Adaptor protein ClpS is an essential regulator of prokaryotic ATP-dependent protease ClpAP, which delivers certain protein substrates with specific amino acid sequences to ClpAP for degradation. However, ClpS also functions as the inhibitor of the ClpAP-mediated protein degradation for other proteins. Here, we constructed the clpS-overexpression Mycobacterium smegmatis strain, and showed for the first time that overexpression of ClpS increased the resistance of M. smegmatis to rifampicin that is one of most widely used antibiotic drugs in treatment of tuberculosis. Using quantitative proteomic technology, we systematically analyzed effects of ClpS overexpression on changes in M. smegmatis proteome, and proposed that the increased rifampicin resistance was caused by ClpS-regulated drug sedimentation and drug metabolism. Our results indicate that the changes in degradation related proteins enhanced drug resistance and quantitative proteomic analysis is an important tool for understanding molecular mechanisms responsible for bacteria drug resistance.
ATP-Dependent Proteases ; metabolism ; Drug Resistance, Bacterial ; Endopeptidase Clp ; metabolism ; Mycobacterium smegmatis ; drug effects ; metabolism ; Proteolysis ; Proteomics ; Rifampin ; pharmacology

BACKGROUND: Acute-on-chronic liver failure (ACLF) is a severe liver disease with complex pathogenesis. Clinical hypoglycemia is common in patients with ACLF and often predicts a worse prognosis. Accumulating evidence suggests that glucose metabolic disturbance, especially gluconeogenesis dysfunction, plays a critical role in the disease progression of ACLF. Lon protease-1 (LONP1) is a novel mediator of energy and glucose metabolism. However, whether gluconeogenesis is a potential mechanism through which LONP1 modulates ACLF remains unknown. METHODS: In this study, we collected liver tissues from ACLF patients, established an ACLF mouse model with carbon tetrachloride (CCl 4 ), lipopolysaccharide (LPS), and D-galactose (D-gal), and constructed an in vitro hypoxia and hyperammonemia-triggered hepatocyte injury model. LONP1 overexpression and knockdown adenovirus were used to assess the protective effect of LONP1 on liver injury and gluconeogenesis regulation. Liver histopathology, biochemical index, mitochondrial morphology, cell viability and apoptosis, and the expression and activity of key gluconeogenic enzymes were detected to explore the underlying protective mechanisms of LONP1 in ACLF. RESULTS: We found that LONP1 and the expressions of gluconeogenic enzymes were downregulated in clinical ACLF liver tissues. Furthermore, LONP1 overexpression remarkably attenuated liver injury, which was characterized by improved liver histopathological lesions and decreased serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in ACLF mice. Moreover, mitochondrial morphology was improved upon overexpression of LONP1. Meanwhile, the expression and activity of the key gluconeogenic enzymes were restored by LONP1 overexpression. Similarly, the hepatoprotective effect was also observed in the hepatocyte injury model, as evidenced by improved cell viability, reduced cell apoptosis, and improved gluconeogenesis level and activity, while LONP1 knockdown worsened liver injury and gluconeogenesis disorders. CONCLUSION We demonstrated that gluconeogenesis dysfunction exists in ACLF, and LONP1 could ameliorate liver injury and improve gluconeogenic dysfunction, which would provide a promising therapeutic target for patients with ACLF.
Animals ; Humans ; Mice ; Acute-On-Chronic Liver Failure/pathology* ; ATP-Dependent Proteases/metabolism* ; Gluconeogenesis ; Hepatocytes/pathology* ; Liver/metabolism* ; Mitochondrial Proteins/metabolism* ; Protease La/metabolism*

FtsH is a membrane-bound, ATP-dependent protease involved in various cellular functions. To understand its roles in Streptococcus pneumoniae and host-pathogen interactions, we inactivated the ftsH gene of D39 strain by inserting a tetracycline-resistance (tet) gene. Several recombinants containing the tet cassette within the ftsH gene were confirmed by Western immunoblotting for the absence of pneumococcal FtsH protein that could cross-react with antiserum raised against Escherichia coli FtsH. Compared with the wild-type D39 strain, the ftsH null mutants grew slowly with encapsulation and alpha-hemolysis on blood agar plates, but failed to grow in liquid media other than Todd Hewitt yeast extract broth. Even fresh cultures of ftsH null mutants appeared gram-negative. When the incubation temperature of liquid cultures was shifted from 37degrees C to 40degrees C, the mutants gradually lysed, whereas the shift to 30degrees C abolished further growth. The mutants also exhibited increased sensitivity to salt and remarkable growth inhibition by optochin. These observations suggest that no functional FtsH protein in pneumococcal cells causes a loss of cell surface integrity, resulting in impairment of cell growth under normal and stressful conditions.
Agar ; ATP-Dependent Proteases ; Blotting, Western ; Escherichia coli ; Host-Pathogen Interactions ; Streptococcus pneumoniae* ; Streptococcus* ; Yeasts

FtsH is a membrane-bound, ATP-dependent protease involved in various cellular functions. To understand its roles in Streptococcus pneumoniae and host-pathogen interactions, we inactivated the ftsH gene of D39 strain by inserting a tetracycline-resistance (tet) gene. Several recombinants containing the tet cassette within the ftsH gene were confirmed by Western immunoblotting for the absence of pneumococcal FtsH protein that could cross-react with antiserum raised against Escherichia coli FtsH. Compared with the wild-type D39 strain, the ftsH null mutants grew slowly with encapsulation and alpha-hemolysis on blood agar plates, but failed to grow in liquid media other than Todd Hewitt yeast extract broth. Even fresh cultures of ftsH null mutants appeared gram-negative. When the incubation temperature of liquid cultures was shifted from 37degrees C to 40degrees C, the mutants gradually lysed, whereas the shift to 30degrees C abolished further growth. The mutants also exhibited increased sensitivity to salt and remarkable growth inhibition by optochin. These observations suggest that no functional FtsH protein in pneumococcal cells causes a loss of cell surface integrity, resulting in impairment of cell growth under normal and stressful conditions.
Agar ; ATP-Dependent Proteases ; Blotting, Western ; Escherichia coli ; Host-Pathogen Interactions ; Streptococcus pneumoniae* ; Streptococcus* ; Yeasts

The gene ftsH encodes a membrane-bound and ATP-dependent protease that is involved in a variety of cellular functions including heat-shock and stress response. Streptococcus pneumoniae DNA encompassing most part of the ftsH gene was cloned in Escherichia coli and sequenced. Due to the unsuccessful cloning as seen in other pneumococcal promoters, the 5'-end of the gene including the upstream promoter region was amplified by inverse polymerase chain reaction and then sequenced by cyclic sequencing. The amino acid sequence that is deduced from the 1,959 bp-long ftsH gene is very similar to FtsH of several gram-positive bacteria and E. coli within the region responsible for the AAA (ATPase associated with diverse cellular activities) function. Except for the N-terminal domain that contains a short extracellular region between two mernbrane-spanning segments, pneumococcal FtsH shows striking sequence similarity to that of a closely related species Lactococcus lactis within the conserved cytoplasmic domain where two ATP-binding motifs, the AAA Signature motif, and a zinc-binding motif are found.
Amino Acid Sequence ; ATP-Dependent Proteases ; Base Sequence* ; Clone Cells* ; Cloning, Organism* ; Cytoplasm ; DNA ; Escherichia coli ; Gram-Positive Bacteria ; Lactococcus lactis ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; Streptococcus pneumoniae* ; Streptococcus* ; Strikes, Employee

Filamentation temperature-sensitive H (FtsH) is an ATP-dependent metalloprotease in prokaryotes and eukaryotes. Homology-based analysis was applied to determine 12 ftsH genes in Arabidopsis genome and 9 members in rice genome. Distribution of these ftsH genes on each chromosome displayed a clear preference for some chromosomes such as chromosome 1, 2, 5 of Arabidopsis and chromosome 1,5 of rice. All 21 FtsH proteins were subcellularly targeted to chloroplast or mitochondria. These members could be phylogenetically assorted as eight groups, of which no ortholog of AtFtsH12 in rice was detected. Paralogs in each group shared similarity higher than 80% and orthologs higher than 70%. This strongly indicated that the members from single group were descended from a common ancestral gene. Four pairs of paralogs, AtftsH1/5, AtftsH2/8, AtftsH7/9 and AftsH3/10 were found in Arabidopsis genome. However, only two pairs of ftsH paralogs, OsftsH3/8 and OsftsH4/5, resided in rice genome. The highly homologous members in each group performed striking conservation of exon-intron boundaries and preference for the variable residues in function domains. By contrast, there was significant difference in base composition and sequence length of introns. The comparative analysis of the ftsH gene families of Arabidopsis and rice provided the basis for characteristic and function research of ftsH genes in other plants.
ATP-Dependent Proteases ; genetics ; Amino Acid Sequence ; Arabidopsis ; genetics ; Arabidopsis Proteins ; genetics ; Comparative Genomic Hybridization ; Genome, Plant ; Metalloproteases ; genetics ; Molecular Sequence Data ; Multigene Family ; Oryza ; genetics ; Protein Subunits ; genetics

OBJECTIVE: To analyze the clinical and genetic characteristics of an infant girl featuring comprehensive developmental backwardness. METHODS: The patient was subjected to clinical examination, gas chromatography mass spectrometry and next-generation sequencing (NGS). RESULTS: The child was insensitive to sound, could not turn over, raise head, laugh or recognize his mother. Laboratory tests were all normal, but metabolic analysis suggested 3-methylglutaconic aciduria due to elevated 3-methylglutaconic acid and 3-methylglutaric acid. NGS has detected two compound heterozygous CLPB variants in the child, namely c.1085G>A and c.1700A>C, which were respectively inherited from her father and mother. Bioinformatic analysis predicted both variants to be pathogenic. The patient was diagnosed with 3-methylglutaconic aciduria type VII (MGCA7). CONCLUSION The MGCA7 in the child was probably caused by CLPB gene variants. NGS has provided a powerful diagnostic tool for this rare disorder.
Endopeptidase Clp ; genetics ; Female ; Genetic Testing ; High-Throughput Nucleotide Sequencing ; Humans ; Infant ; Metabolism, Inborn Errors ; genetics

OBJECTIVETo study the role of Lon gene in tumor cell proliferation, apoptosis and cell stress response.

METHODSSmall interfering RNAs (smRNAs) for Lon gene were designed using Ambion software and synthesized. The recombinant plasmid pSilencer U6 2.1/Lon was constructed with the smRNAs and pSilencer U6 2.1, followed by transfection into MCF7 cells via Lipofectamine(TM) 2000. The positive cLones were detected by RT-PCR 24 h after cell transfection. The transfected MCF7 cells were then subjected to cisplatin treatment, ultraviolet (UV) exposure and heat stress, respectively, after which the cells growth was tested with MTT assay and the measurements were plotted against time or concentration depending on the treatment administered. Apoptosis of MCF7 cells following the treatments was measured with flow cytometry.

RESULTSThe mRNA of Lon gene was downregulated in cells transfected with the recombinant plasmid pSilencer U6 2.1-Lon, and RT-PCR fail to detect the specific band of Lon as could be detected in untransfected and mock-transfected MCF7 cells. MTT assay showed that pSilencer U6 2.1-Lon transfection resulted in reduced cell proliferation capacity. Stress response test revealed that MCF7 cells with Lon gene down-regulation enhanced cell sensitivity for UV and cisplatin, which was not observed for non-transfected or mock transfection group. The same changes were also observed for heat stress exposure at 41 degrees Celsius;, but not at 43 degrees Celsius; or 45 degrees Celsius;. Increased cell apoptosis rate from (1.14-/+0.79)% to (22.47-/+3.15)% occurred following pSilencer U6 2.1-Lon transfection of the cells.

CONCLUSIONSLon gene can be significantly downregulated by introduction of siRNA in MCF7 cells to result in enhanced sensitivity of MCF7 cells to UV, cisplatin and heat stress.

Antineoplastic Agents ; pharmacology ; Apoptosis ; Breast Neoplasms ; genetics ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Cell Survival ; drug effects ; radiation effects ; Cisplatin ; pharmacology ; Dose-Response Relationship, Drug ; Female ; Humans ; Protease La ; genetics ; RNA Interference ; Reverse Transcriptase Polymerase Chain Reaction ; Temperature ; Time Factors ; Ultraviolet Rays