Protein and Peptide Letters - Volume 23, Issue 11, 2016

Recent studies document the importance of endothelial dysfunction (ED) in cancer development and metastasis. We previously reported that vascular oxidative stress and inflammation result in ED in animals bearing brain murine metastatic breast carcinoma (BCa). Substance P (SP), a neuropeptide found in sensory nerve terminals, was reported to enhance anti-tumoral effects of conventional treatments. SP was also reported to have anti-oxidative effects. We therefore examined the effects of continuous exposure to low dose SP on vascular ED observed in metastatic BCa. In this study, cells derived from brain metastasis of 4T1 murine BCa (denoted as 4TBM) were used. Female Balb-c mice 8-10 weeks old were divided into following groups: (1) Control (Hanks' balanced salt solution injected), (2) injected with 4TBM orthotopically, (3) injected with 4TBM orthotopically and then treated with SP via an osmotic mini-pumps (0.1 mM, pumping rate 0.11 μl/hr). Thoracic aorta was removed 20-26 days after injection of tumor cells. Isometric tension studies were performed in response to potassium chloride, phenylephrine, acetylcholine (an endothelium-dependent vasodilator), and sodium nitroprusside (an endothelium-independent vasodilator). On one hand, presence of tumor resulted in significant inhibition of vascular response to ACh in untreated mice. On the other, in vivo SP treatment restored the diminished relaxation response to ACh in thoracic aorta rings obtained from metastatic BCa bearing mice. These findings suggest that SP can inhibit tumor-induced ED which might be partly responsible from reported anti-tumoral effects of SP. Furthermore, protective effects of SP on vascular endothelium may prevent cardiovascular diseases in cancer patients.

Splicing factor, proline- and glutamine-rich protein (SFPQ), was identified in eight human cultivated cell lines by proteomic approaches. The cell proteins have been separated by means of two-dimensional gel electrophoresis in two modifications and identified by matrix-assisted laser desorption ionization mass spectrometry with further tandem mass spectrometry. The analysis of proteins from three human sarcomas cell lines (RD, U-2 OS and SK-UT-1B), three human renal adenocarcinomas cell lines (A-498, 769-P and OKP-GS), and two prostate adenocarcinomas cell lines (DU-145 and PC-3) revealed several electrophoretic isoforms of SFPQ protein. Differences between theoretical and experimental molecular masses and isoelectric points of SFPQ protein have been observed. Detailed investigation of SFPQ peptides by tandem mass spectrometry has detected new phosphorylation state of threonine residue in 168 position of SFPQ isoform in rhabdomyosarcoma cell line. Furthermore, SFPQ has not been identified during proteomic study of several nonmalignant cell lines, including cultured human mesenchymal stromal cells and myoblasts. However, SFPQ has been found in all malignant cell lines in high quantity. In particular, its fractions are abundant in sarcomas cell lines as opposed to nonmalignant mesenchymal cells. It is assumed that high quantity of SFPQ in sarcomas cell lines may affect tumorigenesis.

In NMR experiments, the chemical shift is typically the first parameter measured and is a source of structural information for biomolecules. Indeed, secondary chemical shifts, the difference between the measured chemical shifts and those expected for a randomly oriented sequence of peptides (the "random coil"), are correlated with the secondary structure of proteins; secondary shift analysis is thereby a standard approach in structural biology. For intrinsically disordered or denatured proteins furthermore, secondary chemical shifts reveal the propensity of particular segments to form different secondary structures. However, because the atoms in unfolded proteins all have very similar chemical environments, the chemical shifts measured for a certain atom type vary less than in globular proteins. Since chemical shifts can be measured precisely, the secondary chemical shifts calculated for an unfolded system depend mainly on the particular random coil chemical shift database chosen as a point of reference. Certain databases correct the random coil shift for a given residue based on its neighbors in the amino acid sequence. However, these corrections are typically derived from the analysis of model peptides; there have been relatively few direct and systematic studies of the effect of neighboring residues for specific amino acid sequences in disordered proteins. For the study reported here, we used the intrinsically disordered C-terminal domain of TDP-43, which has a highly repetitive amino-acid sequence, as a model system. We assigned the chemical shifts of this protein at low pH in urea. Our results demonstrate that the identity of the nearest neighbors is decisive in determining the value of the chemical shift for atoms in a random coil arrangement. Based on these observations, we also outline a possible approach to construct a random-coil library of chemical shifts that comprises all possible arrangement of tripeptides from a manageable number of polypeptides.

It has been reported that pulse proteolysis may be used to investigate protein unfolding kinetics in cell lysate. However, the method has not become popular and we could not judge whether or not it is effective for protein folding study. In this work, we examined the folding and unfolding kinetics of a protein and its variants without purification by pulse proteolysis. The unfolding and refolding rates of the unpurified proteins were similar to those of the purified proteins determined by pulse proteolysis and circular dichroism. Furthermore, because we used a super-stable subtilisin as a protease, we could evaluate the kinetics at 50°C. The present work demonstrates the validity of pulse proteolysis for folding and unfolding studies of unpurified proteins.

Recent studies have indicated that PrP23-98, an N-terminal portion of PrP, polymerizes into amyloid-like and proteinase K (PK)-resistant aggregates in the presence of NADPH with copper ions [19], and then that CRT suppressed aggregation of PrP23-98 and also promoted solubilization of the aggregates [18]. As it is interesting to find out whether other chaperones can inhibit aggregation of PrP23-98 in vitro similar to CRT, this study was conducted to determine whether BiP, Grp94, PDI Grp58 and heat shock cognate protein70 (Hsc70) can inhibit aggregation of PrP23-98 in vitro. The present results indicated that Grp94 suppressed aggregation of PrP23-98, but that Grp94 could not mediate solubilization occurred in the aggregates in contrast to CRT. Other chaperons induced aggregation of PrP23-98 in the absence of NADPH.

Plant defensins are plant defence peptides that have many different biological activities, including antifungal, antimicrobial, and insecticidal activities. A cDNA (EgDFS) encoding defensin was isolated from Elaeis guineensis. The open reading frame of EgDFS contained 231 nucleotides encoding a 71-amino acid protein with a predicted molecular weight at 8.69 kDa, and a potential signal peptide. The eight highly conserved cysteine sites in plant defensins were also conserved in EgDFS. The EgDFS sequence lacking 30 amino acid residues at its N-terminus (EgDFSm) was cloned into Escherichia coli BL21 (DE3) pLysS and successfully expressed as a soluble recombinant protein. The recombinant EgDFSm was found to be a thermal stable peptide which demonstrated inhibitory activity against the growth of G. boninense possibly by inhibiting starch assimilation. The role of EgDFSm in oil palm defence system against the infection of pathogen G. boninense was discussed.

The potent multifunctionality of human galectins is based on their modular structure in a not yet fully understood manner. A strategy to dissect the contributions of individual sequence stretches to lectin activity is based on engineering variants of the natural proteins, which are composed of novel combinations of distinct parts. On proof-of-principle level, we here describe the design of a hybrid constituted by the N-terminal tail of chimera-type galectin-3 and the Nterminal carbohydrate recognition domain of tandem-repeat-type galectin-8, its production, purification and its serine phosphorylation characteristic for galectin- 3’s tail. As measured for the respective parental proteins, its binding to (neo)glycoproteins is specific for β-galactosides and inhibitable by lactose, with KD-value closer to galectin-8 than galectin-3. Cell surface staining indicated similarity of the hybrid’s reactivity to O-glycans and sensitivity for sialylation to respective properties of tandem-repeattype galectin-8 and its N-terminal domain. Applied as histochemical tool on tissue sections of murine jejunum and epididymis, intense lactose-inhibitable signals were recorded intracellularly, with a distribution profile akin to that of galectin-3. Tested as agglutinin, the hybrid was potent, excelling wild-type control galectins. The chimera-type design can thus serve as platform for tuning crosslinking activity.

Prostaglandin H synthase (PGHS) catalyzes the biosynthesis of PGG2 and PGH2, the precursor of all prostanoids, from arachidonic acid (AA). PGHS exhibits two enzymatic activities following a branched-chain radical mechanism: 1) a peroxidase activity (POX) that utilizes hydroperoxide through heme redox cycles to generate the critical Tyr385 tyrosyl radical for coupling both enzyme activities; 2) the cyclooxygenase (COX) activity inserting two oxygen molecules into AA to generate endoperoxide/hydroperoxide PGG2 through a series of radical intermediates. Upon the generation of Tyr385 radical, COX catalysis is initiated, with C13 pro-S hydrogen abstraction from AA by Tyr385 radical to generate arachidonyl substrate radical. Oxygen provides a large driving force for the subsequent fast steps leading to the formation of PGG2, including radical redistributions, ring formations, and rearrangements. On the other hand, if the supply of oxygen is severed, equilibrium between arachidonyl radical and tyrosyl radical(s) biases largely towards the latter. In this study, we demonstrate that such equilibrium is shifted by many factors, including temperature, chemical structures of fatty acid substrates and limited supply of oxygen. We also, for the first time, reveal that this equilibrium is significantly affected by co-substrates of POX. The presence of efficient POX co-substrates, which reduces heme to its ferric state, apparently biases the equilibrium towards arachidonyl radical. Therefore a dynamic interplay exists between the two activities of PGHS.

Apolipoprotein (apo)A-I and apoE are the two protein components that have been extensively investigated for their anti-atherogenic properties. Both apolipoproteins possess amphipathic helical structures, responsible for the solubilization of lipids. While apoA-I possesses class A amphipathic helical structures, apoE possesses a 59 residue long amphipathic helical domain linked to a four helix bundle containing the Arg-rich, 10 residue receptor binding domain. An 18 residue model peptide (18A) was designed to mimic the amphipathic helical domains of apoA-I. This and several analogs were able solubilize phospholipids and, when administered into animal models of atherosclerosis, were able to inhibit lesion formation without any effect on plasma cholesterol levels. These analogs were subsequently termed as apoA-I mimetic peptides. When this peptide (18A) was covalently linked to the Arg-rich receptor binding domain of apoE, the resulting peptide Ac-hE18A-NH2, in which hE refers to the 141-150 Arg-rich region of apoE, dramatically reduced plasma cholesterol in several dyslipidemic animal models, resulting in the reduction of lesion formation. This and several other analogs which were able to dramatically decrease plasma cholesterol, analogous to apoE, were termed as apoE mimetic peptides. These observations developed the field of apolipoprotein mimetic peptides which are involved in interacting with lipoproteins and modulating their function. The present review describes progress made in this field which have culminated in clinical trials in humans for both the apoA-I and apoE mimetic peptides.

Many polyketides show biological activities and have thus been applied in clinics, as food additives, and in agriculture. Type II thioesterases (TEIIs) play an important role in polyketide biosynthesis. Most TEIIs belong to α/β-hydrolase family and usually contain a catalytic triad Ser-His-Asp. In polyketide biosynthesis, TEIIs can play an editing role by removal of aberrant non-extendable acyl units in elongation steps, a starter unit selection role by removal of unfavored starter acyl units in initiation steps, and a releasing role by removal of final product in termination steps. Complementation of TEIIs has been observed and applied.

As a graduate course student, I studied the aggregation behavior of the wheat protein gluten, induced by changes in pH and ionic strength, thus beginning my research career in the field of solvent effects. The following 5 years were spent as a post-doctoral reseracher in the U.S., focused on protein-solvent interactions, which, according to my advisor, Dr. Timasheff, was too basic to be supported by academic grants. This study, however, answered a number of questions about how different solvents affect behavior of proteins in solution, e.g., a solubility behavior known as Hofmeister series. This research experience landed me an R position at a startup biotech company, Amgen, where knowledge on solvents played a fundamental role in protein refolding, formulation and chromatography. This demonstrates the unanticipated possibility that an un-funded, basic academic science study earlier can be transitioned later into an industrially-significant applied science.