Progenitor cells in vascular repair

PURPOSE OF REVIEW: A common characteristic of all types of vascular disease is endothelial dysfunction/damage followed by an inflammatory response. Although mature endothelial cells can proliferate and replace damaged cells in the vessel wall, recent findings indicate an impact of stem and progenitor cells in repair process. This review aims to briefly summarize the recent findings in stem/progenitor cell research relating to vascular diseases, focusing on the role of stem/progenitor cells in vascular repair. RECENT FINDINGS: It has been demonstrated that endothelial progenitor cells present in the blood have an ability to repair damaged arterial-wall endothelium. These cells may be derived from a variety of sources, including bone marrow, spleen, liver, fat tissues and the adventitia of the arterial wall. In response to cytokine released from damaged vessel wall and adhered platelets, circulating progenitor cells home in on the damaged areas. It was also reported that the adhered progenitor cells can engraft into endothelium and may differentiate into mature endothelial cells. SUMMARY: Vascular progenitor cells derived from different tissues have an ability to repair damaged vessel, in which the local microenvironment of the progenitors plays a crucial role in orchestrating cell homing and differentiation.     

Detection of trabecular bone microdamage by micro-computed tomography

Microdamage is an important component of bone quality and affects bone remodeling. Improved techniques to assess microdamage without the need for histological sectioning would provide insight into the role of microdamage in trabecular bone strength by allowing the spatial distribution of damage within the trabecular microstructure to be measured. Nineteen cylindrical trabecular bone specimens were prepared and assigned to two groups. The specimens in group I were damaged to 3% compressive strain and labeled with BaSO(4). Group II was not loaded, but was labeled with BaSO(4). Micro-computed tomography (Micro-CT) images of the specimens were obtained at 10 microm resolution. The median intensity of the treated bone tissue was compared between groups. Thresholding was also used to measure the damaged area fraction in the micro-CT scans. The histologically measured damaged area fraction, the median CT intensity, and the micro-CT measured damaged area fraction were all higher in the loaded group than in the unloaded group, indicating that the micro-CT images could differentiate the damaged specimen group from the unloaded specimens. The histologically measured damaged area fraction was positively correlated with the micro-CT measured damaged area fraction and with the median CT intensity of the bone, indicating that the micro-CT images can detect microdamage in trabecular bone with sufficient accuracy to differentiate damage levels between samples. This technique provides a means to non-invasively assess the three-dimensional distribution of microdamage within trabecular bone test specimens and could be used to gain insight into the role of trabecular architecture in microdamage formation.     

Identification of FAAP24, a Fanconi anemia core complex protein that interacts with FANCM

The Fanconi anemia (FA) core complex plays a crucial role in a DNA damage response network with BRCA1 and BRCA2. How this complex interacts with damaged DNA is unknown, as only the FA core protein FANCM (the homolog of an archaeal helicase/nuclease known as HEF) exhibits DNA binding activity. Here, we describe the identification of FAAP24, a protein that targets FANCM to structures that mimic intermediates formed during the replication/repair of damaged DNA. FAAP24 shares homology with the XPF family of flap/fork endonucleases, associates with the C-terminal region of FANCM, and is a component of the FA core complex. FAAP24 is required for normal levels of FANCD2 monoubiquitylation following DNA damage. Depletion of FAAP24 by siRNA results in cellular hypersensitivity to DNA crosslinking agents and chromosomal instability. Our data indicate that the FANCM/FAAP24 complex may play a key role in recruitment of the FA core complex to damaged DNA.     

Essential dynamics of DNA containing a cis.syn cyclobutane thymine dimer lesion.

Conformational properties of a UV-damaged DNA decamer containing a cis.syn cyclobutane thymine dimer (PD) have been investigated by molecular dynamics (MD) simulations. Results from MD simulations of the damaged decamer DNA show a kink of approximately 21.7 degrees at the PD damaged site and a disruption of H bonding between the 5'-thymine of the PD and its complementary adenine. However, no extra-helical flipping of the 3'-adenine complementary to the PD was observed. Comparison to two undamaged DNA decamers, one with the same sequence and the other with an AT replacing the TT sequence, indicates that these properties are specific to the damaged DNA. Essential dynamics (ED) derived from the MD trajectories of the three DNAs show that the backbone phosphate between the two adenines complementary to the PD of the damaged DNA has considerably larger mobility than the rest of the molecule and occurs only in the damaged DNA. As observed in the crystal structure of T4 endonuclease V in a complex with the damaged DNA, the interaction of the enzyme with the damaged DNA can lead to bending along the flexible joint and to induction of adenine flipping into an extra-helical position. Such motions may play an important role in damage recognition by repair enzymes.     

Saccharomyces cerevisiae Ub-conjugating enzyme Ubc4 binds the proteasome in the presence of translationally damaged proteins

Surveillance mechanisms that monitor protein synthesis can promote rapid elimination of misfolded nascent proteins. We showed that the translation elongation factor eEF1A and the proteasome subunit Rpt1 play a central role in the translocation of nascent-damaged proteins to the proteasome. We show here that multiubiquitinated proteins, and the ubiquitin-conjugating (E2) enzyme Ubc4, are rapidly detected in the proteasome following translational damage. However, Ubc4 levels in the proteasome were reduced significantly in a strain that expressed a mutant Rpt1 subunit. Ubc4 and Ubc5 are functionally redundant E2 enzymes that represent ideal candidates for ubiquitinating damaged nascent proteins because they lack significant substrate specificity, are required for the degradation of bulk, damaged proteins, and contribute to cellular stress-tolerance mechanisms. In agreement with this hypothesis, we determined that ubc4Delta ubc5Delta is exceedingly sensitive to protein translation inhibitors. Collectively, these studies suggest a specific role for Ubc4 and Ubc5 in the degradation of cotranslationally damaged proteins that are targeted to the proteasome.     

Human alkyladenine DNA glycosylase uses acid-base catalysis for selective excision of damaged purines

Human alkyladenine DNA glycosylase (AAG) protects against alkylative and oxidative DNA damage, flipping damaged nucleotides out of double-stranded DNA and catalyzing the hydrolytic cleavage of the N-glycosidic bond to release the damaged nucleobase. The crystal structure of AAG bound to a DNA substrate reveals features of the active site that could discriminate between oxidatively damaged or alkylated purines and normal purines. A water molecule bound in the active site adjacent to the anomeric carbon of the N-glycosidic bond is suggestive of direct attack by water, with Glu125 acting as a general base. However, biochemical evidence for this proposed mechanism has been lacking. The structure also fails to explain why smaller pyrimidine nucleosides are excluded as substrates from this relatively permissive active site that catalyzes the excision of a structurally diverse group of damaged purine bases. We have used pH dependencies, site-directed mutagenesis, and a variety of substrates to investigate the catalytic mechanism of AAG. Single-turnover excision of hypoxanthine and 1,N(6)-ethenoadenine follows bell-shaped pH-rate profiles, indicating that AAG-catalyzed excision of these neutral lesions requires the action of both a general acid and a general base. In contrast, the pH-rate profile for the reaction of 7-methylguanine, a positively charged substrate, shows only a single ionization corresponding to a general base. These results suggest that AAG activates neutral lesions by protonation of the nucleobase leaving group. Glu125 must be deprotonated in the active form of the enzyme, consistent with a role as a general base that activates and positions a water nucleophile. Acid-base catalysis can account for much of the 10(8)-fold rate enhancement that is achieved by AAG in the excision of hypoxanthine. The prominent role of nucleobase protonation in catalysis by AAG provides a rationale for its specialization toward damaged purines while effectively excluding pyrimidines.     

The beta -hairpin motif of UvrB is essential for DNA binding, damage processing, and UvrC-mediated incisions.

UvrB plays a major role in recognition and processing of DNA lesions during nucleotide excision repair. The crystal structure of UvrB revealed a similar fold as found in monomeric DNA helicases. Homology modeling suggested that the beta-hairpin motif of UvrB might be involved in DNA binding (Theis, K., Chen, P. J., Skorvaga, M., Van Houten, B., and Kisker, C. (1999) EMBO J. 18, 6899-6907). To determine a role of the beta-hairpin of Bacillus caldotenax UvrB, we have constructed a deletion mutant, Deltabetah UvrB, which lacks residues Gln-97-Asp-112 of the beta-hairpin. Deltabetah UvrB does not form a stable UvrB-DNA pre-incision complex and is inactive in UvrABC-mediated incision. However, Deltabetah UvrB is able to bind to UvrA and form a complex with UvrA and damaged DNA, competing with wild type UvrB. In addition, Deltabetah UvrB shows wild type-like ATPase activity in complex with UvrA that is stimulated by damaged DNA. In contrast to wild type UvrB, the ATPase activity of mutant UvrB does not lead to a destabilization of the damaged duplex. These results indicate that the conserved beta-hairpin motif is a major factor in DNA binding.     

Substrate specificity of a mammalian DNA repair endonuclease that recognizes oxidative base damage.

The substrate specificity of a calf thymus endonuclease on DNA damaged by UV ligh, ionizing radiation, and oxidizing agents was investigated. End-labeled DNA fragments of defined sequence were used as substrates, and the enzyme-generated scission products were analyzed by using DNA sequencing methodologies. The enzyme was shown to incise damaged DNA at pyrimidine sites. The enzyme incised DNA damaged with UV light, ionizing radiation, osmium tetroxide, potassium permanganate, and hydrogen peroxide at cytosine and thymine sites. The substrate specificity of the calf thymus endonuclease was compared to that of Escherichia coli endonuclease III. Similar pyrimidine base damage specificities were found for both enzymes. These results define a highly conserved class of enzymes present in both procaryotes and eucaryotes that may mediate an important role in the repair of oxidative DNA damage.     

Mitochondrial autophagy as a compensatory response to PINK1 deficiency

Macroautophagy (hereafter, autophagy) plays a critical role in maintaining cellular homeostasis by degrading protein aggregates and dysfunctional/damaged organelles. We recently reported that silencing the recessive familial Parkinson disease gene encoding PTEN-induced kinase 1 (PINK1) leads to neuronal cell death accompanied by mitochondrial dysfunction and Drp1-dependent fragmentation. In this model, mitochondrial fission and Beclin 1-dependent autophagy play protective roles, cooperating to sequester and eliminate damaged mitochondria. We discuss the role of superoxide and other reactive oxygen species upstream of mitochondrial depolarization, fission, and autophagy in PINK1 knockdown lines. PINK1 deficiency appears to trigger several compensatory responses that together facilitate clearance of depolarized mitochondria, through a mechanism that is further enhanced by increased expression of parkin. These data offer additional insights that broaden the spectrum of potential interactions between PINK1 and parkin with respect to the regulation of mitochondrial homeostasis and mitophagy.     

Strand-specific binding of RPA and XPA to damaged duplex DNA

The nucleotide excision repair (NER) pathway is a major pathway used to repair bulky adduct DNA damage. Two proteins, xeroderma pigmentosum group A protein (XPA) and replication protein A (RPA), have been implicated in the role of DNA damage recognition in the NER pathway. The particular manner in which these two damage recognition proteins align themselves with respect to a damaged DNA site was assessed using photoreactive base analogues within specific DNA substrates to allow site-specific cross-linking of the damage recognition proteins. Results of these studies demonstrate that both RPA and XPA are in close proximity to the adduct as measured by cross-linking of each protein directly to the platinum moiety. Additional studies demonstrate that XPA contacts both the damaged and undamaged strands of the duplex DNA. Direct evidence is presented demonstrating preferential binding of RPA to the undamaged strand of a duplex damaged DNA molecule.     

Volatiles from damaged plants as major cues in long-range host-searching by the specialist parasitoidCotesia rubecula

The role of volatile stimuli in the long-range host-searching behaviour of the specialist parasitoidCotesia rubecula Marshall (Hymenoptera: Braconidae) was studied. Components from the plant-host-complex Brussels sprouts (Brassica oleracea L. var.gemmifera (DC.) Schulz. cv. ‘Titurel’)-Pieris rapae L. (Lepidoptera: Pieridae) were compared for their attractiveness in dual choice tests in a windtunnel. Stimuli from cabbage plants that were mechanically damaged or damaged byP. rapae caterpillars were more attractive to this parasitoid species than stimuli emitted by the host larvae or their faeces. Parasitoids preferred leaves from the plant-host-complex over artificially damaged leaves. Undamaged cabbage plants were the least attractive to the foraging females. These results indicate that in-flight searching behaviour ofC. rubecula is guided by plant-derived information and that for this specialist species more reliable and specific host-derived cues play a minor role at longer distances.     

巴氏新小绥螨对柑桔全爪螨处理的枳橙叶片挥发物的行为反应

【目的】研究巴氏新小绥螨Neoseiulus barkeri对柑桔全爪螨Panonychus citri及刺吸式昆虫为害柑桔叶片释放的挥发物的行为反应,揭示巴氏新小绥螨的嗅觉反应特点。【方法】采用GC-MS顶空进样法对枳橙叶片常见挥发性化合物、针刺枳橙叶片及其柑桔全爪螨雌成螨取食枳橙叶片挥发物进行定性分析,确定每类化合物的相对保留指数,构建枳橙叶片常见及受害挥发性化合物特征谱库。利用嗅觉测定技术分析巴氏新小绥螨对枳橙叶片挥发物的行为反应。【结果】针刺处理和柑桔全爪螨取食影响枳橙叶片挥发物的组成和含量。两种方法处理时枳橙叶片释放的主要物质为α-蒎烯、水芹烯、4-异丙基甲苯。随着处理加重,增量释放的物质为:cis-π-罗勒烯、月桂烯、柠檬烯、异松油烯,减量表达的物质为2-乙基-1-乙醇和十一烷。在10~(-2)、10~(-4)、10~(-6)和10~(-8)g/m L浓度下,正己醛、正壬醛、乙酸辛酯和正庚醛对巴氏新小绥螨有强烈的引诱作用(P>0.05);月桂烯在10~(-2)、10~(-4)和10~(-6)g/m L浓度下对巴氏新小绥螨有强烈的引诱作用(P>0.05);正壬醇和正辛醇随着浓度增加,对巴氏新小绥螨的引诱作用降低;苯甲醛对巴氏新小绥螨的引诱作用较弱。【结论】巴氏新小绥螨对柑桔全爪螨及刺吸式口器昆虫为害柑桔叶片释放出的挥发物各组分具有不同的行为反应,柑桔及其刺吸式害虫生境中的嗅觉线索在巴氏新小绥螨的寄主定位和生境选择中起着重要作用。     

Destabilization of the MutSα’s protein-protein interface due to binding to the DNA adduct induced by anticancer agent carboplatin via molecular dynamics simulations

DNA mismatch repair (MMR) proteins maintain genetic integrity in all organisms by recognizing and repairing DNA errors. Such alteration of hereditary information can lead to various diseases, including cancer. Besides their role in DNA repair, MMR proteins detect and initiate cellular responses to certain type of DNA damage. Its response to the damaged DNA has made the human MMR pathway a useful target for anticancer agents such as carboplatin. This study indicates that strong, specific interactions at the interface of MutSα in response to the mismatched DNA recognition are replaced by weak, non-specific interactions in response to the damaged DNA recognition. Data suggest a severe impairment of the dimerization of MutSα in response to the damaged DNA recognition. While the core of MutSα is preserved in response to the damaged DNA recognition, the loss of contact surface and the rearrangement of contacts at the protein interface suggest a different packing in response to the damaged DNA recognition. Coupled in response to the mismatched DNA recognition, interaction energies, hydrogen bonds, salt bridges, and solvent accessible surface areas at the interface of MutSα and within the subunits are uncoupled or asynchronously coupled in response to the damaged DNA recognition. These pieces of evidence suggest that the loss of a synchronous mode of response in the MutSα’s surveillance for DNA errors would possibly be one of the mechanism(s) of signaling the MMR-dependent programed cell death much wanted in anticancer therapies. The analysis was drawn from dynamics simulations.     

凋亡细胞的清除及组织稳态维持的研究进展

机体在组织器官受到损伤时,细胞凋亡和机体对凋亡细胞的清除在组织再生中有着密不可分的联系,其背后促进受损组织器官再生的机制一直是研究热点所在。近期研究发现,巨噬细胞在清除凋亡细胞,维持机体稳态以及促进组织器官修复再生中起到了重要作用。本文主要从凋亡的信号通路、巨噬细胞的极化特点以及凋亡细胞与巨噬细胞的相互作用这3个方面对近期研究进行综述。     

The peroxisomal Lon protease LonP2 in aging and disease: functions and comparisons with mitochondrial Lon protease LonP1

Peroxisomes are ubiquitous eukaryotic organelles with the primary role of breaking down very long‐ and branched‐chain fatty acids for subsequent β‐oxidation in the mitochondrion. Like mitochondria, peroxisomes are major sites for oxygen utilization and potential contributors to cellular oxidative stress. The accumulation of oxidatively damaged proteins, which often develop into inclusion bodies (of oxidized, aggregated, and cross‐linked proteins) within both mitochondria and peroxisomes, results in loss of organelle function that may contribute to the aging process. Both organelles possess an isoform of the Lon protease that is responsible for degrading proteins damaged by oxidation. While the importance of mitochondrial Lon (LonP1) in relation to oxidative stress and aging has been established, little is known regarding the role of LonP2 and aging‐related changes in the peroxisome. Recently, peroxisome dysfunction has been associated with aging‐related diseases indicating that peroxisome maintenance is a critical component of ‘healthy aging’. Although mitochondria and peroxisomes are both needed for fatty acid metabolism, little work has focused on understanding the relationship between these two organelles including how age‐dependent changes in one organelle may be detrimental for the other. Herein, we summarize findings that establish proteolytic degradation of damaged proteins by the Lon protease as a vital mechanism to maintain protein homeostasis within the peroxisome. Due to the metabolic coordination between peroxisomes and mitochondria, understanding the role of Lon in the aging peroxisome may help to elucidate cellular causes for both peroxisome and mitochondrial dysfunction.     

XRCC1 stimulates human polynucleotide kinase activity at damaged DNA termini and accelerates DNA single-strand break repair

XRCC1 protein is required for DNA single-strand break repair and genetic stability but its biochemical role is unknown. Here, we report that XRCC1 interacts with human polynucleotide kinase in addition to its established interactions with DNA polymerase-beta and DNA ligase III. Moreover, these four proteins are coassociated in multiprotein complexes in human cell extract and together they repair single-strand breaks typical of those induced by reactive oxygen species and ionizing radiation. Strikingly, XRCC1 stimulates the DNA kinase and DNA phosphatase activities of polynucleotide kinase at damaged DNA termini and thereby accelerates the overall repair reaction. These data identify a novel pathway for mammalian single-strand break repair and demonstrate a concerted role for XRCC1 and PNK in the initial step of processing damaged DNA ends.     

Autophagy and kidney inflammation

Inflammation plays a pivotal role in pathophysiological processes of kidney diseases. Macroautophagy/autophagy plays multiple roles in inflammatory responses, and the regulation of inflammation by autophagy has great potential as a treatment for damaged kidneys. A growing body of evidence suggests autophagy protects kidney from versatile kidney inflammatory insults, including those that are acute, chronic, metabolic, and aging-related. It is noteworthy that, in kidney, mitophagy is active, and damaged lysosomes are removed by autophagy. In this mode, autophagy suppresses inflammation to protect the kidney. Systemic inflammation also affects the kidney via pro-inflammatory cytokines and infiltration of inflammatory cells, and autophagy also has a regulatory role in systemic inflammation. This review focuses on the roles of autophagy in kidney diseases and aging through inflammation, and discusses the potential usage of autophagy as an inflammatory modulator for the treatment of kidney diseases.     

Proteasome-mediated degradation of cotranslationally damaged proteins involves translation elongation factor 1A

Rad23 and Rpn10 play synergistic roles in the recognition of ubiquitinated proteins by the proteasome, and loss of both proteins causes growth and proteolytic defects. However, the physiological targets of Rad23 and Rpn10 have not been well defined. We report that rad23Delta rpn10Delta is unable to grow in the presence of translation inhibitors, and this sensitivity was suppressed by translation elongation factor 1A (eEF1A). This discovery suggested that Rad23 and Rpn10 perform a role in translation quality control. Certain inhibitors increase translation errors during protein synthesis and cause the release of truncated polypeptide chains. This effect can also be mimicked by ATP depletion. We determined that eEF1A interacted with ubiquitinated proteins and the proteasome following ATP depletion. eEF1A interacted with the proteasome subunit Rpt1, and the turnover of nascent damaged proteins was deficient in rpt1. An eEF1A mutant (eEF1A(D156N)) that conferred hyperresistance to translation inhibitors was much more effective at eliminating damaged proteins and was detected in proteasomes in untreated cells. We propose that eEF1A is well suited to detect and promote degradation of damaged proteins because of its central role in translation elongation. Our findings provide a mechanistic foundation for defining how cellular proteins are degraded cotranslationally.     

Protein homeostasis and aging: role of ubiquitin protein ligases

Protein homeostasis is fundamental in normal cellular function and cell survival. The ubiquitin-proteasome system (UPS) plays a central role in maintaining the protein homeostasis network through selective elimination of misfolded and damaged proteins. Impaired function of UPS is implicated in normal aging process and also in several age-related neurodegenerative disorders that are characterized by increased accumulation oxidatively modified proteins and protein aggregates. Growing literature also indicate the potential role of various ubiquitin protein ligases in the regulation of aging process by enhancing the degradation of either central lifespan regulators or abnormally folded and damaged proteins. This review mainly focuses on our current understanding of the importance of UPS function in the regulation of normal aging process.