic sequence stretch. In the second part of this work, analyses on both the transcriptional and translational level have been performed on the halophilic archaeon Halobacterium salinarum, to gain insights into its lifestyle changes leading to cell response when challenged by heat shock. Thereby, quantitative proteomic data obtained from two different approaches regarding the labeling method (ICPL; SILAC), the fractionation of the protein or peptide mixtures (2DE; 1DE-LC), the mass spectrometric analysis (MALDI-TOF/TOF; ESI Q-TOF), and the choice of the growth medium (complex; synthetic) were integrated with data from whole-genome DNA microarrays, real-time quantitative PCR (RTqPCR), and Northern analyses. A number of genes congruently displayed substantial induction after heat shock on both the transcript and protein level as in the case of the thermosome, two AAA-type ATPases, a Dps-like ferritin protein (DpsA), a hsp5-type molecular chaperone, and the transcription initiation factor tfbB. In contrast, the dnaK operon (hsp70) did not exhibit any significant upregulation in either of the approaches. Some genes encoding enzymes of the TCA cycle, of pathways flowing into the latter, and of pathways leading to pyrimidine synthesis, were only translationally induced. Finally, differential transcriptional induction of the transcription initiation factors tfbB and tfbA, determined by RTqPCR, led to the conclusion that they may regulate genes by reciprocal action. The multiplicity of proteomics and transcriptomics methods are complementing one another, covering a bigger area on the one hand, but also confirming some unexpected findings.

Background: Asthma is characterized by airway remodeling, altered mucus production and airway smooth muscle cell (ASMC) contraction causing extensive airway narrowing. In particular, alterations of ASMC contractility seem to be of crucial importance. The elevation of the cytoplasmic Ca(2+) concentration is a key event leading to ASMC contraction and changes in the agonist- induced Ca(2+) increase in ASMC have been reported in asthma. Objective: The aim of this study was to investigate mechanisms underlying these changes. Methods: Murine tracheal smooth muscle cells (MTSMC) from T- bet KO mice and human bronchial smooth muscle cells (HBSMC) incubated with IL-13 and IL-4 served as asthma models. Acetylcholine- induced changes in the cytoplasmic Ca(2+) concentration were recorded using fluorescence microscopy and the expression of Ca(2+) homeostasis regulating proteins was investigated with Western blot analysis. Results: Acetylcholine- induced Ca(2+) transients were elevated in both asthma models. This correlated with an increased Ca(2+) content of the sarcoplasmic reticulum (SR). In MTSMC from T-bet KO mice, the expression of the SR Ca(2+) buffers calreticulin and calsequestrin was higher compared to wild- type mice. In HBSMC incubated with IL-13 or IL-4, the expression of ryanodine receptors, inositol-3-phosphate receptors and sarcoplasmic/ endoplasmic reticulum Ca 2+ ATPases 2 was increased compared to HBSMC without incubation with interleukins. The enlarged acetylcholine- induced Ca(2+) transients could be reversed by blocking inositol-3- phosphate receptors. Conclusions: We conclude that in the murine asthma model the SR Ca(2+) buffer capacity is increased, while in the human asthma model the expression of SR Ca(2+) channels is altered. The investigation of the Ca(2+) homeostasis of ASMC has the potential to provide new therapeutical options in asthma. Copyright (C) 2008 S. Karger AG, Basel.

Chaperonins are highly allosteric double-ring ATPases that mediate cellular protein folding. ATP binding and hydrolysis control opening and closing of the central chaperonin chamber which transiently provides a protected environment for protein folding. During evolution, two distinct strategies to close the chaperonin chamber have emerged. Archaeal and eukaryotic chaperonins contain a built-in lid, whereas bacterial chaperonins use a ring-shaped cofactor as a detachable lid. The present work contributes to the current mechanistical understanding of group II chaperonins by unraveling key functions of the built-in lid. In addition to physically encapsulating the substrate, the lid-forming apical protrusions also play a key role in regulating chaperonin function and ensuring its activity as a “two-stroke” molecular machine. By comparative investigation of two distinct chaperonin systems, namely TRiC and Mm-Cpn, this study uncovers a remarkable degree of mechanistic and functional conservation between group II chaperonins from eukaryotic and archaeal origin, despite their evolutionary distance.

The Mfd (mutation frequency decline) protein is responsible for connecting the cellular processes of transcription and DNA repair in bacteria. Mfd, also termed transcription-repair coupling factor (TRCF), recognizes arrested transcription elongation complexes and catalyzes their dissociation from damaged template DNA in an ATP-dependent manner. Subsequently, Mfd recruits the UvrABC nucleotide excision repair machinery to the damage site. The mechanistic details of this process are not fully understood. X-ray crystallography was used in order to give structural insights into the mechanism of bacterial transcription-coupled repair. During this PhD thesis, the crystal structure of the N terminus (residues 1-333) of Escherichia coli Mfd ("Mfd-N2") was solved. The Mfd N terminus is implicated to function in UvrA-binding. It bears a region with high homology to the nucleotide excision repair protein UvrB. Mfd-N2 is a triangularly shaped molecule of approximately 60×60×30 Å dimensions which contains three structural domains (domains 1A, 1B and 2). Interestingly, the structure of Mfd-N2 very much resembles that of the three N-terminal domains of UvrB. Mfd domain 1A adopts a typical RecA fold. However, it lacks the functional motifs of active ATPases, and we could confirm that the Mfd N-terminus does not possess any ATPase activity. Domain 1B matches the damage-binding domain of the UvrB. Interestingly, Mfd is bereft of the damage-binding motif of UvrB domain 1B, and no DNA binding is associated with this part of Mfd. Domain 2, which possesses the highest sequence homology to UvrB, closely matches the three-dimensional structure of the implicated UvrA-binding domain of UvrB. Highly conserved amino acids between Mfd and UvrB can be found on the surface of domain 2. Using site-directed mutagenesis, several of these residues could be shown to function in the UvrA-Mfd interaction. Remarkably, the corresponding residues in UvrB are required for productive interaction between UvrA and UvrB as well. Taken together, these results suggest that Mfd and UvrB interact with UvrA in a similar manner. Mfd may form an UvrA-recruitment factor at stalled transcription complexes that resembles UvrB architecturally but not catalytically. The molecular similarity between Mfd and UvrB indicates an evolutionary connection between global genome and transcription-coupled nucleotide excision repair in bacteria.

The majority of eukaryotic proteins are degraded by the ubiquitin-proteasome system. In this pathway, cytosolic substrates are first earmarked for degradation by modification with ubiquitin ('ubiquitylation') and subsequently degraded by the 26S pro-teasome, a large protease residing in both the cytosol and the nucleus. ER-resident proteins are similarly degraded but take the route of a specialized pathway coined ER-associated degradation (ERAD). In order to reach the cytosolic ubiquitin/proteasome system, these substrates must first relocate from the ER to the cytosol, possibly with the help of protein conducting membrane channels. Previous work has shown that specific ubiquitin-conjugating enzymes (e.g. Ubc6, Ubc7) and ubiquitin ligases (e.g. Hrd1) con-tribute to ERAD, but how the substrates reach the proteasome remained to be clarified. Besides its function as a quality control system in recognizing and eliminating aberrant proteins, ERAD appears also to play a part in regulatory pathways. This study focuses on the identification of novel components contributing to ERAD. It could be demonstrated that the yeast protein Cdc48 (p97 in mammals), to-gether with its co-factors Ufd1 and Npl4, plays a key role in this process. Cdc48 belongs to the large family of AAA-type ATPases and is believed to function as a chaperone-like enzyme. Previous work has shown that the Cdc48 complex specifically acts on ubiquitylated substrates. This study indicates that the Cdc48 complex takes part in mo-bilization of ERAD substrates from the ER membrane for proteasomal targeting. Fur-thermore, degradation of some ERAD substrates involves the multiubiquitylation factor E4/Ufd2 and proteasome targeting factors of the Rad23 protein family. Another aspect of this work addresses the regulatory functions of ERAD. The fatty acid desaturase Ole1, an integral membrane protein of the ER, was identified as a novel ERAD substrate. Intriguingly, ERAD of Ole1 is specifically regulated since the protein is particularly short lived in the presence of high levels of unsaturated fatty acids, the products of Ole1. Thus, this feedback loop provides an additional mechanism, by which the cell regulates the amount of unsaturated fatty acids. The t-SNARE (syntaxin) protein Ufe1 was characterized as another substrate of ERAD. This protein is required for homotypic membrane fusion of ER vesicles. Notably, Ufe1 degradation is negatively controlled by its binding partner Sly1, a member of the SM (Sec1/Munc18) protein fam-ily. Reciprocal mutations in the Ufe1-Sly1 interaction face result in rapid degradation of Ufe1 by ERAD. Conversely, strong overproduction of Ufe1 was found to be detrimental for cellular growth. These findings suggest that one important function of Sly1 is to con-trol Ufe1 SNARE levels in order to ensure cellular homeostasis. In conclusion, analysis of the degradation of Ole1 and Ufe1 revealed an important contribution of ERAD to es-sential regulatory pathways.

During evolution of photoautotrophic eukaryotes, the nucleus has gained a dominant role in the coordination of the integrated genetic system of the cell consisting of three specifically coevolved genetic compartments. The photosynthetic machinery is encoded by the chloroplast and nuclear genomes. Therefore, biosynthesis and assembly of stochiometric amounts of subunits as well as association of the proteins with corresponding cofactors need to be managed and precisely regulated. To identify novel nuclear-encoded factors involved in the regulation of chloroplast gene expression at different levels, 12 nuclear mutants with high chlorophyll fluorescence (hcf) phenotypes denoting quite diverse defects in the photosynthetic apparatus were selected. Three of them, hcf145, hcf109 and hcf101, were analysed and the affected genes were characterized in more detail. Spectroscopic, fluorimetric and immunological studies have revealed that hcf145 and hcf101 were predominantly affected in photosystem I (PSI), while hcf109 had pleiotropic deficiencies. Remarkably, the dramatic reduction of PSI core complex accumulation in hcf145 was not accompanied by corresponding deficiencies of the outer light-harvesting antenna complex. A comparison of stationary transcript levels with rates of transcription, as estimated by Northern and chloroplast run-on transcription analysis, revealed that the hcf145 mutant is primarily and specifically characterised by a reduced stability of tricistronic chloroplast psaA-psaB-rps14 transcripts. The corresponding operon encodes the two large PSI polypeptides PsaA and PsaB, which form the heterodimeric PSI reaction centre, and the ribosomal protein S14. Chloroplast translation inhibition experiments excluded translational defects as the primary cause of impaired mRNA stability. Defined intervals of the tricistronic transcript were quantified by real-time RT-PCR which established that the psaA region is less stable than the rps14 region in hcf145. Therefore, although up to date, no 5'-3' exoribonucleases have been found in eubacteria (including the ancestors of plants), factor HCF145 appears to be required for the protection of the psaA-psaB-rps14 mRNA against progressive ribonucleolytic degradation starting at the 5' end. In the hcf109 mutant, exclusively plastid transcripts containing UGA stop codons are unstable. The affected gene encodes the first described chloroplast peptide chain release factor AtprfB. Its full-length cDNA, introduced into hcf109 via Agrobacterium-mediated transformation, could functionally complement the mutant. Homology of AtprfB to eubacterial release factors indicates that processes of translational termination in chloroplasts resemble those in eubacteria. The mutant phenotype revealed that translation of all plastid mRNAs containing UGA stop codons is exclusively terminated by AtprfB. However, besides its peptide chain release function, AtprfB appears to acquire yet unknown roles in regulating the stability and translation of the chloroplast mRNAs containing UGA stop codons. These additional regulatory functions could reflect evolutionary constraints which keep the number of plastid TGA stop codons high in vascular plant organelles in contrast to those of algae, mosses and ferns. In contrast to hcf145, steady-state levels and translation of photosynthetic transcripts are not altered in the PSI mutant hcf101. Separation of thylakoid membrane complexes by sucrose gradient centrifugation has uncovered that, similar to hcf145, accumulation of the outer antenna of PSI is not changed in hcf101. Therefore, hcf101 is affected in the assembly of the PSI core complexes. Expression of the HCF101 full-length cDNA in the hcf101 genetic background functionally complemented the mutant. The HCF101 protein encodes a very ancient and universally conserved protein of P-loop ATPases. HCF101 is plastid-localised and represents the first described factor essentially required for the assembly of PSI and other [4Fe-4S]-containing protein complexes in the chloroplast. Relatives of HCF101 are divided into four classes present in all organisms and in all cellular compartments. The antiquity of HCF101 points to the importance of Fe-S cluster biogenesis during the earliest phases of cell evolution. The ubiquity of HCF101 indicates that it is essential for all free-living cells.

To date, mesenchymal cells have only been associated with bone resorption indirectly, and it has been hypothesized that the degradation of bone is associated exclusively with specific functions of osteoclasts. Here we show, in aseptic prosthesis loosening, that aggressive fibroblasts at the bone surface actively contribute to bone resorption and that this is independent of osteoclasts. In two separate models ( a severe combined immunodeficient mouse coimplantation model and a dentin pit formation assay), these cells produce signs of bone resorption that are similar to those in early osteoclastic resorption. In an animal model of aseptic prosthesis loosening (i.e. intracranially self-stimulated rats), it is shown that these fibroblasts acquire their ability to degrade bone early on in their differentiation. Upon stimulation, such fibroblasts readily release acidic components that lower the pH of their pericellular milieu. Through the use of specific inhibitors, pericellular acidification is shown to involve the action of vacuolar type ATPases. Although fibroblasts, as mesenchymal derived cells, are thought to be incapable of resorbing bone, the present study provides the first evidence to challenge this widely held belief. It is demonstrated that fibroblast-like cells, under pathological conditions, may not only enhance but also actively contribute to bone resorption. These cells should therefore be considered novel therapeutic targets in the treatment of bone destructive disorders.

The carcinoembryonic antigen (CEA) gene family belongs to the immunoglobulin supergene family and can be divided into two main subgroups based on sequence comparisons. In humans it is clustered on the long arm of chromosome 19 and consists of approximately 20 genes. The CEA subgroup genes code for CEA and its classical crossreacting antigens, which are mainly membrane-bound, whereas the other subgroup genes encode the pregnancy-specific glycoproteins (PSG), which are secreted. Splice variants of individual genes and differential post-translational modifications of the resulting proteins, e.g., by glycosylation, indicate a high complexity in the number of putative CEA-related molecules. So far, only a limited number of CEA-related antigens in humans have been unequivocally assigned to a specific gene. Rodent CEA-related genes reveal a high sequence divergence and, in part, a completely different domain organization than the human CEA gene family, making it difficult to determine individual gene counterparts. However, rodent CEA-related genes can be assigned to human subgroups based on similarity of expression patterns, which is characteristic for the subgroups. Various functions have been determined for members of the CEA subgroup in vitro, including cell adhesion, bacterial binding, an accessory role for collagen binding or ecto-ATPases activity. Based on all that is known so far on its biology, the clinical outlook for the CEA family has been reassessed.