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1.
Ch. Sultan J. M. Lobaccaro S. Lumbroso S. Missov Ch. Belon M. Bost R. Dumas 《Andrologie》1994,4(3):283-287
Klinfelter syndrome was first described in adult males with gynecomastia, azoospermia and hypergonadotropic hypogonadism. Children with the 47, XXY karyotype demonstrate few clinical findings so Klinefelter syndrome is rarely diagnosed until adult life. Besides children who have been diagnosed during prenatal genetic testing, in infancy a male with 47, XXY (or variants: 46, XY-47, XXY; 48, XXXY; 48, XXYY, 49, XXXXY) may be found while undergoing evaluation of micropenis, hypospadias, cryptorchidism or facial anomalies. The older child may present with learning disabilities, behavior disorders or tall stature. At the time of puberty, the clinical picture includes small testes, gynecomastia and an eunuchoid habitus. Early diagnosis of Klinefelter syndrome must be performed since it has been demonstrated that early treatment with androgens may ameliorate many aspects of the clinical symptoms and attenuate or prevent behavioral and psychiatric disorders associated with 47, XXY males. 相似文献
2.
damo Davi Digenes Siena Isabela Ichihara de Barros Camila Baldin Storti Carlos Alberto Oliveira de Biagi Júnior Larissa Anastacio da Costa Carvalho Silvya Stuchi MariaEngler Josane de Freitas Sousa Wilson Araújo Silva Jr 《Journal of cellular and molecular medicine》2022,26(3):671
Our previous work using a melanoma progression model composed of melanocytic cells (melanocytes, primary and metastatic melanoma samples) demonstrated various deregulated genes, including a few known lncRNAs. Further analysis was conducted to discover novel lncRNAs associated with melanoma, and candidates were prioritized for their potential association with invasiveness or other metastasis‐related processes. In this sense, we found the intergenic lncRNA (ENSG00000230454) and decided to explore its effects in melanoma. For that, we silenced the lncRNA U73166 expression using shRNAs in a melanoma cell line. Next, we experimentally investigated its functions and found that migration and invasion had significantly decreased in knockdown cells, indicating an essential association of lncRNA U73166 for cancer processes. Additionally, using naïve and vemurafenib‐resistant cell lines and data from a patient before and after resistance, we found that vemurafenib‐resistant samples had a higher expression of lncRNA U73166. Also, we retrieved data from the literature that indicates lncRNA U73166 may act as a mediator of RNA processing and cell invasion, probably inducing a more aggressive phenotype. Therefore, our results suggest a relevant role of lncRNA U73166 in metastasis development. We also pointed herein the lncRNA U73166 as a new possible biomarker or target to help overcome clinical vemurafenib resistance. U73166相似文献
3.
4.
Derek M. Shore Gemma L. Baillie Dow H. Hurst Frank Navas III Herbert H. Seltzman Jahan P. Marcu Mary E. Abood Ruth A. Ross Patricia H. Reggio 《The Journal of biological chemistry》2014,289(9):5828-5845
The cannabinoid 1 (CB1) allosteric modulator, 5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)-ethyl]-amide) (), has the paradoxical effect of increasing the equilibrium binding of [3H](−)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxylpropyl]cyclohexan-1-ol (CP55,940, an orthosteric agonist) while at the same time decreasing its efficacy (in G protein-mediated signaling). ORG27569 also decreases basal signaling, acting as an inverse agonist for the G protein-mediated signaling pathway. In ligand displacement assays, ORG27569 can displace the CB1 antagonist/inverse agonist, N-(piperidiny-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide(SR141716A). The goal of this work was to identify the binding site of ORG27569 at CB1. To this end, we used computation, synthesis, mutation, and functional studies to identify the ORG27569-binding site in the CB1 TMH3-6-7 region. This site is consistent with the results of K3.28192A, F3.36200A, W5.43279A, W6.48356A, and F3.25189A mutation studies, which revealed the ORG27569-binding site overlaps with our previously determined binding site of SR141716A but extends extracellularly. Additionally, we identified a key electrostatic interaction between the ORG27569 piperidine ring nitrogen and K3.28192 that is important for ORG27569 to act as an inverse agonist. At this allosteric site, ORG27569 promotes an intermediate conformation of the CB1 receptor, explaining ORG27569''s ability to increase equilibrium binding of CP55,940. This site also explains ORG27569''s ability to antagonize the efficacy of CP55,940 in three complementary ways. 1) ORG27569 sterically blocks movements of the second extracellular loop that have been linked to receptor activation. 2) ORG27569 sterically blocks a key electrostatic interaction between the third extracellular loop residue Lys-373 and D2.63176. 3) ORG27569 packs against TMH6, sterically hindering movements of this helix that have been shown to be important for receptor activation. ORG27569相似文献
5.
The purpose of this table is to provide the community with a citable record of publications of ongoing genome sequencing projects that have led to a publication in the scientific literature. While our goal is to make the list complete, there is no guarantee that we may have omitted one or more publications appearing in this time frame. Readers and authors who wish to have publications added to subsequent versions of this list are invited to provide the bibliographic data for such references to the SIGS editorial office.
Phylum Euryarchaeota
- Halococcus hamelinensis, sequence accession PRJNA80845 [1]
- “Methanocella conradii” HZ254, sequence accession [ CP0032432]
- Thermococcus litoralis NS-C, sequence accession [ AHVB000000003]
Phylum Crenarchaeota
- Candidatus Nitrosopumilus salaria” BD31, sequence accession [ AEXL000000004]
- Candidatus Nitrosoarchaeum limnia, sequence accession [ AHJG000000005]
Phylum Deinococcus-Thermus
- Deinococcus gobiensis, sequence accession [ CP0025366]
Phylum Proteobacteria
- Aggregatibacter actinomycetemcomitans strain ANH9381, sequence accession [ CP0030997]
- Alishewanella jeotgali, sequence accession [ AHTH000000008]
- Enterobacter aerogenes KCTC 2190, sequence accession [ CP0028249]
- Escherichia coli O104:H4, sequence accession [ AFOB0200009210]
- Helicobacter pylori strains 17874, sequence accession PRJNA76569 [11]
- Helicobacter pylori strains P79, sequence accession PRJNA76567 [11]
- Janthinobacterium sp. Strain PAMC 25724, sequence accession [ AHHB0000000012]
- Klebsiella oxytoca KCTC 1686, sequence accession [ CP00321813]
- Klebsiella pneumoniae subsp. pneumoniae HS11286, sequence accession (chromosome), CP003200 (plasmid pKPHS1), CP003223 (plasmid pKPHS2), CP003224 (plasmid pKPHS3), CP003225 (plasmid pKPHS4), CP003226 (plasmid pKPHS5), CP003227 (plasmid pKPHS6) [ CP00322814]
- Oceanimonas sp. GK1, sequence accession [ CP00317115]
- “Pseudogulbenkiania ferrooxidans” Strain 2002, sequence accession [ NZ_ACIS0100000016]
- Pseudomonas extremaustralis 14-3b, sequence accession [ AHIP0000000017]
- Pseudomonas sp. Strain PAMC 25886, sequence accession [ AHHC0000000018]
- Psychrobacter, sequence accession [ AHVZ0000000019]
- Rahnella sp. Strain Y9602, sequence accession [ CP00250520]
- Rhizobium sp. Strain PDO1-076, sequence accession [ AHZC0000000021]
- Rhodospirillum photometricum DSM122, sequence accession [ HE66349322]
- “Rickettsia sibirica sibirica”, sequence accession [ AHIZ0000000023]
- Rickettsia sibirica subsp. mongolitimonae strain HA-91, sequence accession [ AHZB0000000024]
- Salmonella enterica subsp. enterica Serotype Enteritidis Strain LA5, sequence accession [25]
- Salmonella enterica subsp. enterica Serotype Senftenberg Strain SS209, sequence accession [ CAGQ0000000026]
- Salmonella enterica subsp. enterica Serovar Typhi P-stx-12, sequence accession (chromosome) and CP003278 (plasmid) [ CP00327927]
- Sphingomonas echinoides ATCC 14820, sequence accession [ AHIR0000000028]
- Strain HIMB55, sequence accession [ AGIF0000000029]
- Vibrio harveyi CAIM 1792, sequence accession [ AHHQ0000000030]
- Wolbachia Strain wAlbB, sequence accession [ CAGB01000001 to CAGB0100016531]
- Xanthomonas axonopodis pv. punicae Strain LMG 859, sequence accession [ CAGJ01000001 to CAGJ0100021732]
Phylum Tenericutes
- Mycoplasma hyorhinis Strain GDL-1, sequence accession [ CP00323133]
Phylum Firmicutes
- Bacillus subtilis, sequence accession BGSCID 3A27 through BGSCID 28A4 [34]
- Clostridium difficile Strain CD37, sequence accession [ AHJJ0000000035]
- Clostridium perfringens, sequence accession [ AFES0000000036]
- Lactobacillus fructivorans KCTC 3543, sequence accession [ AEQY0000000037]
- Lactococcus lactis IO-1, sequence accession [ AP01228138]
- Lactobacillus plantarum strain NC8, sequence accession [ AGRI0000000039]
- Paenibacillus dendritiformis C454, sequence accession [ AHKH0000000040]
- Paenibacillus sp. Strain Aloe-11, sequence accession [ AGFI0000000041]
- “Peptoniphilus rhinitidis” 1-13T, sequence accession [ BAEW01000001 to BAEW0100005642]
- Streptococcus macedonicus ACA-DC 198, sequence accession and HE613569 [ HE61357043]
- Staphylococcus aureus VC40, sequence accession [ CP00303344]
- Streptococcus infantarius subsp. infantarius Strain CJ18, sequence accession (chromosome), CP003295 (plasmid) [ CP00329645]
- Streptococcus macedonicus ACA-DC 198, sequence accession (chromosome), HE613569 (plasmid pSMA198) [ HE61357046]
Phylum Actinobacteria
- Actinoplanes sp. SE50/110, sequence accession [ CP00317047]
- Amycolatopsis sp. Strain ATCC 39116, sequence accession [48]
- Nocardia cyriacigeorgica GUH-2, sequence accession [ FO08284349]
- Salinibacterium sp., sequence accession [ AHWA0000000050]
- Streptomyces acidiscabies 84-104, sequence accession [ AHBF0000000051]
Non-Bacterial genomes
- Bluetongue Virus Serotype 2, sequence accession (Seg-6) and AJ783905 (Seg-1), JQ681257 (Seg-1), JQ681257 (Seg-2), JQ681258 (Seg-3), JQ681259 (Seg-4), JQ681260 (Seg-5), JQ681261 (Seg-7), JQ6812563 (Seg-8), JQ6812564 (Seg-9), to JQ681262 (Seg-10) [ JQ68126552]
- Virus Serotype 1, sequence accession (Seg-2), AJ585111 (Seg-6), AJ586659 (Seg-1), JQ282770 (Seg-3), JQ282771 (Seg-4), JQ282772 (Seg-5), JQ282773 (Seg-7), JQ282774 (Seg-8), JQ282775 (Seg-9), and JQ282776 (Seg-10) [ JQ28277752]
- Chloroplast genome of Erycina pusilla, sequence accession JF_746994 [53]
- Danio rerio, sequence accession [ JQ43410154]
- Enterococcal Bacteriophage SAP6, sequence accession [ JF73112855]
- Eubenangee virus, sequence accession through JQ070376 [ JQ07038556]
- Fujian/411-like viruses, sequence accession [ CY087969 to CY08856857]
- Hantavirus Variant of Rio Mamoré Virus, Maripa Virus, sequence accession (segment S), JQ611712 (segment M), and JQ611713 (segment L) [ JQ61171458]
- Pata virus, sequence accession through JQ070386 [ JQ07039559]
- Porcine Circovirus 2, sequence accession [ JQ41380860]
- Porcine Reproductive and Respiratory Syndrome Virus, sequence accession [ JQ32627161]
- Streptococcus mutans Phage M102AD, sequence accession [ DQ38616262]
- Tilligery virus, sequence accession through JQ070366 [ JQ07037563]
6.
In this short report, the genome-wide homologous recombination events were re-evaluated for classical swine fever virus (CSFV) strain . We challenged a previous study which suggested only one recombination event in AF407339 based on 25 CSFV genomes. Through our re-analysis on the 25 genomes in the previous study and the 41 genomes used in the present study, we argued that there should be possibly at least two clear recombination events happening in AF407339 through genome-wide scanning. The reasons for identifying only one recombination event in the previous study might be due to the limited number of available CSFV genome sequences at that time and the limited usage of detection methods. In contrast, as identified by most detection methods using all available CSFV genome sequences, two major recombination events were found at the starting and ending zones of the genome AF407339, respectively. The first one has two parents AF407339 (minor) and AF333000 (major) with beginning and ending breakpoints located at 19 and 607 nt of the genome respectively. The second one has two parents AY554397 (minor) and AF531433 (major) with beginning and ending breakpoints at 8397 and 11,078 nt of the genome respectively. Phylogenetic incongruence analysis using neighbor-joining algorithm with 1000 bootstrapping replicates further supported the existence of these two recombination events. In addition, we also identified additional 18 recombination events on the available CSFV strains. Some of them may be trivial and can be ignored. In conclusion, CSFV might have relatively high frequency of homologous recombination events. Genome-wide scanning of identifying recombination events should utilize multiple detection methods so as to reduce the risk of misidentification. GQ902941相似文献
7.
Bacterial regeneration of ammonium and phosphate as affected by the carbon:nitrogen:phosphorus ratio of organic substrates 总被引:10,自引:2,他引:8
Yasuhiko Tezuka 《Microbial ecology》1990,19(3):227-238
The effect of carbonnitrogenphosphorus (CNP) ratio of organic substrates on the regeneration of ammonium and phosphate was investigated by growing natural assemblages of freshwater bacteria in mineral media supplemented with the simple organic C, N, and P sources (glucose, asparagine, and sodium glycerophosphate, respectively) to give 25 different substrate CNP ratios. Both ammonium and phosphate were regenerated when CN and NP atomic ratios of organic substrates were 101 and 161, respectively. Only ammonium was regenerated when CN and NP ratios were 101 and 10–201, respectively. On the other hand, neither ammonium nor phosphate was regenerated when CN and NP ratios were 151 and 51, respectively. In no case was phosphate alone regenerated. As bacteria were able to alter widely the CNP ratio of their biomass, the growth yield of bacteria appeared primarily dependent on the substrate carbon concentration, irrespective of a wide variation in the substrate CNP ratio. 相似文献
8.
The purpose of this table is to provide the community with a citable record of publications of ongoing genome sequencing projects that have led to a publication in the scientific literature. While our goal is to make the list complete, there is no guarantee that we may have omitted one or more publications appearing in this time frame. Readers and authors who wish to have publications added to subsequent versions of this list are invited to provide the bibliographic data for such references to the SIGS editorial office.
- Phylum Crenarchaeota
- Thermoproteus tenax, strain Kra1, DSM 2078T sequence accession [ FN8698591]
- Phylum Euryarchaeota
- Haloarcula hispanica CGMCC 1.2049, sequence accession (chromosome I), CP002921 (chromosome II), and CP002922 (plasmid pHH400) [ CP0029232]
- Methanococcus maripaludis, strain X1 (unculturable) sequence accession [ CP0029133]
- Phylum Proteobacteria
- Acinetobacter baumannii strain 1656-2, sequence accession [ CP0019214]
- Arcobacter butzleri strain ED-1, sequence accession , AP012047, and AP012048 [ AP0120495]
- Brucella suis strain 1330, sequence accession and CP002997 [ CP0029986]
- Campylobacter fetus subsp. venerealis NCTC 10354, sequence accession [ AFGH010000007]
- “Chromobacterium sp.” strain C-61, sequence accession to CAEE01000001 [ CAEE010011188]
- Cronobacter sakazakii strain E899, sequence accession [ AFMO000000009]
- “Desulfovibrio sp.” strain A2, sequence accession [ AGFG0100000010]
- “Erythrobacter sp.” strain NAP1, sequence accession [ NZ_AAMW0000000011]
- Escherichia coli strain XH140A, sequence accession [ AFVX0100000012]
- Escherichia coli strain XH001, sequence accession [ AFYG0100000013]
- Haemophilus haemolyticus strain , sequence accession M19107 [ AFQN0000000014]
- Haemophilus haemolyticus strain , sequence accession M19501 [ AFQO0000000014]
- Haemophilus haemolyticus strain , sequence accession M21127 [ AFQP0000000014]
- Haemophilus haemolyticus strain , sequence accession M21621 [ AFQQ0000000014]
- Haemophilus haemolyticus strain , sequence accession M21639 [ AFQR0000000014]
- Idiomarina sp.” strain A28L, sequence accession [ AFPO01000001 to AFPO0100002815]
- Ketogulonicigenium vulgare” strain WSH-001, sequence accession (chromosome), CP002018 (plasmid pKVU_100), and CP002019 (plasmid pKVU_200) [ CP00202016]
- Methylobacter tundripaludum strain SV96, sequence accession [ AEGW0000000017]
- Pseudogulbenkiania sp.” strain NH8B, sequence accession [ AP01222418]
- Pseudomonas aeruginosa NCGM1179, sequence accession through DF126593 [ DF12661319]
- Pseudomonas putida strain B001, sequence accession to CAED01000001 [ CAEE0100026220]
- Pseudomonas putida strain B6-2, sequence accession [ AGCS0100000021]
- Pseudomonas stutzeri CGMCC 1.1803, sequence accession [ CP00288122]
- Ralstonia solanacearum phylotype IB, strain Y45, sequence accession [ AFWL0100000023]
- Rheinheimera sp.” strain A13L, sequence accession through AFHI01000001 [ AFHI0100007224]
- Sphingobium yanoikuyae strain XLDN2-5, sequence accession [ AFXE0100000025]
- Vibrio cholerae strain Amazonia, sequence accession [ AFSV0100000026]
- Phylum Firmicutes
- Bacillus coagulans strain XZL4, sequence accession [ AFWM0100000027]
- Bacillus megaterium strain WSH-002, sequence accession (chromosome), plasmids CP003017 (plasmid pBME_100), CP003018 (plasmid pBME_200), and CP003019 (plasmid pBME_300) [ CP00302028]
- Bacillus pumilus strain S-1, sequence accession [ AGBY0000000029]
- “Desulfosporosinus sp.” strain OT, sequence accession [ AGAF0100000030]
- Lentibacillus jeotgali strain Grbi, sequence accession [ AGAV0100000031]
- Leuconostoc carnosum KCTC 3525, sequence accession [ BACM0100000032]
- Listeria ivanovii subsp. ivanovii strain PAM 55, sequence accession [ FR68725333]
- Paenibacillus riograndensis strain SBR5, sequence accession [ AGBD0100000034]
- Sporolactobacillus inulinus strain CASD, sequence accession [ AFVQ0000000035]
- Streptococcus pseudopneumoniae strain IS7493, sequence accession and CP002925 [ CP00292636]
- Streptococcus salivarius strain 57.I, sequence accession and CP002888 [ CP00288937]
- Streptococcus salivarius strain M18, sequence accession [ AGBV0100000038]
- Streptococcus suis SS12, sequence accession [ CP00264039]
- Streptococcus suis D9, sequence accession [ CP00264139]
- Streptococcus suis D12, sequence accession [ CP00264439]
- Streptococcus suis ST1, sequence accession [ CP00265139]
- Weissella thailandensis strain fsh4-2, sequence accession through HE575133 [ HE57518240]
- Phylum Tenericutes
- Mycoplasma anatis strain 1340, sequence accession [ AFVJ0000000041]
- Mycoplasma capricolum subsp. capripneumoniae strain M1601, sequence accession [ AENG0100000042]
- Mycoplasma putrefaciens Type strain KS1, sequence accession [ CP00302143]
- Corynebacterium pseudotuberculosis strain PAT10, sequence accession [ CP00292444]
- Phylum Actinobacteria
- Bifidobacterium animalis subsp. lactis strain BLC1, sequence accession [ CP00303945]
- Bifidobacterium breve strain DPC 6330, sequence accession [ AFXX0100000046]
- Brachybacterium squillarum strain M-6-3, sequence accession [ AGBX0100000047]
- “Citricoccus sp.” strain CH26A, sequence accession [ AFXQ0100000048]
- Corynebacterium glutamicum strain S9114, sequence accession [ AFYA0100000049]
- Dietzia alimentaria strain 72, sequence accession [ AGFF0100000050]
- Mycobacterium colombiense CECT 3035, sequence accession [ AFVW0000000051]
- Mycobacterium tuberculosis NCGM2209, sequence accession and DF126614 [ DF12661552]
- Rhodococcus erythropolis strain XP, sequence accession [ AGCF0100000053]
- Serinicoccus profundi MCCC 1A05965T, sequence accession [ AFYF0000000054]
- Phylum Spirochaetes
- Leptospira interrogans, sequence accession (CI), CP001221 (CII) [ CP00122255]
- Phylum Bacteroidetes
- Bacteroides faecis Type strain MAJ27T, sequence accession [ AGDG0100000056]
- Bizionia argentinensis, Type strain JUB59T sequence accession [ AFXZ0100000057]
- Flavobacterium branchiophilum strain FL-15, sequence accession [ FQ85918358]
- “Flavobacteriaceae” strain S85, sequence accession [ AFPK0000000059]
- Phylum Thermotogae
- “Thermotoga sp.” strain RQ2, sequence accession [ CP00096960]
Non-Bacterial genomes
- Aspergillus kawachii IFO 4308, sequence accession through DF126447, BACL01000001 through BACL01001641, DF126592 [ AP01227261]
- Cajanus cajan pigeonpea, sequence accession PRJNA72815 [62]
- Coxsackievirus A22, sequence accession [ JN54251063]
- Gordonia phage GRU1, sequence accession [ JF92379764]
- Gordonia phage GTE5, sequence accession [ JF92379664]
- Heterocephalus glaber naked mole rat, sequence accession , AFSB00000000 [ AFSB0100000065]
- Human Adenovirus Prototype 17, sequence accession [ HQ91040766]
- Macaca mulatta lasiota rhesus macaque, sequence accession [ AEHL0000000067]
- Macaca mulatta mulatta rhesus macaque, sequence accession [ AEHK0000000067]
- Porcine epidemic diarrhea virus, sequence accession [ JN54722868]
9.
10.
11.
12.
Flavia Pichiorri Hiroshi Okumura Tatsuya Nakamura Preston N. Garrison Pierluigi Gasparini Sung-Suk Suh Teresa Druck Kelly A. McCorkell Larry D. Barnes Carlo M. Croce Kay Huebner 《The Journal of biological chemistry》2009,284(2):1040-1049
We have previously shown that Fhit tumor suppressor protein interacts with
Hsp60 chaperone machinery and ferredoxin reductase (Fdxr) protein.
Fhit-effector interactions are associated with a Fhit-dependent increase in
Fdxr stability, followed by generation of reactive oxygen species and
apoptosis induction under conditions of oxidative stress. To define Fhit
structural features that affect interactions, downstream signaling, and
biological outcomes, we used cancer cells expressing Fhit mutants with amino
acid substitutions that alter enzymatic activity, enzyme substrate binding, or
phosphorylation at tyrosine 114. Gastric cancer cell clones stably expressing
mutants that do not bind substrate or cannot be phosphorylated showed
decreased binding to Hsp60 and Fdxr and reduced mitochondrial localization.
Expression of Fhit or mutants that bind interactor proteins results in
oxidative damage and accumulation of cells in G2/M or
sub-G1 fractions after peroxide treatment; noninteracting mutants
are defective in these biological effects. Gastric cancer clones expressing
noncomplexing Fhit mutants show reduction of Fhit tumor suppressor activity,
confirming that substrate binding, interaction with heat shock proteins,
mitochondrial localization, and interaction with Fdxr are important for Fhit
tumor suppressor function.Fhit protein is a powerful tumor suppressor that is frequently lost or
reduced in cancer cells because of rearrangement of the exquisitely DNA
damage-sensitive fragile FHIT gene. Restoration of Fhit expression
suppresses tumorigenicity of cancer cells of various types, and the ability to
induce apoptosis in cancer cells in vitro is reduced by specific Fhit
mutations (1,
2).Through studies of signal pathways affected by Fhit expression, by searches
for Fhit protein effectors, and by in vitro analyses of Fhit
activity, we and others have defined Fhit enzymatic activity in vitro
(3), apoptotic activity in
cells and tumors
(4–6),
and most recently identification of a Fhit protein complex that affects Fhit
stability, mitochondrial localization, and interaction with ferredoxin
reductase (Fdxr)5
(7). The complex includes Hsp60
and Hsp10 that mediate Fhit stability and may affect import into mitochondria,
where Fhit interacts with Fdxr, which is responsible for transferring
electrons from NADPH to cytochrome P450 via ferredoxin. Virally mediated Fhit
restoration in Fhit-deficient cancer cells increases production of
intracellular reactive oxygen species (ROS), followed by increased apoptosis
of cancer cells under oxidative stress conditions; conversely, Fhit-negative
cells escape apoptosis, likely carrying oxidative DNA damage that contributes
to accumulation of mutations.The Fhit protein sequence, showing high homology to the histidine triad
(HIT) family of proteins, suggested that the protein product would hydrolyze
diadenosine tetraphosphate or diadenosine triphosphate (Ap3A)
(8), and in vitro
studies showed that Ap3A was cleaved into ADP and AMP by Fhit. The
catalytic histidine triad within Fhit was essential for catalytic activity
(3), and a Fhit mutant that
substituted Asn for His at the central histidine (H96N mutant) was
catalytically inactive, although it bound substrate well
(3). Early tumor suppression
studies showed that cancer cells stably transfected with wild type (WT) or
H96N mutant Fhit were suppressed for tumor growth in nude mice. This suggested
the hypothesis that the Fhit-substrate complex sends the tumor suppression
signal (9,
10). To test this hypothesis,
a series of FHIT alleles was designed to reduce substrate-binding
and/or hydrolytic rates and was characterized by quantitative cell-death
assays on cancer cells virally infected with each allele. The allele series
covered defects as great as 100,000-fold in kcat and
increases as large as 30-fold in Km. Mutants with
2–7-fold increases in Km had significantly reduced
apoptotic indices and the mutant with a 30-fold increase in
Km retained little apoptotic function. Thus, the
proapoptotic function of Fhit, which is likely associated with tumor
suppressor function, is limited by substrate binding and is unrelated to
substrate hydrolysis (11).Fhit, a homodimeric protein of 147 amino acids, is a target of tyrosine
phosphorylation by the Src family protein kinases, which can phosphorylate
Tyr-114 of Fhit in vitro and in vivo
(12). After co-expression of
Fhit with the Elk tyrosine kinase in Escherichia coli to generate
phosphorylated forms of Fhit, unphosphorylated, mono-, and diphosphorylated
Fhit were purified, and enzyme kinetics studies showed that monophosphorylated
Fhit exhibited monophasic kinetics with Km and
kcat values ∼2- and ∼7-fold lower, respectively,
than for unphosphorylated Fhit. Diphosphorylated Fhit exhibited biphasic
kinetics; one site had Km and kcat
values ∼2- and ∼140-fold lower, respectively, than for
unphosphorylated Fhit; the second site had a Km
∼60-fold higher and a kcat ∼6-fold lower than for
unphosphorylated Fhit (13).
Thus, it was possible that the alterations in Km and
kcat values for phosphorylated forms of Fhit might favor
formation and lifetime of the Fhit-Ap3A complex and enhance tumor
suppressor activity (see Fhit forms
Kinetic parameters
% Sub-G1
Direct binding
Subcellular location
Co-IP in vivo
8-OHdG
Apoptosis
Tumor suppressor
Km (mm) kcat (s–1) A549 MKN74 Hsp60 Fdxr Hsp60 Fdxr
Fhit WT
1.6 +/– 0.19
2.7 +/– 0.95
43
24
Yes
Yes
Cyt & mito
Yes
Yes
Yes
Yes
Yes
Catalyt mutants H96D
Up 2-fold
Down >2 × 104 29
NT
NT
NT
Cyt & mito
Yes
Yes
NT
Yes
NT
H96N
Up 2-fold
Down >5 × 105
31
14.4
NT
NT
Cyt & mito
Yes
Yes
Yes
Yes
Yes
Loop mutants Y114A
Up 23-fold
Down 2-fold
3.7
NT
NT
NT
Cyt
+/–
+/–
+/–
No
No
Y114D
NT
NT
2.9
6
NT
NT
Cyt
+/–
+/–
–
No
–/+
Y114E
NT
NT
NT
NT
NT
NT
Cyt & mito
–/+
–/+
–
No
NT
Y114F
Up 5-fold
Up 1.1-fold
11.5
3
NT
NT
Cyt & mito
–/+
–/+
–
No
No
Y114W
Up 5-fold
Up 1.4-fold
NT
NT
NT
NT
Cyt & mito
–/+
–
–
NT
NT
del113–117
Up 10-fold
Down 38-fold
5
NT
NT
NT
NT
NT
NT
–
No
NT
Other mutants L25W
Up 7-fold
Down 4-fold
15
NT
NT
NT
Cyt
–
–
–
NT
–/+
I10W,L25W
Up 32-fold
Down 6-fold
11
NT
NT
NT
NT
NT
NT
NT
NT
NT
F5W
Up 3.3 fold
NT
NT
5
NT
NT
NT
NT
NT
+/–
No
NT
Purified pFhit pFhit
Down 0.4-fold
Down 7-fold
NA
NA
–/+
Yes
NA
NA
NA
NA
NA
NA
ppFhit
Down 0.4-fold
Down > 100-fold
NA
NA
–/+
Yes
NA
NA
NA
NA
NA
NA
Up 60-fold
Down 6-fold