An arachidonic acid generation/export system involved in the regulation of cholesterol transport in mitochondria of steroidogenic cells

Recent studies demonstrated the importance of the mitochondrial ATP in the regulation of a novel long-chain fatty acid generation/export system in mitochondria of diabetic rat heart. In steroidogenic systems, mitochondrial ATP and intramitochondrial arachidonic acid (AA) generation are important for steroidogenesis. Here, we report that mitochondrial ATP is necessary for the generation and export of AA, steroid production and steroidogenic acute regulatory protein induction supported by cyclic 3'-5'-adenosine monophosphate in steroidogenic cells. These results demonstrate that ATP depletion affects AA export and provide new evidence of the existence of the fatty acid generation and export system involved in mitochondrial cholesterol transport.     

Improvement of glycine biosynthesis from one-carbon compounds and ammonia catalyzed by the glycine cleavage system in vitro

Glycine cleavage system (GCS) plays a central role in one-carbon (C1) metabolism and receives increasing interest as a core part of the recently proposed reductive glycine pathway (rGlyP) for assimilation of CO₂ and formate. Despite decades of research, GCS has not yet been well understood and kinetic data are barely available. This is to a large degree because of the complexity of GCS, which is composed of four proteins (H, T, P, and L) and catalyzes reactions involving different substrates and cofactors. In vitro kinetics of reconstructed microbial multi-enzyme glycine cleavage/synthase system is desired to better implement rGlyP in microorganisms like Escherichia coli for the use of C1 resources. Here, we examined in vitro several factors that may affect the rate of glycine synthesis via the reverse GCS reaction. We found that the ratio of GCS component proteins has a direct influence on the rate of glycine synthesis, namely higher ratios of P protein and especially H protein to T and L proteins are favorable, and the carboxylation reaction catalyzed by P protein is a key step determining the glycine synthesis rate, whereas increasing the ratio of L protein to other GCS proteins does not have significant effect and the ratio of T protein to other GCS proteins should be kept low. The effect of substrate concentrations on glycine synthesis is quite complex, showing interdependence with the ratios of GCS component proteins. Furthermore, adding the reducing agent dithiothreitol to the reaction mixture not only results in great tolerance to high concentration of formaldehyde, but also increases the rate of glycine synthesis, probably due to its functions in activating P protein and taking up the role of L protein in the non-enzymatic reduction of H_ox to H_red. Moreover, the presence of some monovalent and divalent metal ions can have either positive or negative effect on the rate of glycine synthesis, depending on their type and their concentration.     

Evidences against vesicle-dependent trafficking and involvement of extracellular proteasomes into cell-to-cell communications

The ubiquitin proteasome system is involved in the regulation of most basic intracellular processes, and deregulation of this system can results in certain kinds of human diseases. Proteolytic core this system, the 20S proteasome, has been found in physiological fluids of both healthy humans and patients suffering from a variety of inflammatory, autoimmune, and neoplastic diseases. The concentration of these extracellular proteasomes has been found to correlate with the diseased state, being of a prognostic significance. The transport mechanisms and functions of these proteasomes, however, are largely unclear. Previous studies revealed that the transport of extracellular proteasomes may occur via microvesicles and exosomes, which led to the hypothesis that extracellular proteasomes are implicated in cell-to-cell communication process. Here we show that microvesicles and exosomes, two major known types of intercellular vehicles, contain no detectable proteasomes. Moreover, neither affinity purified nor naturally released into conditioned medium by donor cells 20S proteasomes could penetrate recipient HeLa cells. Taken together, these results suggest that extracellular proteasomes are unlikely to be involved in the cell-to-cell communication and that their release by cells serve other biological purposes.     

Reduction of IL-2 fragmentation during manufacturing of a novel immunocytokine by DoE process optimization

Interleukin-2 (IL-2) is a potent molecule in cancer therapy. Clinical application, however, is limited due to its strong side effects during the treatment. We developed an IL-2 variant (IL-2v) immunocytokine to circumvent the drawbacks of the current IL-2 therapy. During the production of the IL-2v immunocytokine in Chinese hamster ovary (CHO) cells, molecules with fragmented IL-2v and therefore reduced cytokine activity can be observed. To control product fragmentation different production process conditions were investigated. By shifting temperature or pH after the cell growth phase to lower values, fragmented species can be reduced from 10% to 12% to about 4%. However, with the adopted process conditions, the effective titer is decreased concomitantly. Moreover, fermentation length and inoculation cell density are parameters to adjust fragmentation and effective titer. A suitable method for efficient process optimization is the design of experiment approach. With this procedure, novel optimal values for temperature, pH value, harvest day, and inoculation cell densities were proposed and tested subsequently. In comparison to the former process, the improved process reduces fragmentation by 66% while keeping the effective titer comparable. In summary, these findings will help to control fragmentation in CHO production processes of different IL-2v or IL-2 containing therapeutic proteins.     

Structure of an engineered intein reveals thiazoline ring and provides mechanistic insight

We have engineered an intein which spontaneously and reversibly forms a thiazoline ring at the native N-terminal Lys-Cys splice junction. We identified conditions to stablize the thiazoline ring and provided the first crystallographic evidence, at 1.54 Å resolution, for its existence at an intein active site. The finding bolsters evidence for a tetrahedral oxythiazolidine splicing intermediate. In addition, the pivotal mutation maps to a highly conserved B-block threonine, which is now seen to play a causative role not only in ground-state destabilization of the scissile N-terminal peptide bond, but also in steering the tetrahedral intermediate toward thioester formation, giving new insight into the splicing mechanism. We demonstrated the stability of the thiazoline ring at neutral pH as well as sensitivity to hydrolytic ring opening under acidic conditions. A pH cycling strategy to control N-terminal cleavage is proposed, which may be of interest for biotechnological applications requiring a splicing activity switch, such as for protein recovery in bioprocessing.     

Design,synthesis and molecular docking of new N-4-piperazinyl ciprofloxacin-triazole hybrids with potential antimicrobial activity

New N-4-piperazinyl ciprofloxacin-triazole hybrids 6a-o were prepared and characterized. The in vitro antimycobacterial activity revealed that compound 6a experienced promising antimycobacterial activity against Mycobactrium smegmatis compared with the reference isoniazide (INH). Additionally, compound 6a exhibited broad spectrum antibacterial activity against all the tested strains either Gram-positive or Gram-negative bacteria compared with the reference ciprofloxacin. Also, compounds 6g and 6i displayed considerable antifungal activity compared with the reference ketoconazole. DNA cleavage assay of the highly active compounds 6c and 6h showed a good correlation between the Mycobactrium cleaved DNA gyrase assay and their in vitro antimycobactrial activity. Moreover, molecular modeling studies were done for the designed ciprofloxacin derivatives to predict their binding modes towards Topoisomerase II enzyme (PDB: 5bs8).     

Synthesis of a new disulfide Fmoc monomer for creating biologically susceptible linkages in peptide nucleic acid oligomers

Peptide nucleic acids (PNA) are one of many synthetic mimics of DNA and RNA that have found applications as biological probes, as nano-scaffold components, and in diagnostics. In an effort to use PNA as constructs for cellular delivery we investigated the possibility of installing a biologically susceptible disulfide bond in the backbone of a PNA oligomer. Here we report the synthesis of a new abasic Fmoc monomer containing a disulfide bond that can be incorporated into a PNA oligomer (DS-PNA) using standard solid phase peptide synthesis. The disulfide bond survives cleavage from the resin and DS-PNA forms duplexes with complementary PNA oligomers. Initial studies aimed at determining if the disulfide bond is cleavable to reducing agents while in a duplex are explored using UV thermal analysis and HPLC.     

Maternal effect of low temperature on handedness determination in C. elegans embryos

C. elegans embryos, larvae, and adults exhibit several left-right asymmetries with an invariant dextral handedness, which first becomes evident in the embryo at the 6-cell stage. Reversed (sinistral) handedness was not observed among > 10,000 N2 adults reared at 16°C or 20°C under standard conditions. However, among the progeny of adults reproducing at 10°C, the frequency of animals with sinistral handedness was increased to ∼0.5%. Cold pulse experiments indicated that the critical period for this increase was in early oogenesis, several hours before the first appearance of left-right asymmetry in the embryo. Hermaphrodites reared at 10°C and mated with males reared at 20°C produced sinistral outcross as well as sinistral self-progeny, indicating that the low temperature effect on oocytes was sufficient to cause reversals. Increased frequency of reversal was also observed among animals developed from embryos lacking the egg shell. Possible mechanisms for the control of embryonic handedness are discussed in the context of these results, including the hypothesis that handedness could be dictated by the chirality of a gametic component. © 1996 Wiley-Liss, Inc.     

Neuronal postdevelopmentally acting SAX-7S/L1CAM can function as cleaved fragments to maintain neuronal architecture in Caenorhabditis elegans

Whereas remarkable advances have uncovered mechanisms that drive nervous system assembly, the processes responsible for the lifelong maintenance of nervous system architecture remain poorly understood. Subsequent to its establishment during embryogenesis, neuronal architecture is maintained throughout life in the face of the animal’s growth, maturation processes, the addition of new neurons, body movements, and aging. The Caenorhabditis elegans protein SAX-7, homologous to the vertebrate L1 protein family of neural adhesion molecules, is required for maintaining the organization of neuronal ganglia and fascicles after their successful initial embryonic development. To dissect the function of sax-7 in neuronal maintenance, we generated a null allele and sax-7S-isoform-specific alleles. We find that the null sax-7(qv30) is, in some contexts, more severe than previously described mutant alleles and that the loss of sax-7S largely phenocopies the null, consistent with sax-7S being the key isoform in neuronal maintenance. Using a sfGFP::SAX-7S knock-in, we observe sax-7S to be predominantly expressed across the nervous system, from embryogenesis to adulthood. Yet, its role in maintaining neuronal organization is ensured by postdevelopmentally acting SAX-7S, as larval transgenic sax-7S(+) expression alone is sufficient to profoundly rescue the null mutants’ neuronal maintenance defects. Moreover, the majority of the protein SAX-7 appears to be cleaved, and we show that these cleaved SAX-7S fragments together, not individually, can fully support neuronal maintenance. These findings contribute to our understanding of the role of the conserved protein SAX-7/L1CAM in long-term neuronal maintenance and may help decipher processes that go awry in some neurodegenerative conditions.     

The Emergence of the Spike Furin Cleavage Site in SARS-CoV-2

Compared with other SARS-related coronaviruses (SARSr-CoVs), SARS-CoV-2 possesses a unique furin cleavage site (FCS) in its spike. This has stimulated discussion pertaining to the origin of SARS-CoV-2 because the FCS has been observed to be under strong selective pressure in humans and confers the enhanced ability to infect some cell types and induce cell–cell fusion. Furthermore, scientists have demonstrated interest in studying novel cleavage sites by introducing them into SARSr-CoVs. We review what is known about the SARS-CoV-2 FCS in the context of its pathogenesis, origin, and how future wildlife coronavirus sampling may alter the interpretation of existing data.