Characterization of eukaryotic-like kinase activity in Escherichia coli using the gene-protein database

Abstract The gene-protein database was used to obtain the two-dimensional polyacrylamide gel coordinates of proteins phosphorylated in extracts of Escherichia coli including those phosphorylated by eukaryotic-like kinase activities. These suggest that the phosphoproteins correspond to, or co-migrate with, the product of an open reading frame at 1.3 min (Orf80), Enzyme 1 of the phosphoenolpyruvate-dependent phosphotransferase system (Ptsl), the tRNA synthetase for histidine (HisS), and proteins involved in the response to carbon starvation and quinone treatment.     

The detection of two antigenic groups among Renibacterium salmoninarum isolates

The analysis of the membrane proteins and their antigenic properties in a group of 14 geographically diverse strains of Renibacterium salmoninarum revealed the existence of antigenic diversity within this species. Eleven isolates, including the type strain ATCC 33209, shared a similar protein profile with a major component of 57 kDa whereas three strains showed a common pattern with a major protein of 30 kDa. The quantitative agglutination tests and Western blotting assays seem to indicate the existence of serological heterogeneity, with two distinct groups being detected.     

Evidence of differential renal dysfunctions during exercise in men

Post-exercise proteinuria is a common phenomenon in healthy subjects. Previous studies have used albumin (Alb) and β₂-microglobulin (β₂-m) molecules as representatives of high- and low-molecular-weight proteins. Recently, more specific markers of the human kidney proximal tubule have been used to identify the precise site of alterations. Active male subjects underwent two strenuous runs, one 400-m run and one 3000-m run. Urine was collected from the subjects before and after each event. Total protein (TP), Alb, α₁-microglobulin (α₁-m), β₂-m, intestinal alkaline phosphatase (IAP), tissue-nonspecific alkaline phosphatase (TNAP) and N-acetyl-β-d-glucosaminidase (NAG) were determined for each sample. The short-distance run (400 m) resulted in the largest increases (P ≤ 0.05) in TP (31-fold), Alb (100-fold) and β₂-m (164-fold) as compared to the long-distance run (3000-m). The α₁-m excretion rates were increased to a lesser extent by the exercises. The IAP activity was slightly increased (+90%) by the 400-m run while the TNAP and NAG activities showed a 6.8-fold and a 3.6-fold increase, respectively, after this event. Smaller increases were recorded for the long-distance run (P = 0.05). To conclude, the present investigation showed that: (1) post-exercise proteinuria is related to the absolute intensity of exercise; (2) the impairment of protein reabsorption is revealed better by changes in Alb and β₂-m; (3) changes in TNAP and NAG activities could reveal biochemical modifications that occur in the proximal tubule, particularly at the S1-S2 segment. Accepted: 31 January 1997     

Tight junction biogenesis during early development

The tight junction (TJ) is an essential component of the differentiated epithelial cell required for polarised transport and intercellular integrity and signalling. Whilst much can be learnt about how the TJ is constructed and maintained and how it functions using a wide range of cellular systems, the mechanisms of TJ biogenesis within developmental models must be studied to gain insight into this process as an integral part of epithelial differentiation. Here, we review TJ biogenesis in the early mammalian embryo, mainly considering the mouse but also including the human and other species, and, briefly, within the amphibian embryo. We relate TJ biogenesis to inherent mechanisms of cell differentiation and biosynthesis occurring during cleavage of the egg and the formation of the first epithelium. We also evaluate a wide range of exogenous cues, including cell-cell interactions, protein kinase C signalling, gap junctional communication, Na⁺/K⁺-ATPase and cellular energy status, that may contribute to TJ biogenesis in the embryo and how these may shape the pattern of early morphogenesis.     

Advances in the development of human protein microarrays

Introduction: High-content protein microarrays in principle enable the functional interrogation of the human proteome in a broad range of applications, including biomarker discovery, profiling of immune responses, identification of enzyme substrates, and quantifying protein-small molecule, protein-protein and protein-DNA/RNA interactions. As with other microarrays, the underlying proteomic platforms are under active technological development and a range of different protein microarrays are now commercially available. However, deciphering the differences between these platforms to identify the most suitable protein microarray for the specific research question is not always straightforward.

Areas covered: This review provides an overview of the technological basis, applications and limitations of some of the most commonly used full-length, recombinant protein and protein fragment microarray platforms, including ProtoArray Human Protein Microarrays, HuProt Human Proteome Microarrays, Human Protein Atlas Protein Fragment Arrays, Nucleic Acid Programmable Arrays and Immunome Protein Arrays.

Expert commentary: The choice of appropriate protein microarray platform depends on the specific biological application in hand, with both more focused, lower density and higher density arrays having distinct advantages. Full-length protein arrays offer advantages in biomarker discovery profiling applications, although care is required in ensuring that the protein production and array fabrication methodology is compatible with the required downstream functionality.     

Efficient removal of toxic phthalate by immobilized serine-type aldehyde-tagged esterase G

Esterase G (EstG) from dibutyl phthalate (DBP)-degrading Sphingobium sp. SM42 was immobilized on amine-functionalized supports through aldehyde tag technology. Two different sulfatase motif tags, either LCTPSR (cysteine-type) or MSAPAR (serine-type), each of which is recognized by a specific formylglycine generating enzyme (FGE), were fused to the C-terminus of EstG. The cysteine-specific FGE was derived from Pseudomonas putida KT2440 while Klebsiella sp. SLS5 provided serine-specific FGE. The EstG with serine-type aldehyde tag showed a greater immobilization yield and higher specific activity by 4.8-fold and 1.8-fold, respectively. The immobilized EstG retained over 90% of its original activity after seven cycles of usage, and exhibited significantly improved thermostability by retaining 66% activity after 1 h incubation at 60 °C. Additionally, nearly 100% and over 30% of the DBP in 10 mM and 100 mM solutions, respectively, was degraded by the immobilized EstG within 18 h.     

Residue Histidine 50 Plays a Key Role in Protecting α-Synuclein from Aggregation at Physiological pH

α-Synuclein (αSyn) aggregation is involved in the pathogenesis of Parkinson disease (PD). Recently, substitution of histidine 50 in αSyn with a glutamine, H50Q, was identified as a new familial PD mutant. Here, nuclear magnetic resonance (NMR) studies revealed that the H50Q substitution causes an increase of the flexibility of the C-terminal region. This finding provides direct evidence that this PD-causing mutant can mediate long range effects on the sampling of αSyn conformations. In vitro aggregation assays showed that substitution of His-50 with Gln, Asp, or Ala promotes αSyn aggregation, whereas substitution with the positively charged Arg suppresses αSyn aggregation. Histidine carries a partial positive charge at neutral pH, and so our result suggests that positively charged His-50 plays a role in protecting αSyn from aggregation under physiological conditions.     

Insulin Receptor Substrate 2-mediated Phosphatidylinositol 3-kinase Signaling Selectively Inhibits Glycogen Synthase Kinase 3β to Regulate Aerobic Glycolysis

Insulin receptor substrate 1 (IRS-1) and IRS-2 are cytoplasmic adaptor proteins that mediate the activation of signaling pathways in response to ligand stimulation of upstream cell surface receptors. Despite sharing a high level of homology and the ability to activate PI3K, only Irs-2 positively regulates aerobic glycolysis in mammary tumor cells. To determine the contribution of Irs-2-dependent PI3K signaling to this selective regulation, we generated an Irs-2 mutant deficient in the recruitment of PI3K. We identified four tyrosine residues (Tyr-649, Tyr-671, Tyr-734, and Tyr-814) that are essential for the association of PI3K with Irs-2 and demonstrate that combined mutation of these tyrosines inhibits glucose uptake and lactate production, two measures of aerobic glycolysis. Irs-2-dependent activation of PI3K regulates the phosphorylation of specific Akt substrates, most notably glycogen synthase kinase 3β (Gsk-3β). Inhibition of Gsk-3β by Irs-2-dependent PI3K signaling promotes glucose uptake and aerobic glycolysis. The regulation of unique subsets of Akt substrates by Irs-1 and Irs-2 may explain their non-redundant roles in mammary tumor biology. Taken together, our study reveals a novel mechanism by which Irs-2 signaling preferentially regulates tumor cell metabolism and adds to our understanding of how this adaptor protein contributes to breast cancer progression.