The translational values of TRIM family in pan-cancers: From functions and mechanisms to clinics.

Cancer is the second leading cause of human death across the world. Tripartite motif (TRIM) family, with E3 ubiquitin ligase activities in majority of its members, is reported to be involved in multiple cellular processes and signaling pathways. TRIM proteins have critical effects in the regulation of biological behaviors of cancer cells. Here, we discussed the current understanding of the molecular mechanism of TRIM proteins regulation of cancer cells. We also comprehensively reviewed published studies on TRIM family members as oncogenes or tumor suppressors in the oncogenesis, development, and progression of a variety of types of human cancers. Finally, we highlighted that certain TRIM family members are potential molecular biomarkers for cancer diagnosis and prognosis, and potential therapeutic targets.

Suppression of Reactive Oxygen Species Accumulation Accounts for Paradoxical Bacterial Survival at High Quinolone Concentration.

When bacterial cells are exposed to increasing concentrations of quinolone-class antibacterials, survival drops, reaches a minimum, and then recovers, sometimes to 100%. Despite decades of study, events underlying this paradoxical high-concentration survival remain obscure. Since reactive oxygen species (ROS) have been implicated in antimicrobial lethality, conditions generating paradoxical survival were examined for diminished ROS accumulation. Escherichia coli cultures were treated with various concentrations of nalidixic acid, followed by measurements of survival, rate of protein synthesis, and ROS accumulation. The last measurement used a dye (carboxy-H2DCFDA) that fluoresces in the presence of ROS; fluorescence was assessed by microscopy (individual cells) and flow cytometry (batch cultures). High, nonlethal concentrations of nalidixic acid induced lower levels of ROS than moderate, lethal concentrations. Sublethal doses of exogenous hydrogen peroxide became lethal and eliminated the nalidixic acid-associated paradoxical survival. Thus, quinolone-mediated lesions needed for ROS-executed killing persist at high, nonlethal quinolone concentrations, thereby implicating ROS as a key factor in cell death. Chloramphenicol suppressed nalidixic acid-induced ROS accumulation and blocked lethality, further supporting a role for ROS in killing. Nalidixic acid also inhibited protein synthesis, with extensive inhibition at high concentrations correlating with lower ROS accumulation and paradoxical survival. A catalase deficiency, which elevated ROS levels, overcame the inhibitory effect of chloramphenicol on nalidixic acid-mediated killing, emphasizing the importance of ROS. The data collectively indicate that ROS play a dominant role in the lethal action of narrow-spectrum quinolone-class compounds; a drop in ROS levels accounted for the quinolone tolerance observed at very high concentrations.

Fluoroquinolone-Gyrase-DNA Cleaved Complexes.

The quinolones are potent antibacterials that act by forming complexes with DNA and either gyrase or topoisomerase IV. These ternary complexes, called cleaved complexes because the DNA moiety is broken, block replication, transcription, and bacterial growth. Cleaved complexes readily form in vitro when gyrase, plasmid DNA, and quinolone are combined and incubated; complexes are detected by the linearization of plasmid DNA, generally assayed by gel electrophoresis. The stability of the complexes can be assessed by treatment with EDTA, high temperature, or dilution to dissociate the complexes and reseal the DNA moiety. Properties of the complexes are sensitive to quinolone structure and to topoisomerase amino acid substitutions associated with quinolone resistance. Consequently, studies of cleaved complexes can be used to identify improvements in quinolone structure and to understand the biochemical basis of target-based resistance. Cleaved complexes can also be detected in quinolone-treated bacterial cells by their ability to rapidly block DNA replication and to cause chromosome fragmentation; they can even be recovered from lysed cells following CsCl density-gradient centrifugation. Thus, in vivo and cell-fractionation tests are available for assessing the biological relevance of work with purified components.

Contribution of reactive oxygen species to thymineless death in Escherichia coli.

Nutrient starvation usually halts cell growth rather than causing death. Thymine starvation is exceptional, because it kills cells rapidly. This phenomenon, called thymineless death (TLD), underlies the action of several antibacterial, antimalarial, anticancer, and immunomodulatory agents. Many explanations for TLD have been advanced, with recent efforts focused on recombination proteins and replication origin (oriC) degradation. Because current proposals account for only part of TLD and because reactive oxygen species (ROS) are implicated in bacterial death due to other forms of harsh stress, we investigated the possible involvement of ROS in TLD. Here, we show that thymine starvation leads to accumulation of both single-stranded DNA regions and intracellular ROS, and interference with either event protects bacteria from double-stranded DNA breakage and TLD. Elevated levels of single-stranded DNA were necessary but insufficient for TLD, whereas reduction of ROS to background levels largely abolished TLD. We conclude that ROS contribute to TLD by converting single-stranded DNA lesions into double-stranded DNA breaks. Participation of ROS in the terminal phases of TLD provides a specific example of how ROS contribute to stress-mediated bacterial self-destruction.

Suppression of gyrase-mediated resistance by C7 aryl fluoroquinolones.

Fluoroquinolones form drug-topoisomerase-DNA complexes that rapidly block transcription and replication. Crystallographic and biochemical studies show that quinolone binding involves a water/metal-ion bridge between the quinolone C3-C4 keto-acid and amino acids in helix-4 of the target proteins, GyrA (gyrase) and ParC (topoisomerase IV). A recent cross-linking study revealed a second drug-binding mode in which the other end of the quinolone, the C7 ring system, interacts with GyrA. We report that addition of a dinitrophenyl (DNP) moiety to the C7 end of ciprofloxacin (Cip-DNP) reduced protection due to resistance substitutions in Escherichia coli GyrA helix-4, consistent with the existence of a second drug-binding mode not evident in X-ray structures of drug-topoisomerase-DNA complexes. Several other C7 aryl fluoroquinolones behaved in a similar manner with particular GyrA mutants. Treatment of E. coli cultures with Cip-DNP selectively enriched an uncommon variant, GyrA-A119E, a change that may impede binding of the dinitrophenyl group at or near the GyrA-GyrA interface. Collectively the data support the existence of a secondary quinolone-binding mode in which the quinolone C7 ring system interacts with GyrA; the data also identify C7 aryl derivatives as a new way to obtain fluoroquinolones that overcome existing GyrA-mediated quinolone resistance.

Ribosomal elongation factor 4 promotes cell death associated with lethal stress.

UNLABELLED: Ribosomal elongation factor 4 (EF4) is highly conserved among bacteria, mitochondria, and chloroplasts. However, the EF4-encoding gene, lepA, is nonessential and its deficiency shows no growth or fitness defect. In purified systems, EF4 back-translocates stalled, posttranslational ribosomes for efficient protein synthesis; consequently, EF4 has a protective role during moderate stress. We were surprised to find that EF4 also has a detrimental role during severe stress: deletion of lepA increased Escherichia coli survival following treatment with several antimicrobials. EF4 contributed to stress-mediated lethality through reactive oxygen species (ROS) because (i) the protective effect of a ΔlepA mutation against lethal antimicrobials was eliminated by anaerobic growth or by agents that block hydroxyl radical accumulation and (ii) the ΔlepA mutation decreased ROS levels stimulated by antimicrobial stress. Epistasis experiments showed that EF4 functions in the same genetic pathway as the MazF toxin, a stress response factor implicated in ROS-mediated cell death. The detrimental action of EF4 required transfer-messenger RNA (tmRNA, which tags truncated proteins for degradation and is known to be inhibited by EF4) and the ClpP protease. Inhibition of a protective, tmRNA/ClpP-mediated degradative activity would allow truncated proteins to indirectly perturb the respiratory chain and thereby provide a potential link between EF4 and ROS. The connection among EF4, MazF, tmRNA, and ROS expands a pathway leading from harsh stress to bacterial self-destruction. The destructive aspect of EF4 plus the protective properties described previously make EF4 a bifunctional factor in a stress response that promotes survival or death, depending on the severity of stress. IMPORTANCE: Translation elongation factor 4 (EF4) is one of the most conserved proteins in nature, but it is dispensable. Lack of strong phenotypes for its genetic knockout has made EF4 an enigma. Recent biochemical work has demonstrated that mild stress may stall ribosomes and that EF4 can reposition stalled ribosomes to resume proper translation. Thus, EF4 protects cells from moderate stress. Here we report that EF4 is paradoxically harmful during severe stress, such as that caused by antimicrobial treatment. EF4 acts in a pathway that leads to excessive accumulation of reactive oxygen species (ROS), thereby participating in a bacterial self-destruction that occurs when cells cannot effectively repair stress-mediated damage. Thus, EF4 has two opposing functions-at low-to-moderate levels of stress, the protein is protective by allowing stress-paused translation to resume; at high-levels of stress, EF4 helps bacteria self-destruct. These data support the existence of a bacterial live-or-die response to stress.

Bypassing fluoroquinolone resistance with quinazolinediones: studies of drug-gyrase-DNA complexes having implications for drug design.

Widespread fluoroquinolone resistance has drawn attention to quinazolinediones (diones), fluoroquinolone-like topoisomerase poisons that are unaffected by common quinolone-resistance mutations. To better understand differences between quinolones and diones, we examined their impact on the formation of cleaved complexes (drug-topoisomerase-DNA complexes in which the DNA moiety is broken) with gyrase, one of two bacterial targets of the drugs. Formation of cleaved complexes, measured by linearization of a circular DNA substrate, required lower concentrations of quinolone than dione. The reverse reaction, detected as resealing of DNA breaks in cleaved complexes, required higher temperatures and EDTA concentrations for quinolones than diones. The greater stability of quinolone-containing complexes was attributed to the unique ability of the quinolone C3/C4 keto acid to complex with magnesium and form a previously described drug-magnesium-water bridge with GyrA-Ser83 and GyrA-Asp87. A nearby substitution in GyrA (G81C) reduced activity differences between quinolone and dione, indicating that resistance due to this variation derives from perturbation of the magnesium-water bridge. To increase dione activity, we examined a relatively small, flexible C-7-3-(aminomethyl)pyrrolidinyl substituent, which is distal to the bridging C3/C4 keto acid substituent of quinolones. The 3-(aminomethyl)pyrrolidinyl group at position C-7 was capable of forming binding interactions with GyrB-Glu466, as indicated by inspection of crystal structures, computer-aided docking, and measurement of cleaved-complex formation with mutant and wild-type GyrB proteins. Thus, modification of dione C-7 substituents constitutes a strategy for obtaining compounds active against common quinolone-resistant mutants.

Fluoroquinolone-gyrase-DNA complexes: two modes of drug binding.

DNA gyrase and topoisomerase IV control bacterial DNA topology by breaking DNA, passing duplex DNA through the break, and then resealing the break. This process is subject to reversible corruption by fluoroquinolones, antibacterials that form drug-enzyme-DNA complexes in which the DNA is broken. The complexes, called cleaved complexes because of the presence of DNA breaks, have been crystallized and found to have the fluoroquinolone C-7 ring system facing the GyrB/ParE subunits. As expected from x-ray crystallography, a thiol-reactive, C-7-modified chloroacetyl derivative of ciprofloxacin (Cip-AcCl) formed cross-linked cleaved complexes with mutant GyrB-Cys(466) gyrase as evidenced by resistance to reversal by both EDTA and thermal treatments. Surprisingly, cross-linking was also readily seen with complexes formed by mutant GyrA-G81C gyrase, thereby revealing a novel drug-gyrase interaction not observed in crystal structures. The cross-link between fluoroquinolone and GyrA-G81C gyrase correlated with exceptional bacteriostatic activity for Cip-AcCl with a quinolone-resistant GyrA-G81C variant of Escherichia coli and its Mycobacterium smegmatis equivalent (GyrA-G89C). Cip-AcCl-mediated, irreversible inhibition of DNA replication provided further evidence for a GyrA-drug cross-link. Collectively these data establish the existence of interactions between the fluoroquinolone C-7 ring and both GyrA and GyrB. Because the GyrA-Gly(81) and GyrB-Glu(466) residues are far apart (17 Å) in the crystal structure of cleaved complexes, two modes of quinolone binding must exist. The presence of two binding modes raises the possibility that multiple quinolone-enzyme-DNA complexes can form, a discovery that opens new avenues for exploring and exploiting relationships between drug structure and activity with type II DNA topoisomerases.

Production and characterization of recombinant 9 and 15 kDa granulysin by fed-batch fermentation in Pichia pastoris.

Granulysin is a cytolytic, proinflammatory protein produced by human cytolytic T-lymphocytes and natural killer cells. Granulysin has two stable isoforms with molecular weight of 9 and 15 kDa; the 9-kDa form is a result of proteolytic maturation of the 15-kDa precursor. Recombinant 9-kDa granulysin exhibits cytolytic activity against a variety of microbes, such as bacteria, parasites, fungi, yeast and a variety of tumor cell lines. However, it is difficult to produce granulysin in large quantities by traditional methods. In this study, we developed a simple and robust fed-batch fermentation process for production and purification of recombinant 9- and 15-kDa granulysin using Pichia pastoris in a basal salt medium at high cell density. The granulysin yield reaches at least 100 mg/l in fermentation, and over 95 % purity was achieved with common His-select affinity and ion exchange chromatography. Functional analysis revealed that the yeast-expressed granulysin displayed dose-dependent target cytotoxicity. These results suggest that fermentation in P. pastoris provides a sound strategy for large-scale recombinant granulysin production that may be used in clinical applications and basic research.