Cyanide binding and heme cavity conformational transitions in Drosophila melanogaster hexacoordinate hemoglobin

The reason for the presence of hemoglobin-like molecules in insects, such as Drosophila melanogaster, that live in fully aerobic environments has yet to be determined. Heme endogenous hexacoordination (where HisE7 and HisF8 axial ligands to the heme Fe atom are both provided by the protein) is a recently discovered mechanism proposed to modulate O(2) affinity in hemoglobins from different species. Previous results have shown that D. melanogaster hemoglobin 1 (product of the glob1 gene) displays heme endogenous hexacoordination in both the ferrous and ferric states. Here we present kinetic data characterizing the exogenous cyanide ligand binding process, and the three-dimensional structure (at 1.4 A resolution) of the ensuing cyano-met D. melanogaster hemoglobin. Comparison with the crystal structure of the endogenously hexacoordinated D. melanogaster hemoglobin shows that the transition to the cyano-met form is supported by conformational readjustment in the CD-D-E region of the protein, which removes HisE7 from the heme. The structural and functional features of D. melanogaster hemoglobin are examined in light of previous results achieved for human and mouse neuroglobins and for human cytoglobin, which display heme endogenous hexacoordination. The study shows that, despite the rather constant value for cyanide association rate constants for the ferric hemoproteins, different distal site conformational readjustments and/or heme sliding mechanisms are displayed by the known hexacoordinate hemoglobins as a result of exogenous ligand binding.     

Seasonal changes in the thermal tolerances of the toad Rhinella arenarum (Bufonidae) in the Monte Desert of Argentina

We studied the thermal tolerances of Rhinella arenarum during the dry and wet seasons of the Monte Desert in San Juan Province, Argentina. This toad had differences in CT_max between dry and wet seasons, and the CT_max values were higher in the wet season (Austral summer). Operative temperature, body temperature, environmental maximal temperature, and relative humidity were related to CT_max, suggesting seasonal acclimatization of R. arenarum. Additionally, the CT_max recorded for R. arenarum was 36.2 °C, and the maximum ambient temperature recorded during the toads' activity time was 37 °C. Also, the CT_min recorded for R. arenarum was 5.3 °C and the minimum environmental temperature recorded was 7.2 °C. The wide thermal tolerance range recorded and the relationship between tolerance limits and the environmental extremes indicate that seasonal acclimatization is an effective mechanism by which toads can raise their thermal tolerance, allowing them to survive in the challenging conditions of the Monte Desert. Additional studies are needed to understand the relationship between the thermal tolerance of this desert amphibian and the environmental parameters that influence its thermal physiology.     

FK506 binding protein 8 peptidylprolyl isomerase activity manages a late stage of cystic fibrosis transmembrane conductance regulator (CFTR) folding and stability

Cystic fibrosis (CF) is caused by mutations in the apical chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) with 90% of patients carrying at least one deletion of the F508 (ΔF508) allele. This mutant form of CFTR is characterized by a folding and trafficking defect that prevents exit from the endoplasmic reticulum. We previously reported that ΔF508 CFTR can be recovered in a complex with Hsp90 and its co-chaperones as an on-pathway folding intermediate, suggesting that Δ508 CF disease arises due to a failure of the proteostasis network (PN), which manages protein folding and degradation in the cell. We have now examined the role of FK506-binding protein 8 (FKBP8), a component of the CFTR interactome, during the biogenesis of wild-type and ΔF508 CFTR. FKBP8 is a member of the peptidylprolyl isomerase family that mediates the cis/trans interconversion of peptidyl prolyl bonds. Our results suggest that FKBP8 is a key PN factor required at a post-Hsp90 step in CFTR biogenesis. In addition, changes in its expression level or alteration of its activity by a peptidylprolyl isomerase inhibitor alter CFTR stability and transport. We propose that CF is caused by the sequential failure of the prevailing PN pathway to stabilize ΔF508-CFTR for endoplasmic reticulum export, a pathway that can be therapeutically managed.     

Mechanism of action of therapeutic monoclonal antibodies: Promises and pitfalls of in vitro and in vivo assays

Therapeutic monoclonal antibodies (mAbs) are mostly used in cancer, as anti-infectious agents and as immunomodulatory drugs, and are amongst the most active area of research and development in the pharmaceutical industry. This class of drugs comprises unconjugated antibodies or antibody fragments, antibody-drug conjugates, radio-immunoconjugates and bispecific/trispecific molecules. A better understanding of the mechanism of action of successful mAbs is fundamental for the selection of more active and less toxic mAbs of new generation. Furthermore reliable screening of new compounds at an early stage of preclinical development, for both efficacy and toxicity, should allow the selection of the best molecules at an early stage, and improve the rate of success of this class of drugs. Here we review the major methods that are employed for testing the activity of therapeutic mAbs in vitro and in vivo in small animal models and point out to some of the pitfalls in these assays.     

Flaviviral helicase: insights into the mechanism of action of a motor protein

Motor proteins are involved in crucial cell activities, such as cargo transport or nucleic acid remodeling, by converting the free energy of ATP hydrolysis into motion or mechanical work. Flavivirus helicase is a motor protein involved in dsRNA separation during viral replication, thus essential for virus infection. Since a clear vision of the protein activity, in particular of the relationship between ATP cycling and dynamics, is missing, we carried over a molecular dynamics study on Dengue virus helicase in its ATP bound and unbound states. Our simulations show different opening levels of the ssRNA access site to the helicase core. Specifically, we show that ATP induces a closed state into the ssRNA access site, likely involved in the helicase unwinding activity.     

Oriented immobilization of the tobacco etch virus protease for the cleavage of fusion proteins

The tobacco etch virus (TEV) protease is a useful tool for the removal of fusion tags from recombinant proteins. The difficulty in obtaining this enzyme led us to look for an optimal method for its use. In this work, we produced both the wild-type and the S219V mutant TEV proteases fused to the Streptag II affinity sequence (Streptag II-TEV(WT), and Streptag II-TEV(S219V), respectively). The two enzymes were affinity immobilized on a streptavidin-agarose matrix and compared to their respective free forms. Both immobilized Streptag II-TEV(WT) and Streptag II-TEV(S219V) were active on the 74-kDa Streptag II substrate with a retained activity of 83.5% and 81%, respectively compared to their free corresponding forms. The slight enzyme activity decrease caused by the immobilization was balanced by the enhanced stability and the successful repetitive use of the proteolytic columns. Thus, the wild-type and the mutant immobilized proteases were used, during a period of 18 months, for nine batch reactions with retention of 38% and 51% of their initial activities, respectively. The present results demonstrate that immobilized TEV protease on streptavidin-agarose is an attractive and efficient tool for fusion protein cleavage, especially when the target protein is fused to a streptagged fusion partner. Using this strategy, the total process can be shortened by performing the cleavage and the recovery of the purified target protein in one step.     

Reduction of the background magnetic field inhibits ability of Drosophila melanogaster to survive ionizing radiation

The effects of exposure to an environment where the background magnetic field (BMF) has been reduced were studied on wild‐type Drosophila melanogaster by measuring its ability to survive a single exposure to ionizing radiation (IR) during its larval stage. The experimental design presented shows a timeframe, IR dose, and BMF parameters that will cause a significant and reproducible reduction of survival on this insect model. These results suggest that BMFs may play a fundamental role in the recovery or harm of a biological system that is exposed to single doses of IR. Bioelectromagnetics 33:706–709, 2012. © 2012 Wiley Periodicals, Inc.     

Discovery of P2X3 selective antagonists for the treatment of chronic pain

Purinergic receptor P2X3 has been linked to analgesia in a number of pre-clinical models of pain, and is expressed in the human pain perception pathway. Only few P2X3-selective antagonists have been reported to date. This Letter describes the SAR and in vivo analgesic profile of a novel scaffold of selective P2X3 antagonists.