Molecular chaperone function of stress inducible Hsp70 is critical for intracellular multiplication of Toxoplasma gondii

Intracellular pathogens like Toxoplasma gondii often target proteins and pathways critical for host cell survival and stress response. Molecular chaperones encoded by the evolutionary conserved Heat shock proteins (Hsps) maintain proteostasis and are vital to cell survival following exposure to any form of stress. A key protein of this family is Hsp70, an ATP-driven molecular chaperone, which is stress inducible and often indiscernible in normal cells. Role of this protein with respect to intracellular survival and multiplication of protozoan parasite like T. gondii remains to be examined. We find that T. gondii infection upregulates expression of host Hsp70. Hsp70 selective inhibitor 2-phenylethynesulfonamide (PES) attenuates intracellular T. gondii multiplication. Biotinylated PES confirms selective interaction of this small molecule inhibitor with Hsp70. We show that PES acts by disrupting Hsp70 chaperone function which leads to dysregulation of host autophagy. Silencing of host Hsp70 underscores its importance for intracellular multiplication of T. gondii, however, attenuation achieved using PES is not completely attributable to host Hsp70 indicating the presence of other intracellular targets of PES in infected host cells. We find that PES is also able to target T. gondii Hsp70 homologue which was shown using PES binding assay. Detailed molecular docking analysis substantiates PES targeting of TgHsp70 in addition to host Hsp70. While establishing the importance of protein quality control in infection, this study brings to the fore a unique opportunity of dual targeting of host and parasite Hsp70 demonstrating how structural conservation of these proteins may be exploited for therapeutic design.     

Comparative phylogenetic analysis of genome-wide Mlo gene family members from Glycine max and Arabidopsis thaliana

Powdery mildew locus O (Mlo) gene family is one of the largest seven transmembrane protein-encoding gene families. The Mlo proteins act as negative regulators of powdery mildew resistance and a loss-of-function mutation in Mlo is known to confer broad-spectrum resistance to powdery mildew. In addition, the Mlo gene family members are known to participate in various developmental and biotic and abiotic stress response-related pathways. Therefore, a genome-wide similarity search using the characterized Mlo protein sequences of Arabidopsis thaliana was carried out to identify putative Mlo genes in soybean (Glycine max) genome. This search identified 39 Mlo domain containing protein-encoding genes that were distributed on 15 of the 20 G. max chromosomes. The putative promoter regions of these Mlo genes contained response elements for different external stimuli, including different hormones and abiotic stresses. Of the 39 GmMlo proteins, 35 were rich (8.7–13.1 %) in leucine, while five were serine-rich (9.2–11.9 %). Furthermore, all the GmMlo members were localized in the plasma membrane. Phylogenetic analysis of the GmMlo and the AtMlo proteins classified them into three main clusters, and the cluster I comprised two sub-clusters. Multiple sequence alignment visualized the location of seven transmembrane domains, and a conserved CaM-binding domain. Some of the GmMlo proteins (GmMlo10, 20, 22, 23, 32, 36, 37) contained less than seven transmembrane domains. The motif analysis yielded 27 motifs; out of these, motif 2, the only motif present in all the GmMlos, was highly conserved and three amino acid residues were essentially invariant. Five of the GmMlo members were much smaller in size; presumably they originated through deletion following a gene duplication event. The presence of a large number of GmMlo members in the G. max genome may be due to its paleopolyploid nature and the large genome size as compared to that of Arabidopsis. The findings of this study may further help in characterization and isolation of individual GmMlo members.     

Antibody VH and VL recombination using phage and ribosome display technologies reveals distinct structural routes to affinity improvements with VH-VL interface residues providing important structural diversity

In vitro selection technologies are an important means of affinity maturing antibodies to generate the optimal therapeutic profile for a particular disease target. Here, we describe the isolation of a parent antibody, KENB061 using phage display and solution phase selections with soluble biotinylated human IL-1R1. KENB061 was affinity matured using phage display and targeted mutagenesis of VH and VL CDR3 using NNS randomization. Affinity matured VHCDR3 and VLCDR3 library blocks were recombined and selected using phage and ribosome display protocol. A direct comparison of the phage and ribosome display antibodies generated was made to determine their functional characteristics.     

Guanidino compound metabolism in arginine-free diet induced hyperammonemia.

Guanidino compounds, intermediates of arginine metabolism, are altered in many pathological conditions especially those involving the urea cycle. Arginine and creatine play an important role in nitrogen metabolism whereas other guanidino compounds such as guanidinosuccinic acid and N-acetylarginine are toxins. Our objective was to investigate the relationship between guanidino compounds and hyperammonemia. Young and adult ferrets were fed a single meal of either an arginine-containing diet (ACD) or an arginine-free diet (AFD). Guanidino compounds were determined by HPLC in the plasma, liver, kidney and brain 3 h after feeding the specified diet. Only young ferrets fed AFD developed hyperammonemia. Plasma and kidney arginine was decreased whereas guanidinosuccinic acid was increased in young ferrets fed AFD. Hepatic creatine and kidney and brain guanidinoacetic acid were significantly decreased in this group. These results indicate that AFD-induced hyperammonemia produced decreased methylation activity in the liver and transamidination activity in kidney. Elevated guanidinosuccinate levels coupled with deficient hepatic creatine synthesis may play a role in the pathophysiology of hyperammonemia.     

Purification and properties of alpha-aminoadipate aminotransferase from rat kidney.

Previous studies with rat kidney preparations indicated that alpha-aminoadipate aminotransferase (AadAT) and kynurenine aminotransferase (KAT) activities are associated with a single protein. However, recent studies from our laboratory demonstrated that AadAT and KAT activities belong to two different proteins. AadAT from rat kidney supernatant fraction was purified by affinity chromatography to electrophoretic homogeneity. This rapid and efficient procedure improved the yield and the degree of purification over previously published methods and separated AadAT from KAT. The molecular weight of the enzyme was estimated to be 89,000 by Sephadex G-200 gel filtration chromatography. SDS-PAGE indicated that the enzyme is composed of two apparently identical subunits. Absorption spectra and the kinetic properties of AadAT are reported.     

Scale-down of penicillin production in Penicillium chrysogenum

In large-scale production reactors the combination of high broth viscosity and large broth volume leads to insufficient liquid-phase mixing, resulting in gradients in, for example, the concentrations of substrate and oxygen. This often leads to differences in productivity of the full-scale process compared with laboratory scale. In this scale-down study of penicillin production, the influence of substrate gradients on process performance and cell physiology was investigated by imposing an intermittent feeding regime on a laboratory-scale culture of a high yielding strain of Penicillium chrysogenum. It was found that penicillin production was reduced by a factor of two in the intermittently fed cultures relative to constant feed cultivations fed with the same amount of glucose per hour, while the biomass yield was the same. Measurement of the levels of the intermediates of the penicillin biosynthesis pathway, along with the enzyme levels, suggested that the reduction of the flux through the penicillin pathway is mainly the result of a lower influx into the pathway, possibly due to inhibitory levels of adenosine monophosphate and pyrophosphate and lower activating levels of adenosine triphosphate during the zero-substrate phase of each cycle of intermittent feeding.     

Formation of Free Fatty Acid and Ceramide During Brain Handling: Lability of Sphingomyelin

Intact brain and brain homogenates readily form free fatty acids and ceramides, even in the cold during subcellular isolation procedures. The fatty acid formation is slightly stimulated by chelators and might be due to phospholipid hydrolysis by lysosomal phospholipases. The ceramide formation is accompanied by loss of sphingomyelin and is apparently due to the action of neutral, metal ion-activated sphingomyelinase. The latter reaction is inhibited by EDTA whereas both degradative processes are inhibited by mercuriphenylsulfonate, the thiol-reacting inhibitor. Cerebroside does not seem to be a source of accumulated ceramide.     

Homologues of neisserial heme oxygenase in gram-negative bacteria: degradation of heme by the product of the pigA gene of Pseudomonas aeruginosa

The oxidative cleavage of heme to release iron is a mechanism by which some bacterial pathogens can utilize heme as an iron source. The pigA gene of Pseudomonas aeruginosa is shown to encode a heme oxygenase protein, which was identified in the genome sequence by its significant homology (37%) with HemO of Neisseria meningitidis. When the gene encoding the neisserial heme oxygenase, hemO, was replaced with pigA, we demonstrated that pigA could functionally replace hemO and allow for heme utilization by neisseriae. Furthermore, when pigA was disrupted by cassette mutagenesis in P. aeruginosa, heme utilization was defective in iron-poor media supplemented with heme. This defect could be restored both by the addition of exogenous FeSO4, indicating that the mutant did not have a defect in iron metabolism, and by in trans complementation with pigA from a plasmid with an inducible promoter. The PigA protein was purified by ion-exchange chromotography. The UV-visible spectrum of PigA reconstituted with heme showed characteristics previously reported for other bacterial and mammalian heme oxygenases. The heme-PigA complex could be converted to ferric biliverdin in the presence of ascorbate, demonstrating the need for an exogenous reductant. Acidification and high-performance liquid chromatography analysis of the ascorbate reduction products identified a major product of biliverdin IX-beta. This differs from the previously characterized heme oxygenases in which biliverdin IX-alpha is the typical product. We conclude that PigA is a heme oxygenase and may represent a class of these enzymes with novel regiospecificity.