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[EN] The biological purpose of plant stem cells is to maintain themselves while providing new pools of differentiated cells that form organs and rejuvenate or replace damaged tissues. Protein homeostasis or proteostasis is required for cell function and viability. However, the link between proteostasis and plant stem cell identity remains unknown. In contrast to their differentiated counterparts, we find that root stem cells can prevent the accumulation of aggregated proteins even under proteotoxic stress conditions such as heat stress or proteasome inhibition. Notably, root stem cells exhibit enhanced expression of distinct chaperones that maintain proteome integrity. Particularly, intrinsic high levels of the T-complex protein-1 ring complex/chaperonin containing TCP1 (TRiC/CCT) complex determine stem cell maintenance and their remarkable ability to suppress protein aggregation. Overexpression of CCT8, a key activator of TRiC/CCT assembly, is sufficient to ameliorate protein aggregation in differentiated cells and confer resistance to proteotoxic stress in plants. Taken together, our results indicate that enhanced proteostasis mechanisms in stem cells could be an important requirement for plants to persist under extreme environmental conditions and reach extreme long ages. Thus, proteostasis of stem cells can provide insights to design and breed plants tolerant to environmental challenges caused by the climate change.

[EN] The biological purpose of plant stem cells is to maintain themselves while providing new pools of differentiated cells that form organs and rejuvenate or replace damaged tissues. Protein homeostasis or proteostasis is required for cell function and viability. However, the link between proteostasis and plant stem cell identity remains unknown. In contrast to their differentiated counterparts, we find that root stem cells can prevent the accumulation of aggregated proteins even under proteotoxic stress conditions such as heat stress or proteasome inhibition. Notably, root stem cells exhibit enhanced expression of distinct chaperones that maintain proteome integrity. Particularly, intrinsic high levels of the T-complex protein-1 ring complex/chaperonin containing TCP1 (TRiC/CCT) complex determine stem cell maintenance and their remarkable ability to suppress protein aggregation. Overexpression of CCT8, a key activator of TRiC/CCT assembly, is sufficient to ameliorate protein aggregation in differentiated cells and confer resistance to proteotoxic stress in plants. Taken together, our results indicate that enhanced proteostasis mechanisms in stem cells could be an important requirement for plants to persist under extreme environmental conditions and reach extreme long ages. Thus, proteostasis of stem cells can provide insights to design and breed plants tolerant to environmental challenges caused by the climate change.

The biological purpose of plant stem cells is to maintain themselves while providing new pools of differentiated cells that form organs and rejuvenate or replace damaged tissues. Protein homeostasis or proteostasis is required for cell function and viability. However, the link between proteostasis and plant stem cell identity remains unknown. In contrast to their differentiated counterparts, we find that root stem cells can prevent the accumulation of aggregated proteins even under proteotoxic stress conditions such as heat stress or proteasome inhibition. Notably, root stem cells exhibit enhanced expression of distinct chaperones that maintain proteome integrity. Particularly, intrinsic high levels of the T-complex protein-1 ring complex/chaperonin containing TCP1 (TRiC/CCT) complex determine stem cell maintenance and their remarkable ability to suppress protein aggregation. Overexpression of CCT8, a key activator of TRiC/CCT assembly, is sufficient to ameliorate protein aggregation in differentiated cells and confer resistance to proteotoxic stress in plants. Taken together, our results indicate that enhanced proteostasis mechanisms in stem cells could be an important requirement for plants to persist under extreme environmental conditions and reach extreme long ages. Thus, proteostasis of stem cells can provide insights to design and breed plants tolerant to environmental challenges caused by the climate change.

Ageing is driven by a loss of cellular integrity(1). Given the major role of ubiquitin modifications in cell function(2), here we assess the link between ubiquitination and ageing by quantifying whole-proteome ubiquitin signatures in Caenorhabditis elegans. We find a remodelling of the ubiquitinated proteome during ageing, which is ameliorated by longevity paradigms such as dietary restriction and reduced insulin signalling. Notably, ageing causes a global loss of ubiquitination that is triggered by increased deubiquitinase activity. Because ubiquitination can tag proteins for recognition by the proteasome(3), a fundamental question is whether deficits in targeted degradation influence longevity. By integrating data from worms with a defective proteasome, we identify proteasomal targets that accumulate with age owing to decreased ubiquitination and subsequent degradation. Lowering the levels of age-dysregulated proteasome targets prolongs longevity, whereas preventing their degradation shortens lifespan. Among the proteasomal targets, we find the IFB-2 intermediate filament(4) and the EPS-8 modulator of RAC signalling(5). While increased levels of IFB-2 promote the loss of intestinal integrity and bacterial colonization, upregulation of EPS-8 hyperactivates RAC in muscle and neurons, and leads to alterations in the actin cytoskeleton and protein kinase JNK. In summary, age-related changes in targeted degradation of structural and regulatory proteins across tissues determine longevity.

Ageing is driven by a loss of cellular integrity(1). Given the major role of ubiquitin modifications in cell function(2), here we assess the link between ubiquitination and ageing by quantifying whole-proteome ubiquitin signatures in Caenorhabditis elegans. We find a remodelling of the ubiquitinated proteome during ageing, which is ameliorated by longevity paradigms such as dietary restriction and reduced insulin signalling. Notably, ageing causes a global loss of ubiquitination that is triggered by increased deubiquitinase activity. Because ubiquitination can tag proteins for recognition by the proteasome(3), a fundamental question is whether deficits in targeted degradation influence longevity. By integrating data from worms with a defective proteasome, we identify proteasomal targets that accumulate with age owing to decreased ubiquitination and subsequent degradation. Lowering the levels of age-dysregulated proteasome targets prolongs longevity, whereas preventing their degradation shortens lifespan. Among the proteasomal targets, we find the IFB-2 intermediate filament(4) and the EPS-8 modulator of RAC signalling(5). While increased levels of IFB-2 promote the loss of intestinal integrity and bacterial colonization, upregulation of EPS-8 hyperactivates RAC in muscle and neurons, and leads to alterations in the actin cytoskeleton and protein kinase JNK. In summary, age-related changes in targeted degradation of structural and regulatory proteins across tissues determine longevity.

The biological purpose of plant stem cells is to maintain themselves while providing new pools of differentiated cells that form organs and rejuvenate or replace damaged tissues. Protein homeostasis or proteostasis is required for cell function and viability. However, the link between proteostasis and plant stem cell identity remains unknown. In contrast to their differentiated counterparts, we find that root stem cells can prevent the accumulation of aggregated proteins even under proteotoxic stress conditions such as heat stress or proteasome inhibition. Notably, root stem cells exhibit enhanced expression of distinct chaperones that maintain proteome integrity. Particularly, intrinsic high levels of the T-complex protein-1 ring complex/chaperonin containing TCP1 (TRiC/CCT) complex determine stem cell maintenance and their remarkable ability to suppress protein aggregation. Overexpression of CCT8, a key activator of TRiC/CCT assembly, is sufficient to ameliorate protein aggregation in differentiated cells and confer resistance to proteotoxic stress in plants. Taken together, our results indicate that enhanced proteostasis mechanisms in stem cells could be an important requirement for plants to persist under extreme environmental conditions and reach extreme long ages. Thus, proteostasis of stem cells can provide insights to design and breed plants tolerant to environmental challenges caused by the climate change.

The biological purpose of plant stem cells is to maintain themselves while providing new pools of differentiated cells that form organs and rejuvenate or replace damaged tissues. Protein homeostasis or proteostasis is required for cell function and viability. However, the link between proteostasis and plant stem cell identity remains unknown. In contrast to their differentiated counterparts, we find that root stem cells can prevent the accumulation of aggregated proteins even under proteotoxic stress conditions such as heat stress or proteasome inhibition. Notably, root stem cells exhibit enhanced expression of distinct chaperones that maintain proteome integrity. Particularly, intrinsic high levels of the T-complex protein-1 ring complex/chaperonin containing TCP1 (TRiC/CCT) complex determine stem cell maintenance and their remarkable ability to suppress protein aggregation. Overexpression of CCT8, a key activator of TRiC/CCT assembly, is sufficient to ameliorate protein aggregation in differentiated cells and confer resistance to proteotoxic stress in plants. Taken together, our results indicate that enhanced proteostasis mechanisms in stem cells could be an important requirement for plants to persist under extreme environmental conditions and reach extreme long ages. Thus, proteostasis of stem cells can provide insights to design and breed plants tolerant to environmental challenges caused by the climate change.

UBR5 is an E3 ubiquitin ligase involved in distinct processes such as transcriptional regulation and development. UBR5 is highly upregulated in embryonic stem cells (ESCs), whereas its expression decreases with differentiation, suggesting a role for UBR5 in ESC function. However, little is known about how UBR5 regulates ESC identity. Here, we define the protein interactome of UBR5 in ESCs and find interactions with distinct components of the H/ACA ribonucleoprotein complex, which is required for proper maturation of ribosomal RNA (rRNA). Notably, loss of UBR5 induces an abnormal accumulation of rRNA processing intermediates, resulting in diminished ribosomal levels. Consequently, lack of UBR5 triggers an increase in p53 levels and a concomitant decrease in cellular proliferation rates. Thus, our results indicate a link between UBR5 and rRNA maturation.

UBR5 is an E3 ubiquitin ligase involved in distinct processes such as transcriptional regulation and development. UBR5 is highly upregulated in embryonic stem cells (ESCs), whereas its expression decreases with differentiation, suggesting a role for UBR5 in ESC function. However, little is known about how UBR5 regulates ESC identity. Here, we define the protein interactome of UBR5 in ESCs and find interactions with distinct components of the H/ACA ribonucleoprotein complex, which is required for proper maturation of ribosomal RNA (rRNA). Notably, loss of UBR5 induces an abnormal accumulation of rRNA processing intermediates, resulting in diminished ribosomal levels. Consequently, lack of UBR5 triggers an increase in p53 levels and a concomitant decrease in cellular proliferation rates. Thus, our results indicate a link between UBR5 and rRNA maturation.

A moderate reduction in body temperature can induce a remarkable lifespan extension. Here we examine the link between cold temperature, germline fitness and organismal longevity. We show that low temperature reduces age-associated exhaustion of germline stem cells (GSCs) in Caenorhabditis elegans, a process modulated by thermosensory neurons. Notably, robust self-renewal of adult GSCs delays reproductive ageing and is required for extended lifespan at cold temperatures (10 degrees C, 15 degrees C). These cells release prostaglandin E2 (PGE2) to induce cbs-1 expression in the intestine, increasing the somatic production of hydrogen sulfide, a gaseous signalling molecule that prolongs lifespan. Loss of adult GSCs reduces intestinal cbs-1 expression and cold-induced longevity, whereas application of exogenous PGE2 rescues these phenotypes. Importantly, tissue-specific intestinal overexpression of cbs-1 mimics cold-temperature conditions and extends longevity even at warm temperatures (25 degrees C). Thus, our results indicate that GSCs communicate with somatic tissues to coordinate extended reproductive capacity with longevity.

A moderate reduction in body temperature can induce a remarkable lifespan extension. Here we examine the link between cold temperature, germline fitness and organismal longevity. We show that low temperature reduces age-associated exhaustion of germline stem cells (GSCs) in Caenorhabditis elegans, a process modulated by thermosensory neurons. Notably, robust self-renewal of adult GSCs delays reproductive ageing and is required for extended lifespan at cold temperatures (10 degrees C, 15 degrees C). These cells release prostaglandin E2 (PGE2) to induce cbs-1 expression in the intestine, increasing the somatic production of hydrogen sulfide, a gaseous signalling molecule that prolongs lifespan. Loss of adult GSCs reduces intestinal cbs-1 expression and cold-induced longevity, whereas application of exogenous PGE2 rescues these phenotypes. Importantly, tissue-specific intestinal overexpression of cbs-1 mimics cold-temperature conditions and extends longevity even at warm temperatures (25 degrees C). Thus, our results indicate that GSCs communicate with somatic tissues to coordinate extended reproductive capacity with longevity.

Hong Man Cho,1 Joon Soo Ha,2 Jae Woong Seo,1 Hyun Ju Lee,1 Sun Do Kim,1 Hyochoon Lee,1 Hyung Bae Park31Department of Orthopaedic Surgery, Gwangju Veterans Hospital, Gwangju, Korea; 2Department of Orthopaedics, King’s College Hospital, London, UK; 3Department of Biology, College of Chonbuk National University, Jeonju, KoreaBackground: Transfer fractures in the lower limbs of bedridden and chair-bound nursing home patients can result from trauma induced by the usual lifting, moving, turning, or transferring maneuvers. Treatment entails immobilization for pain control and position change; however, splints/hard casts increase the risk of pressure sores. Therefore, we evaluated the use of a sponge cast.Materials and methods: Between March 2011 and October 2017, 17 patients with a lower limb transfer fracture due to transferring maneuvers in a nursing home were recruited. We evaluated the improvement in pseudo-motion and divided the patients as having bony union, fibrous union, or remaining pseudo-motion. We also investigated the occurrence of pressure sores due to immobilization up until the final follow-up.Results: Femur fractures occurred in 15 patients and lower leg fractures in two. Six of the 15 femur fractures were periprosthetic (four hip arthroplasty and two knee arthroplasty). Pseudo-motion was improved in 15 of 17 cases, within an average of 17.3 weeks for the improvement (14–23 weeks; bony union: 11 cases and fibrous union: four cases). Pseudo-motion remained in two cases: one periprosthetic fracture around the knee arthroplasty and the other, a femur neck fracture. No pressure sores occurred.Conclusions: A sponge cast appears to be one of the effective treatment options available for bedridden or chair-bound patients with a lower limb fracture due to its low risk of complications and satisfactory clinical results.Keywords: transfer fracture, long bone fracture, conservative treatment, bedridden, immobile patients

Dysregulated miRNA expression and mutation of genes involved in miRNA biogenesis have been reported in motor neuron diseases including spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Therefore, identifying molecular mechanisms governing miRNA expression is important to understand these diseases. Here, we report that expression of DROSHA, which is a critical enzyme in the microprocessor complex and essential for miRNA biogenesis, is reduced in motor neurons from an SMA mouse model. We show that DROSHA is degraded by neuronal activity induced autophagy machinery, which is also dysregulated in SMA. Blocking neuronal activity or the autophagy-lysosome pathway restores DROSHA levels in SMA motor neurons. Moreover, reducing DROSHA levels enhances axonal growth. As impaired axonal growth is a well described phenotype of SMA motor neurons, these data suggest that DROSHA reduction by autophagy may mitigate the phenotype of SMA. In summary, these findings suggest that autophagy regulates RNA metabolism and neuronal growth via the DROSHA/miRNA pathway and this pathway is dysregulated in SMA.

Induced pluripotent stem cells (iPSCs) undergo unlimited self-renewal while maintaining their potential to differentiate into post-mitotic cells with an intact proteome. As such, iPSCs suppress the aggregation of polyQ-expanded huntingtin (HTT), the mutant protein underlying Huntington's disease (HD). Here we show that proteasome activity determines HTT levels, preventing polyQ-expanded aggregation in iPSCs from HD patients (HD-iPSCs). iPSCs exhibit high levels of UBR5, a ubiquitin ligase required for proteasomal degradation of both normal and mutant HTT. Conversely, loss of UBR5 increases HTT levels and triggers polyQ-expanded aggregation in HD-iPSCs. Moreover, UBR5 knockdown hastens polyQ-expanded aggregation and neurotoxicity in invertebrate models. Notably, UBR5 overexpression induces polyubiquitination and degradation of mutant HTT, reducing polyQ-expanded aggregates in HD-cell models. Besides HTT levels, intrinsic enhanced UBR5 expression determines global proteostasis of iPSCs preventing the aggregation of misfolded proteins ensued from normal metabolism. Thus, our findings indicate UBR5 as a modulator of super-vigilant proteostasis of iPSCs.

Dysregulated miRNA expression and mutation of genes involved in miRNA biogenesis have been reported in motor neuron diseases including spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Therefore, identifying molecular mechanisms governing miRNA expression is important to understand these diseases. Here, we report that expression of DROSHA, which is a critical enzyme in the microprocessor complex and essential for miRNA biogenesis, is reduced in motor neurons from an SMA mouse model. We show that DROSHA is degraded by neuronal activity induced autophagy machinery, which is also dysregulated in SMA. Blocking neuronal activity or the autophagy-lysosome pathway restores DROSHA levels in SMA motor neurons. Moreover, reducing DROSHA levels enhances axonal growth. As impaired axonal growth is a well described phenotype of SMA motor neurons, these data suggest that DROSHA reduction by autophagy may mitigate the phenotype of SMA. In summary, these findings suggest that autophagy regulates RNA metabolism and neuronal growth via the DROSHA/miRNA pathway and this pathway is dysregulated in SMA.

Induced pluripotent stem cells (iPSCs) undergo unlimited self-renewal while maintaining their potential to differentiate into post-mitotic cells with an intact proteome. As such, iPSCs suppress the aggregation of polyQ-expanded huntingtin (HTT), the mutant protein underlying Huntington's disease (HD). Here we show that proteasome activity determines HTT levels, preventing polyQ-expanded aggregation in iPSCs from HD patients (HD-iPSCs). iPSCs exhibit high levels of UBR5, a ubiquitin ligase required for proteasomal degradation of both normal and mutant HTT. Conversely, loss of UBR5 increases HTT levels and triggers polyQ-expanded aggregation in HD-iPSCs. Moreover, UBR5 knockdown hastens polyQ-expanded aggregation and neurotoxicity in invertebrate models. Notably, UBR5 overexpression induces polyubiquitination and degradation of mutant HTT, reducing polyQ-expanded aggregates in HD-cell models. Besides HTT levels, intrinsic enhanced UBR5 expression determines global proteostasis of iPSCs preventing the aggregation of misfolded proteins ensued from normal metabolism. Thus, our findings indicate UBR5 as a modulator of super-vigilant proteostasis of iPSCs.

Embryonic stem cells (ESCs) exhibit a striking ability to replicate continuously in the absence of senescence. Despite the knowledge gained into ESC biology and cell reprogramming, the mechanisms that regulate their pluripotency, self-renewal, and differentiation remain largely unknown. Recently, cumulative evidence has highlighted the importance of protein homeostasis, or proteostasis, in the maintenance of ESC function. These findings indicate that ESCs exhibit intrinsic differences in the regulation and activity of key nodes of the proteostasis network such as global protein synthesis, folding, and degradation rates. Here, we review new insights into proteostasis of ESCs and the questions raised by these findings. In addition, we discuss the potential of these discoveries to be applied into aging and cancer research.

While the transcriptional network of human embryonic stem cells (hESCs) has been extensively studied, relatively little is known about how post-transcriptional modulations determine hESC function. RNA-binding proteins play central roles in RNA regulation, including translation and turnover. Here we show that the RNA-binding protein CSDE1 (cold shock domain containing E1) is highly expressed in hESCs to maintain their undifferentiated state and prevent default neural fate. Notably, loss of CSDE1 accelerates neural differentiation and potentiates neurogenesis. Conversely, ectopic expression of CSDE1 impairs neural differentiation. We find that CSDE1 post-transcriptionally modulates core components of multiple regulatory nodes of hESC identity, neuroectoderm commitment and neurogenesis. Among these key pro-neural/neuronal factors, CSDE1 binds fatty acid binding protein 7 (FABP7) and vimentin (VIM) mRNAs, as well as transcripts involved in neuron projection development regulating their stability and translation. Thus, our results uncover CSDE1 as a central post-transcriptional regulator of hESC identity and neurogenesis.

Embryonic stem cells (ESCs) exhibit a striking ability to replicate continuously in the absence of senescence. Despite the knowledge gained into ESC biology and cell reprogramming, the mechanisms that regulate their pluripotency, self-renewal, and differentiation remain largely unknown. Recently, cumulative evidence has highlighted the importance of protein homeostasis, or proteostasis, in the maintenance of ESC function. These findings indicate that ESCs exhibit intrinsic differences in the regulation and activity of key nodes of the proteostasis network such as global protein synthesis, folding, and degradation rates. Here, we review new insights into proteostasis of ESCs and the questions raised by these findings. In addition, we discuss the potential of these discoveries to be applied into aging and cancer research.

While the transcriptional network of human embryonic stem cells (hESCs) has been extensively studied, relatively little is known about how post-transcriptional modulations determine hESC function. RNA-binding proteins play central roles in RNA regulation, including translation and turnover. Here we show that the RNA-binding protein CSDE1 (cold shock domain containing E1) is highly expressed in hESCs to maintain their undifferentiated state and prevent default neural fate. Notably, loss of CSDE1 accelerates neural differentiation and potentiates neurogenesis. Conversely, ectopic expression of CSDE1 impairs neural differentiation. We find that CSDE1 post-transcriptionally modulates core components of multiple regulatory nodes of hESC identity, neuroectoderm commitment and neurogenesis. Among these key pro-neural/neuronal factors, CSDE1 binds fatty acid binding protein 7 (FABP7) and vimentin (VIM) mRNAs, as well as transcripts involved in neuron projection development regulating their stability and translation. Thus, our results uncover CSDE1 as a central post-transcriptional regulator of hESC identity and neurogenesis.

Human embryonic stem cells can replicate indefinitely while maintaining their undifferentiated state and, therefore, are immortal in culture. This capacity may demand avoidance of any imbalance in protein homeostasis (proteostasis) that would otherwise compromise stem cell identity. Here we show that human pluripotent stem cells exhibit enhanced assembly of the TRiC/CCT complex, a chaperonin that facilitates the folding of 10% of the proteome. We find that ectopic expression of a single subunit (CCT8) is sufficient to increase TRiC/CCT assembly. Moreover, increased TRiC/CCTcomplex is required to avoid aggregation of mutant Huntingtin protein. We further show that increased expression of CCT8 in somatic tissues extends Caenorhabditis elegans lifespan in a TRiC/CCT-dependent manner. Ectopic expression of CCT8 also ameliorates the age-associated demise of proteostasis and corrects proteostatic deficiencies in worm models of Huntington's disease. Our results suggest proteostasis is a common principle that links organismal longevity with hESC immortality.

Human embryonic stem cells can replicate indefinitely while maintaining their undifferentiated state and, therefore, are immortal in culture. This capacity may demand avoidance of any imbalance in protein homeostasis (proteostasis) that would otherwise compromise stem cell identity. Here we show that human pluripotent stem cells exhibit enhanced assembly of the TRiC/CCT complex, a chaperonin that facilitates the folding of 10% of the proteome. We find that ectopic expression of a single subunit (CCT8) is sufficient to increase TRiC/CCT assembly. Moreover, increased TRiC/CCTcomplex is required to avoid aggregation of mutant Huntingtin protein. We further show that increased expression of CCT8 in somatic tissues extends Caenorhabditis elegans lifespan in a TRiC/CCT-dependent manner. Ectopic expression of CCT8 also ameliorates the age-associated demise of proteostasis and corrects proteostatic deficiencies in worm models of Huntington's disease. Our results suggest proteostasis is a common principle that links organismal longevity with hESC immortality.

The C-family (cbb(3)) of heme-copper oxygen reductases are proton-pumping enzymes terminating the aerobic respiratory chains of many bacteria, including a number of human pathogens. The most common form of these enzymes contains one copy each of 4 subunits encoded by the ccoNOQP operon. In the cbb3 from Rhodobacter capsulatus, the enzyme is assembled in a stepwise manner, with an essential role played by an assembly protein CcoH. Importantly, it has been proposed that a transient interaction between the transmembrane domains of CcoP and CcoH is essential for assembly. Here, we test this proposal by showing that a genetically engineered form of cbb(3) from Vibrio cholerae (CcoNOQP(X)) that lacks the hydrophilic domain of CcoP, where the two heme c moieties are present, is fully assembled and stable. Single-turnover kinetics of the reaction between the fully reduced CcoNOQP(X) and O-2 are essentially the same as the wild type enzyme in oxidizing the 4 remaining redox-active sites. The enzyme retains approximately 10% of the steady state oxidase activity using the artificial electron donor TMPD, but has no activity using the physiological electron donor cytochrome c(4), since the docking site for this cytochrome is presumably located on the absent domain of CcoP. Residue E49 in the hydrophobic domain of CcoP is the entrance of the K-C-channel for proton input, and the E49A mutation in the truncated enzyme further reduces the steady state activity to less than 3%. Hence, the same proton channel is used by both the wild type and truncated enzymes.

We have studied the in vivo role of SLIRP in regulation of mitochondrial DNA (mtDNA) gene expression and show here that it stabilizes its interacting partner protein LRPPRC by protecting it from degradation. Although SLIRP is completely dependent on LRPPRC for its stability, reduced levels of LRPPRC persist in the absence of SLIRP in vivo. Surprisingly, Slirp knockout mice are apparently healthy and only display a minor weight loss, despite a 50-70% reduction in the steady-state levels of mtDNA-encoded mRNAs. In contrast to LRPPRC, SLIRP is dispensable for polyadenylation of mtDNA-encoded mRNAs. Instead, deep RNA sequencing (RNAseq) of mitochondrial ribosomal fractions and additional molecular analyses show that SLIRP is required for proper association of mRNAs to the mitochondrial ribosome and efficient translation. Our findings thus establish distinct functions for SLIRP and LRPPRC within the LRPPRC-SLIRP complex, with a novel role for SLIRP in mitochondrial translation. Very surprisingly, our results also demonstrate that mammalian mitochondria have a great excess of transcripts under basal physiological conditions in vivo.

We have studied the in vivo role of SLIRP in regulation of mitochondrial DNA (mtDNA) gene expression and show here that it stabilizes its interacting partner protein LRPPRC by protecting it from degradation. Although SLIRP is completely dependent on LRPPRC for its stability, reduced levels of LRPPRC persist in the absence of SLIRP in vivo. Surprisingly, Slirp knockout mice are apparently healthy and only display a minor weight loss, despite a 50-70% reduction in the steady-state levels of mtDNA-encoded mRNAs. In contrast to LRPPRC, SLIRP is dispensable for polyadenylation of mtDNA-encoded mRNAs. Instead, deep RNA sequencing (RNAseq) of mitochondrial ribosomal fractions and additional molecular analyses show that SLIRP is required for proper association of mRNAs to the mitochondrial ribosome and efficient translation. Our findings thus establish distinct functions for SLIRP and LRPPRC within the LRPPRC-SLIRP complex, with a novel role for SLIRP in mitochondrial translation. Very surprisingly, our results also demonstrate that mammalian mitochondria have a great excess of transcripts under basal physiological conditions in vivo.

The respiratory chains of nearly all aerobic organisms are terminated by proton-pumping heme-copper oxygen reductases (HCOs). Previous studies have established that C-family HCOs contain a single channel for uptake from the bacterial cytoplasm of all chemical and pumped protons, and that the entrance of the K-C-channel is a conserved glutamate in subunit III. However, the majority of the K-C-channel is within subunit I, and the pathway from this conserved glutamate to subunit I is not evident. In the present study, molecular dynamics simulations were used to characterize a chain of water molecules leading from the cytoplasmic solution, passing the conserved glutamate in subunit III and extending into subunit I. Formation of the water chain, which controls the delivery of protons to the K-C-channel, was found to depend on the conformation of Y241(Vc), located in subunit I at the interface with subunit III. Mutations of Y241(Vc) (to A/F/H/S) in the Vibrio cholerae cbb(3) eliminate catalytic activity, but also cause perturbations that propagate over a 28-angstrom distance to the active site heme b(3). The data suggest a linkage between residues lining the KC-channel and the active site of the enzyme, possibly mediated by transmembrane helix alpha 7, which contains both Y241(Vc) and the active site crosslinked Y255(Vc), as well as two Cu-B histidine ligands. Other mutations of residues within or near helix alpha 7 also perturb the active site, indicating that this helix is involved in modulation of the active site of the enzyme., AuthorCount:11

Heme-copper oxidases (HCuOs) catalyse the reduction of oxygen, using the liberated free energy to maintain a proton-motive force across the membrane. In the mitochondrial-like A-type HCuOs, binding of heavy metal ions at the surface of the protein inhibits proton transfer. In bacterial C-type oxidases, the entry point to the proton pathway is on an accessory subunit unrelated to any subunit in A-type HCuOs. Despite this, we show here that heavy metal ions such as Zn2+ inhibit O-2-reduction very similarly in C-type as in A-type HCuOs, and furthermore that the binding site shares the same Glu-His motif. (C) 2013 Federation of European Biochemical Societies., AuthorCount:2

Heme-copper oxidases (HCuOs) terminate the respiratory chain in mitochondria and most bacteria. They are transmembrane proteins that catalyse the reduction of oxygen and use the liberated free energy to maintain a proton-motive force across the membrane. The HCuO superfamily has been divided into the oxygen-reducing A-. B- and C-type oxidases as well as the bacterial NO reductases (NOR), catalysing the reduction of NO in the denitrification process. Proton transfer to the catalytic site in the mitochondrial-like A family occurs through two well-defined pathways termed the D- and K-pathways. The B, C, and NOR families differ in the pathways as well as the mechanisms for proton transfer to the active site and across the membrane. Recent structural and functional investigations, focussing on proton transfer in the B, C and NOR families will be discussed in this review. This article is part of a Special Issue entitled: Respiratory Oxidases., 4

Aim: To investigate the effect of calcium concentration on the bone tissue response to Ca-incorporated titanium implants. Materials and methods: Two titanium surfaces containing 4.2% and 6.6% calcium were prepared using the micro-arc oxidation process. The implants were inserted in the tibia of nine New Zealand White rabbits. After 6 weeks of healing, the bone response to the implants was quantitatively compared by biomechanical and histomorphometrical measurements. Results: Ca 4.2% and Ca 6.6% containing implants revealed no distinctive differences in their qualitative surface chemistry; chemical bonding state of Ca in titanium oxide was mainly calcium titanates. No significant differences were observed between two implants in peak removal torque and shear strength comparisons (P>0.05). Histomorphometrical analyses presented no significant differences in bonemetal contact, bone area and newly formed bone measurements between two implants (P>0.05). Conclusions: From biomechanical and histomorphometrical measurements, the two calcium concentrations in this study did not differ significantly with respect to their influence on the bone tissue response. This similar bone response in rabbit tibiae may be explained by the similarity of the qualitative Ca chemistry in titanium surfaces., Funding Agencies:Ministry of Education, Science and Technology of Korea Hjalmar Svensson Research FoundationSylvans Foundation

Heme-copper oxidases (HCuOs) are the last components of the respiratory chain in mitochondria and many bacteria. They catalyze O(2) reduction and couple it to the maintenance of a proton-motive force across the membrane in which they are embedded. In the mitochondrial-like, A family of HCuOs, there are two well established proton transfer pathways leading from the cytosol to the active site, the D and the K pathways. In the C family (cbb(3)) HCuOs, recent work indicated the use of only one pathway, analogous to the K pathway. In this work, we have studied the functional importance of the suggested entry point of this pathway, the Glu-25 (Rhodobacter sphaeroides cbb(3) numbering) in the accessory subunit CcoP (E25(P)). We show that catalytic turnover is severely slowed in variants lacking the protonatable Glu-25. Furthermore, proton uptake from solution during oxidation of the fully reduced cbb(3) by O(2) is specifically and severely impaired when Glu-25 was exchanged for Ala or Gln, with rate constants 100-500 times slower than in wild type. Thus, our results support the role of E25(P) as the entry point to the proton pathway in cbb(3) and that this pathway is the main proton pathway. This is in contrast to the A-type HCuOs, where the D (and not the K) pathway is used during O(2) reduction. The cbb(3) is in addition to O(2) reduction capable of NO reduction, an activity that was largely retained in the E25(P) variants, consistent with a scenario where NO reduction in cbb(3) uses protons from the periplasmic side of the membrane., 3

In this work we have investigated the effect of a pathogenic mitochondrial DNA mutation found in human colon cells, at a functional-molecular level. The mutation results in the amino-acid substitution Tyr19His in subunit I of the human CytcO and it is associated with respiratory deficiency. It was introduced into Rhodobacter sphaeroides, which carries a cytochrome c oxidase (cytochrome aa(3)) that serves as a model of the mitochondrial counterpart. The residue is situated in the middle of a pathway that is used to transfer substrate protons as well as protons that are pumped across the membrane. The Tyr33His (equivalent residue in the bacterial CytcO) structural variant of the enzyme was purified and its function was investigated. The results show that in the structurally altered CytcO the activity decreased due to slowed proton transfer; proton transfer from an internal proton donor, the highly-conserved Glu286, to the catalytic site was slowed by a factor of similar to 5, while reprotonation of the Glu from solution was slowed by a factor of similar to 40. In addition, in the structural variant proton pumping was completely impaired. These results are explained in terms of introduction of a barrier for proton transfer through the D pathway and changes in the coordination of water molecules surrounding the Glu286 residue. The study offers an explanation, at the molecular level, to the link between a specific amino-acid substitution and a pathogenic phenotype identified in human colon cells., authorCount :4

One key role of many cellular membranes is to hold a transmembrane electrochemical ion gradient that stores free energy, which is used, for example, to generate ATP or to drive transmembrane transport processes. In mitochondria and many bacteria, the gradient is maintained by proton-transport proteins that are part of the respiratory (electron-transport) chain. Even though our understanding of the structure and function of these proteins has increased significantly, very little is known about the specific role of functional protein-membrane and membrane-mediated protein-protein interactions. Here, we have investigated the effect of membrane incorporation on proton-transfer reactions within the membrane-bound proton pump cytochrome c oxidase. The results show that the membrane acts to accelerate proton transfer into the enzyme's catalytic site and indicate that the intramolecular proton pathway is wired via specific amino acid residues to the two-dimensional space defined by the membrane surface. We conclude that the membrane not only acts as a passive barrier insulating the interior of the cell from the exterior solution, but also as a component of the energy-conversion machinery., authorCount :4

Cytochrome c oxidase (CytcO), the final electron acceptor in the respiratory chain, catalyzes the reduction of O-2 to H2O while simultaneously pumping protons across the inner mitochondrial or bacterial membrane to maintain a transmembrane electrochemical gradient that drives, for example, ATP synthesis. In this work mutations that were predicted to alter proton translocation and enzyme activity in preliminary computational studies are characterized with extensive experimental and computational analysis. The mutations were introduced in the D pathway, one of two proton-uptake pathways, in CytcO from Rhodobacter sphaeroides. Serine residues 200 and 201, which are hydrogen-bonded to crystallographically resolved water molecules halfway up the D pathway, were replaced by more bulky hydrophobic residues (Ser200lle, Ser200Val/Ser201Val, and Ser200Val/Ser201Tyr) to query the effects of changing the local structure on enzyme activity as well as proton uptake, release, and intermediate transitions. In addition, the effects of these mutations on internal proton transfer were investigated by blocking proton uptake at the pathway entrance (Asp132Asn replacement in addition to the above-mentioned mutations). Even though the overall activities of all mutant CytcO's were lowered, both the Ser200lle and Ser200Val/Ser201Val variants maintained the ability to pump protons. The lowered activities were shown to be due to slowed oxidation kinetics during the P-R -> F and F -> O transitions (P-R is the "peroxy" intermediate formed at the catalytic site upon reaction of the four-electron-reduced CytcO with O-2, F is the oxoferryl intermediate, and O is the fully oxidized CytcO). Furthermore, the P-R -> F transition is Shown to be essentially pH independent up to pH 12 (i.e., the apparent pK(a) of Glu286 is increased from 9.4 by at least 3 pK(a) units) in the Ser200Val/Ser201Val mutant. Explicit simulations of proton transport in the mutated enzymes revealed that the solvation dy, authorCount :7

Cytochrome c oxidase utilizes the energy from electron transfer and reduction of oxygen to water and pumps protons across the membrane, generating a proton motive force. A large body of biochemical work has shown that all the pumped protons enter the enzyme through the D-channel, which is apparent in X-ray structures as a chain of water molecules connecting D132 at the cytoplasmic surface of the enzyme to E286, near the enzyme active site. The exit pathway utilized by pumped protons beyond this point and leading to the bacterial periplasm is not known. Also not known is the proton loading site (or sites) which undergoes changes in pKa in response to the chemistry at the enzyme active site and drives the proton pump mechanism. In this paper, we examine the role of R481, a highly conserved arginine that forms an ion pair with the D-propionate of heme a3. The R481H, R481N, R481Q, and R481L mutants were examined. The R481H mutant oxidase is approximately 18% active and pumps protons with approximately 40% of the stoichiometry of the wild type. The R481N, R481Q, and R481L mutants each retain only approximately 5% of the steady-state activity, and this is shown to be due to inhibition of steps in the reaction of O(2) with the reduced enzyme. Neither the R481N mutant nor the R481Q mutant oxidases pump protons, but remarkably, the R481L mutant does pump protons with the same efficiency as the R481H mutant. Since the proton pump is clearly operating in the R481L mutant, these results rule out an essential role in the proton pump mechanism for R481 or its hydrogen bond partner, the D-propionate of heme a3.

OBJECTIVES: This study assessed the influences of various factors that are related to youth smoking such as gender, age and type of school, and we wanted to provide supporting data for tailored and effective policy initiatives to reduce adolescent smoking. METHODS: A self-report survey was conducted on 14,910 teen-age students who were selected based on the nationwide distribution of students in large and small cities and counties, the gender ratio of the students and the ratio of students attending various type of school at 38 middle and high schools in six representative areas of each province. The survey was handled and managed by a health education teacher at each school. Binary and multinomial logistic regression was used in the analyses. RESULTS: Smoking by adolescents was associated with gender, age and even height. Male high school students tended to smoke more than female high school students, but this differences was not significant for middle school students. The older the adolescents were, the more likely that they smoked, except for the female high school students. Height was meaningful for all adolescents, except for the boys at the vocational high schools. Monthly allowance was significant for all adolescents. School factors such as type of school and the students' school performance were also crucial factors. Attending a vocational high school was strongly related to smoking, especially for girls. Students' school performance and the perceived level of stress were strongly associated with smoking, especially for boys. Home factors such as the relationship with parents and conversation time with family members were closely related to smoking behavior. Knowledge about the health hazard of smoking was also found to be strongly related to adolescent smoking. CONCLUSIONS: In conclusion, demographic factors, school factors, home surroundings and the perception on the harmfulness of smoking are strongly related to adolescent smoking behavior, but these diff

We report on an observing campaign in March 2001 to monitor the brightness of the later arriving Q0957+561 B image in order to compare with the previously published brightness observations of the (first arriving) A image. The 12 participating observatories provided 3543 image frames which we have analyzed for brightness fluctuations. From our classical methods for time delay determination, we find a 417.09 +/- 0.07 day time delay which should be free of effects due to incomplete sampling. During the campaign period, the quasar brightness was relatively constant and only small fluctuations were found; we compare the structure function for the new data with structure function estimates for the 1995--6 epoch, and show that the structure function is statistically non-stationary. We also examine the data for any evidence of correlated fluctuations at zero lag. We discuss the limits to our ability to measure the cosmological time delay if the quasar's emitting surface is time resolved, as seems likely., Comment: AAS LaTeX, 5 PostScript figures

We report on an observing campaign in March 2001 to monitor the brightness of the later arriving Q0957+561 B image in order to compare with the previously published brightness observations of the (first arriving) A image. The 12 participating observatories provided 3543 image frames which we have analyzed for brightness fluctuations. From our classical methods for time delay determination, we find a 417.09 +/- 0.07 day time delay which should be free of effects due to incomplete sampling. During the campaign period, the quasar brightness was relatively constant and only small fluctuations were found; we compare the structure function for the new data with structure function estimates for the 1995--6 epoch, and show that the structure function is statistically non-stationary. We also examine the data for any evidence of correlated fluctuations at zero lag. We discuss the limits to our ability to measure the cosmological time delay if the quasar's emitting surface is time resolved, as seems likely., Comment: AAS LaTeX, 5 PostScript figures

An observing campaign with 10 participating observatories has undertaken to monitor the optical brightness of the Q0957 gravitationally lensed quasar for 10 consecutive nights in January 2000. The resulting A image brightness curve has significant brightness fluctuations and makes a photometric prediction for the B image light curve for a second campaign planned for 12-21 March 2001. The ultimate purpose is to determine the gravitational lens time delay to a fraction of an hour, and to seek evidence for rapid microlensing., Comment: 8 pages, AASTeX 4.0, accepted by the Astrophysical Journal

An observing campaign with 10 participating observatories has undertaken to monitor the optical brightness of the Q0957 gravitationally lensed quasar for 10 consecutive nights in January 2000. The resulting A image brightness curve has significant brightness fluctuations and makes a photometric prediction for the B image light curve for a second campaign planned for 12-21 March 2001. The ultimate purpose is to determine the gravitational lens time delay to a fraction of an hour, and to seek evidence for rapid microlensing., Comment: 8 pages, AASTeX 4.0, accepted by the Astrophysical Journal