Protein trafficking in response to DNA damage

Human cells are prone to a range of natural environmental stresses and administered agents that damage or modify DNA, resulting in a cellular response typified by either cell death, or a cell cycle arrest, to permit repair of the genomic damage. DNA damage often elicits movement of proteins from one subcellular location to another, and the redistribution of proteins involved in genomic maintenance into distinct nuclear DNA repair foci is well documented. In this review, we discuss the DNA damage-induced trafficking of proteins to and from other distinct subcellular organelles including the nucleolus, mitochondria, Golgi complex and centrosome. The extent of intracellular transport suggests a dynamic and possibly co-ordinated role for protein trafficking in the DNA damage response.     

NHERF2/NHERF3 Protein Heterodimerization and Macrocomplex Formation Are Required for the Inhibition of NHE3 Activity by Carbachol

NHERF1, NHERF2, and NHERF3 belong to the NHERF (Na⁺/H⁺ exchanger regulatory factor) family of PSD-95/Discs-large/ZO-1 (PDZ) scaffolding proteins. Individually, each NHERF protein has been shown to be involved in the regulation of multiple receptors or transporters including Na⁺/H⁺ exchanger 3 (NHE3). Although NHERF dimerizations have been reported, results have been inconsistent, and the physiological function of NHERF dimerizations is still unknown. The current study semiquantitatively compared the interaction strength among all possible homodimerizations and heterodimerizations of these three NHERF proteins by pulldown and co-immunoprecipitation assays. Both methods showed that NHERF2 and NHERF3 heterodimerize as the strongest interaction among all NHERF dimerizations. In vivo NHERF2/NHERF3 heterodimerization was confirmed by FRET and FRAP (fluorescence recovery after photobleach). NHERF2/NHERF3 heterodimerization is mediated by PDZ domains of NHERF2 and the C-terminal PDZ domain recognition motif of NHERF3. The NHERF3-4A mutant is defective in heterodimerization with NHERF2 and does not support the inhibition of NHE3 by carbachol. This suggests a role for NHERF2/NHERF3 heterodimerization in the regulation of NHE3 activity. In addition, both PDZ domains of NHERF2 could be simultaneously occupied by NHERF3 and another ligand such as NHE3, α-actinin-4, and PKCα, promoting formation of NHE3 macrocomplexes. This study suggests that NHERF2/NHERF3 heterodimerization mediates the formation of NHE3 macrocomplexes, which are required for the inhibition of NHE3 activity by carbachol.     

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.     

Impact of ASHA Training on Active Case Detection of Visceral Leishmaniasis in Bihar,India

Background

One of the major challenges for management of visceral leishmaniasis (VL) is early diagnosis of cases to improve treatment outcome and reduce transmission. We have therefore investigated active case detection of VL with the help of accredited social health activists (ASHA). ASHAs are women who live in the community and receive performance-based incentives for overseeing maternal and other health-related issues in their village.

Methods and Principal Finding

Through conducting interviews with 400 randomly selected ASHAs from four primary health care centers (PHCs), it was observed that their level of knowledge about visceral leishmaniasis (VL) regarding transmission, diagnosis, and treatment was limited. The baseline data indicated that less than 10% of VL cases seeking treatment at the PHCs were referred by ASHAs. To increase the knowledge and the referral rate of VL cases by ASHAs, training sessions were carried out during the monthly ASHA meetings at their respective PHCs. Following a single training session, the referral rate increased from less than 10% to over 27% and the overall knowledge about VL substantially improved. It was not possible, however, to demonstrate that ASHA training reduced the time that individuals had fever before treatment at the PHC.

Conclusions

Training ASHAs to identify VL cases in villages for early diagnosis and treatment at the local PHC is feasible and should be undertaken routinely to improve knowledge about VL.     

Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates

The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione. In contrast, a partially accessible site with a positively charged environment promoted hydrolysis of the succinimide ring in the linker, thereby preventing this exchange reaction. The site with partial solvent-accessibility and neutral charge displayed both properties. In a mouse mammary tumor model, the stability and therapeutic activity of the antibody conjugate were affected positively by succinimide ring hydrolysis and negatively by maleimide exchange with thiol-reactive constituents in plasma. Thus, the chemical and structural dynamics of the conjugation site can influence antibody conjugate performance by modulating the stability of the antibody-linker interface.     

Effect of (+)-usnic acid on mitochondrial functions as measured by mitochondria-specific oligonucleotide microarray in liver of B6C3F1 mice

Usnic acid is a lichen metabolite used as a weight-loss dietary supplement due to its uncoupling action on mitochondria. However, its use has been associated with severe liver disorders in some individuals. Animal studies conducted thus far evaluated the effects of usnic acid on mitochondria primarily by measuring the rate of oxygen consumption and/or ATP generation. To obtain further insight into usnic acid-mediated effects on mitochondria, we examined the expression levels of 542 genes associated with mitochondrial structure and functions in liver of B6C3F₁ female mice using a mitochondria-specific microarray. Beginning at 8 weeks of age, mice received usnic acid at 0, 60, 180, and 600 ppm in ground, irradiated 5LG6 diet for 14 days. Microarray analysis showed a significant effect of usnic acid on the expression of several genes only at the highest dose of 600 ppm. A prominent finding of the study was a significant induction of genes associated with complexes I through IV of the electron transport chain. Moreover, several genes involved in fatty acid oxidation, the Krebs cycle, apoptosis, and membrane transporters were over-expressed. Usnic acid is a lipophilic weak acid that can diffuse through mitochondrial membranes and cause a proton leak (uncoupling). The up-regulation of complexes I–IV may be a compensatory mechanism to maintain the proton gradient across the mitochondrial inner membrane. In addition, induction of fatty acid oxidation and the Krebs cycle may be an adaptive response to uncoupling of mitochondria.     

The mechanism of heme transfer from the cytoplasmic heme binding protein PhuS to the delta-regioselective heme oxygenase of Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa has evolved two outer membrane receptor-mediated uptake systems (encoded by the phu and has operons) by which it can utilize the hosts heme and hemeproteins as a source of iron. PhuS is a cytoplasmic heme binding protein encoded within the phu operon and has previously been shown to function in the trafficking of heme to the iron-regulated heme oxygenase (pa-HO). While the heme association rate for PhuS was similar to that of myoglobin, a markedly higher rate of heme dissociation (approximately 10(5) s(-1)) was observed, in keeping with a function in heme-trafficking. Additionally, the transfer of heme from PhuS to pa-HO was shown to be specific and unidirectional when compared to transfer to the non-iron regulated heme oxygenase (BphO), in which heme distribution between the two proteins merely reflects their relative intrinsic affinities for heme. Furthermore, the rate of transfer of heme from holo-PhuS to pa-HO of 0.11 +/- 0.01 s(-1) is 30-fold faster than that to apo-myoglobin, despite the significant higher binding affinity of apo-myoglobin for heme (kH = 1.3 x 10(-8) microM) than that of PhuS (0.2 microM). This data suggests that heme transfer to pa-HO is independent of heme affinity and is consistent with temperature dependence studies which indicate the reaction is driven by a negative entropic contribution, typical of an ordered transition state, and supports the notion that heme transfer from PhuS to pa-HO is mediated via a specific protein-protein interaction. In addition, pH studies, and reactions conducted in the presence of cyanide, suggest the involvement of spin transition during the heme transfer process, whereby the heme undergoes spin change from 6-c LS to 6-c HS either in PhuS or pa-HO. On the basis of the magnitudes of the activation parameters obtained in the presence of cyanide, whereby both complexes are maintained in a 6-c LS state, and the biphasic kinetics of heme transfer from holo-PhuS to pa-HO-wt, supports the notion that the spin-state crossover occur within holo-PhuS prior to the heme transfer step. Alternatively, the lack of the biphasic kinetic with pa-HO-G125V, 6-c LS, and with comparable rate of heme transfer as pa-HO is supportive of a mechanism in which the spin-change could occur within pa-HO. The present data suggests either or both of the two pathways proposed for heme transfer may occur under the present experimental conditions. The dissection of which pathway is physiologically relevant is the focus of ongoing studies.     

bisFabs: Tools for rapidly screening hybridoma IgGs for their activities as bispecific antibodies

Bispecific antibodies are a growing class of therapeutic molecules. Many of the current bispecific formats require DNA engineering to convert the parental monoclonal antibodies into the final bispecific molecules. We describe here a method to generate bispecific molecules from hybridoma IgGs in 3–4 d using chemical conjugation of antigen-binding fragments (Fabs) (bisFabs). Proteolytic digestion conditions for each IgG isotype were analyzed to optimize the yield and quality of the final conjugates. The resulting bisFabs showed no significant amounts of homodimers or aggregates. The predictive value of murine bisFabs was tested by comparing the T-cell redirected cytotoxic activity of a panel of antibodies in either the bisFab or full-length IgG formats. A variety of antigens with different structures and expression levels was used to extend the comparison to a wide range of binding geometries and antigen densities. The activity observed for different murine bisFabs correlated with those observed for the full-length IgG format across multiple different antigen targets, supporting the use of bisFabs as a screening tool. Our method may also be used for the screening of bispecific antibodies with other mechanisms of action, allowing for a more rapid selection of lead therapeutic candidates.     

Bioturbation potential of chironomid larvae for the sediment–water phosphorus exchange in simulated pond systems of varied nutrient enrichment

Chironomid larvae (2.0 individuals/cm²) were introduced in sediment–water microcosms of 3.0 l capacity to assess the impact of bioturbation on phosphorus flux across sediment–water interface, under different nutrient-enriched conditions. Recruitment of chironomid resulted in 21% and 19% increase in aquatic orthophosphate and nitrate quanta, respectively, with concomitant decrease in nutrient concentration in the sediment compared to macrofauna-free controls under mesotrophic condition. It implied that cost of fertilizer for biological production could be curtailed by at least 19–21% by recovering nutrients stored in the sediment pool. Bioturbation-induced orthophosphate flux under chironomid impacted mesotrophic treatment was 2.3- and 1.8-fold greater than that under bioturbated eutrophic treatment, suggesting that the macrofaunal impact was reduced in the presence of higher nutrient load perhaps due to physicochemical stressors under eutrophic condition. Nevertheless, chironomid larvae can further accelerate nutrient enrichment in the eutrophic system that may invite a “snow ball effect” towards a hypereutrophic one. The counts of both heterotrophic and phosphate solubilizing bacteria show strong positive correlation with orthophosphate concentration in water and the correlation also exists between organic carbon concentration in sediment and phosphate in overlying water. This implied that the accelerated phosphate flux was the result of coordinated eco-engineering activities of chironomid larvae and microbe-mediated mineralization of organic matter.