Does trypsin cut before proline?

Trypsin is the most commonly used enzyme in mass spectrometry for protein digestion with high substrate specificity. Many peptide identification algorithms incorporate these specificity rules as filtering criteria. A generally accepted "Keil rule" is that trypsin cleaves next to arginine or lysine, but not before proline. Since this rule was derived two decades ago based on a small number of experimentally confirmed cleavages, we decided to re-examine it using 14.5 million tandem spectra (2 orders of magnitude increase in the number of observed tryptic cleavages). Our analysis revealed a surprisingly large number of cleavages before proline. We examine several hypotheses to explain these cleavages and argue that trypsin specificity rules used in peptide identification algorithms should be modified to "legitimatize" cleavages before proline. Our approach can be applied to analyze any protease, and we further argue that specificity rules for other enzymes should also be re-evaluated based on statistical evidence derived from large MS/MS data sets.     

Is proline accumulation per se correlated with stress tolerance or is proline homeostasis a more critical issue?

Proline has been recognized as a multi‐functional molecule, accumulating in high concentrations in response to a variety of abiotic stresses. It is able to protect cells from damage by acting as both an osmotic agent and a radical scavenger. Proline accumulated during a stress episode is degraded to provide a supply of energy to drive growth once the stress is relieved. Proline homeostasis is important for actively dividing cells as it helps to maintain sustainable growth under long‐term stress. It also underpins the importance of the expansion of the proline sink during the transition from vegetative to reproductive growth and the initiation of seed development. Its role in the reproductive tissue is to stabilize seed set and productivity. Thus, to cope with abiotic stress, it is important to develop strategies to increase the proline sink in the reproductive tissue. We give a holistic account of proline homeostasis, taking into account the regulation of proline synthesis, its catabolism, and intra‐ and intercellular transport, all of which are vital components of growth and development in plants challenged by stress.     

Discovery and SAR studies of methionine–proline anilides as dengue virus NS2B-NS3 protease inhibitors

A series of methionine–proline dipeptide derivatives and their analogues were designed, synthesized and assayed against the serotype 2 dengue virus NS2B-NS3 protease, and methionine–proline anilides 1 and 2 were found to be the most active DENV 2 NS2B-NS3 competitive inhibitors with K_i values of 4.9 and 10.5 μM. The structure and activity relationship and the molecular docking revealed that l-proline, l-methionine and p-nitroaniline in 1 and 2 are the important characters in blocking the active site of NS2B-NS3 protease. Our current results suggest that the title dipeptidic scaffold represents a promising structural core to discover a new class of active NS2B-NS3 competitive inhibitors.     

Efficient cryopreservation of adventitious shoots of Begonia x erythrophylla using encapsulation–dehydration requires pretreatment with both ABA and proline

Despite the widespread use of tissue culture as a means of propagating begonias and concerns regarding the preservation of germplasm, little information is available on the cryopreservation of these commercially important plants. For this reason studies were conducted to develop an encapsulation–dehydration method for the cryopreservation of adventitious shoots of the rhizomatous begonia, Begonia x erythrophylla. Adventitious shoots of B. x erythrophylla were found to be sensitive to dehydration and very sensitive to freezing. While pre-treatment with 0.75 M sucrose significantly increased the percentage of encapsulated shoots surviving dehydration, pre-treatment with sucrose did not afford cryoprotection without prior dehydration. Addition of abscisic acid and proline to the pre-treatment medium significantly improved the percentage of shoots surviving freezing. Pre-treatment of shoots with a medium containing, 0.75 M sucrose, 3.8 μM abscisic acid and 2.15 mM proline resulted in greater than 50% of shoots surviving freezing.     

Combating non-Hodgkin lymphoma by targeting both CD20 and HLA-DR through CD20–243 CrossMab

Although rituximab has revolutionized the treatment of hematological malignancies, the acquired resistance is one of the prime obstacles for cancer treatment, and development of novel CD20-targeting antibodies with potent anti-tumor activities and specificities is urgently needed. Emerging evidence has indicated that lysosomes can be considered as an “Achilles heel” for cancer cells, and might serve as an effective way to kill resistant cancer cells. HLA-DR antibody L243 has been recently reported to elicit potent lysosome-mediated cell death in lymphoma and leukemia cells, suggesting that HLA-DR could be used as a potential target against lymphoma. In this study, we generated a bispecific immunoglobulin G-like antibody targeting both CD20 and HLA-DR (CD20–243 CrossMab) through CrossMab technology. We found that the CrossMab could induce remarkably high levels of complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity and anti-proliferative activity. Notably, although HLA-DR is expressed on normal and malignant cells, the CrossMab exhibited highly anti-tumor specificity, showing efficient eradication of hematological malignancies both in vitro and in vivo. Our data indicated that combined targeting of CD20 and HLA-DR could be an effective approach against malignancies, suggesting that CD20–243 CrossMab would be a promising therapeutic agent against lymphoma.     

Combating non-Hodgkin lymphoma by targeting both CD20 and HLA-DR through CD20–243 CrossMab

Although rituximab has revolutionized the treatment of hematological malignancies, the acquired resistance is one of the prime obstacles for cancer treatment, and development of novel CD20-targeting antibodies with potent anti-tumor activities and specificities is urgently needed. Emerging evidence has indicated that lysosomes can be considered as an “Achilles heel” for cancer cells, and might serve as an effective way to kill resistant cancer cells. HLA-DR antibody L243 has been recently reported to elicit potent lysosome-mediated cell death in lymphoma and leukemia cells, suggesting that HLA-DR could be used as a potential target against lymphoma. In this study, we generated a bispecific immunoglobulin G-like antibody targeting both CD20 and HLA-DR (CD20–243 CrossMab) through CrossMab technology. We found that the CrossMab could induce remarkably high levels of complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity and anti-proliferative activity. Notably, although HLA-DR is expressed on normal and malignant cells, the CrossMab exhibited highly anti-tumor specificity, showing efficient eradication of hematological malignancies both in vitro and in vivo. Our data indicated that combined targeting of CD20 and HLA-DR could be an effective approach against malignancies, suggesting that CD20–243 CrossMab would be a promising therapeutic agent against lymphoma.     

Reciprocal regulation of Δ 1-pyrroline-5-carboxylate synthetase and proline dehydrogenase genes controls proline levels during and after osmotic stress in plants

Plants generally accumulate free proline under osmotic stress conditions. Upon removal of the osmotic stress, the proline levels return to normal. In order to understand the mechanisms involved in regulating the levels of proline, we cloned and characterized a proline dehydrogenase (PDH) cDNA from Arabidopsis thaliana (AtPDH). The 1745?bp cDNA contains a major open reading frame encoding a peptide of 499 amino acids. The deduced amino acid sequence has high homology with both Saccharomyces cerevisiae and Drosophila melanogaster proline oxidases and contains a putative mitochondrial targeting sequence. When expressed in yeast, the AtPDH cDNA complemented a yeast put1 mutation and exhibited proline oxidase activity. We also determined the free proline contents and the Δ¹-pyrroline-5-carboxylate synthetase (P5CS) and PDH mRNA levels under different osmotic stress and recovery conditions. The results demonstrated that the removal of free proline during the recovery from salinity or dehydration stress involves an induction of the PDH gene while the activity of P5CS declines. The reciprocal regulation of P5CS and PDH genes appears to be a key mechanism in the control of the levels of proline during and after osmotic stress. The PDH gene was also significantly induced by exogenously applied proline. The induction of PDH by proline, however, was inhibited by salt stress.     

Does abscisic acid influence proline accumulation in stressed leaves?

Root treatments of barley (Hordeum distichum L.) plants with 10^-7 to 10^-4 M abscisic acid (ABA) caused an increase in proline content, especially at higher concentrations, within 2–3 h. Even 3 h after the removal of ABA from the medium the plants continued to accumulate proline. The higher the concentration of the ABA, the higher was the proline level at 6 h. When the highest ABA concentration, 10^-4 M, was tested with polyethylene glycol (PEG) (-5.0 bars) in the medium, the ABA treatment resulted in a higher proline content than in control plants. The treatments PEG alone and PEG + ABA resulted in heavy accumulation of proline, especially, 3 h after releasing the plants from the stress. The proline content in PEG+ABA-treated plants was always higher than plants treated with PGE or ABA alone. In peas (Pisum sativum L. cv Alaska) the same trend occurred although to a lesser degree. These findings indicate an influence of ABA on proline accumulation in water-stressed plants.Abbreviations ABA abscisic acid - PEG polyethylene glycol - RWC relative water content     

Bioactivity of nitrolinoleate: effects on adhesion molecules and CD40–CD40L system

The vascular effects of nitrolinoleate (LNO₂), an endogenous product of linoleic acid (LA) nitration by nitric oxide-derived species and a potential nitrosating agent, were investigated on rat endothelial-leukocyte interactions. Confocal microscopy analysis demonstrated that LNO₂ was capable to deliver free radical nitric oxide (^·NO) into cells, 5 min after its administration to cultured cells, with a peak of liberation at 30 min. THP-1 monocytes incubated with LNO₂ for 5 min presented nitrosation of CD40, leading to its inactivation. Other anti-inflammatory actions of LNO₂ were observed in vivo by intravital microscopy assays. LNO₂ decreased the number of adhered leukocytes in postcapillary venules of the mesentery network. In addition to this, LNO₂ reduced mRNA and protein expression of β2-integrin in circulating leukocytes, as well as VCAM-1 in endothelial cells isolated from postcapillary venules, confirming its antiadhesive effects on both cell types. Moreover, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a nitric oxide scavenger, partially abolished the inhibitory action of LNO₂ on leukocyte-endothelium interaction, suggesting that the antiadhesion effects of LNO₂ involve a dual role in leukocyte adhesion, acting as a nitric oxide donor as well as through nitric oxide-independent mechanisms. In conclusion, LNO₂ inhibited adhesion molecules expression and promoted ^·NO inactivation of the CD40–CD40L system, both important processes of the inflammatory response.     

The role of proline residues in the folding kinetics of the bovine pancreatic trypsin inhibitor derivative RCAM(14–38)

The role of proline residues in the folding of the trypsin inhibitor derivative RCAM(14–38) has been studied by testing for slow-folding species of the unfolded protein, which could result from the introduction of wrong proline isomers after unfolding. The unfolded protein at 25 °C contains chiefly fast-folding (U_F) molecules: they refold with a time constant of 40 milliseconds at pH 6.8 in 1.9 m-guanidinium chloride. At least one minor slow-folding (U_s) species has been found, using fluorescence to monitor refolding. The reaction in which this U_s species is formed after unfolding shows the properties expected for the cis: Irans isomerization of a proline residue. When refolding is monitored by tyrosine absorbance, two minor slow reactions are found. The faster reaction is in the same time range (15 s at 25 °C) as that studied by fluorescence, and the slower reaction is quite slow (200 s at 25 °C). It is not known whether the slower reaction results from a second U_s species. There are four trans proline residues in bovine pancreatic trypsin inhibitor: the proportion of slow-folding molecules (not more than 25% at 25 °C) is smaller than expected if every proline residue can produce a U_s species and if the cis to trans ratio of each residue after unfolding is at least 0.1:0.9.Criteria based on folding kinetics are given for classifying the types of folding reaction shown by unfolded molecules containing a single wrong proline isomer. Levitt (1980) has classified three types of proline residues according to the energy difference (small, intermediate or large) between the native protein and the predicted minimum energy structure containing a wrong proline isomer. He suggests that these three types of proline residues can be recognized by the types of folding reactions they produce. Only type II (intermediate) folding reactions have thus far been characterized by the criteria introduced here. We point out that the type of folding reaction depends also on the folding conditions, and a possible explanation for this effect is given.     

The effect of CD137–CD137 ligand interaction on phospholipase C signaling pathway in human endothelial cells

We previously reported the emerging role of CD137–CD137L interaction in inflammation and atherosclerosis. The mechanism of CD137–CD137L interaction may be related to a variety of signaling pathways. The most important signaling pathway involves the activation of phospholipase C(PLC) which induces the diacylglycerol–protein kinase C(DAG–PKC) and the inositol trisphosphate-intracellular free calcium (IP₃-[Ca²⁺]i) pathway. In the current study, we investigated whether CD137–CD137L interaction can stimulate the PLC signaling pathway in human umbilical vein endothelial cells (HUVEC). The diacylglycerol (DAG) and inositol trisphosphate (IP₃) levels in HUVEC were measured by radioenzymatic assay. The activity of protein kinase (PKC) was detected by its ability to transfer phosphate from [γ-³²P]ATP to lysine-rich histone. The [Ca²⁺]i concentrations were measured by flow cytometric analysis. The DAG level and PKC activity were increased in a concentration-dependent, biphasic manner in HUVEC induced by anti-CD137. PKC activity was mainly in the cytosol at rest, and then translocated to the membrane when stimulated by anti-CD137. Similarly, rapid IP₃ formation induced by anti-CD137 coincided with the peak of the DAG level. Moreover, anti-CD137 induced peak [Ca²⁺]i responses including the rapid transient phase and the sustained phase. However, anti-CD137L suppressed the activation of the DAG–PKC and IP₃-[Ca²⁺]i signaling pathway, which was stimulated by anti-CD137 in HUVEC. In conclusion, the data suggested that CD137–CD137L interaction induces robust activation of the PLC signaling pathway in HUVEC.     

CD4 and CD8 T cell responses to tumour-associated Epstein–Barr virus antigens in nasopharyngeal carcinoma patients

Nasopharyngeal carcinoma (NPC), an Epstein–Barr virus (EBV)-associated tumour common in Southern Chinese populations, is a potentially important target for T cell-based immunotherapy. The tumour cells are HLA class I- and II-positive and express a limited subset of EBV latent proteins, namely the nuclear antigen EBNA1 and the latent membrane proteins LMP2 and (in some cases) LMP1. To ask whether the tumour develops in the presence of a potentially protective host response or in its absence, we set out to determine the prevailing levels of CD4+ and CD8+ T cell memory to these proteins in NPC patients at tumour diagnosis. We first screened healthy Chinese donors against Chinese strain EBNA1, LMP1 and LMP2 sequences in Elispot assays of interferon-γ release and identified the immunodominant CD4+ and CD8+ epitope peptides presented by common Chinese HLA alleles. Then, comparing 60 patients with >70 healthy controls on peptide epitope mini-panels, we found that T cell memory to CD4 epitopes in all three proteins was unimpaired in the blood of patients at diagnosis. In most cases NPC patients also showed detectable responses to CD8 epitopes relevant to their HLA type, the one consistent exception being the absence in patients of a B*4001-restricted response to LMP2. We infer that NPC arises in patients whose prevailing levels of T cell memory to tumour-associated EBV proteins is largely intact; the therapeutic goal must therefore be to re-direct the existing memory repertoire more effectively against antigen-expressing tumour cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Coordination abilities of tetrapeptides containing proline and tyrosine—A spectrophotometric and potentiometric study

The synthesis of three tetrapeptides, Gly-Pro-Gly-Tyr. Gly-Pro-Tyr-Gly. and Tyr-Pro-Gly-Gly, are described. All contain proline as the second amino acid subunit to act as a break point in metal complex formation.The proton and copper(II) complex formation constants have been measured at 22°C and l = 0.10 mol dm^?3 (KNO₃). The copper(II) complexes have also been studied spectrophotometrically over the pH range of 6–11 by absorption spectroscopy (800–200 nm), circular dichroism spectroscopy, and electron paramagnetic resonance spectroscopy. The experimental data have been combined to determine the complex species present as a function of pH and the coordination centers used.     

Selective depletion of FOXP3high cells by Fas–Fas-L–induced apoptosis occurs in CD4+CD25+-enriched populations during repeated expansion

Background aimsExpansion of anti-CD25 bead-isolated human Tregs culture has paradoxically resulted in reduced suppressive activity, but the mechanism(s) responsible for these observations are poorly defined.MethodsMagnetic-bead isolated human CD25⁺ cells were expanded with anti-CD3/CD28 beads and high doses of rhIL-2. Detection of Fas and Fas ligand (Fas-L) expression, activation of Caspase 8, cell proliferation and cytokine production was evaluated by multi-color fluorescence-activated cell sorting analysis. The role of Fas–Fas-L–mediated cell death was dissected through the use of agonist or antagonist monoclonal antibodies directed at Fas and Fas-L.ResultsRepeated expansion of bead-enriched CD4⁺CD25⁺ cells generated a cellular product with markedly reduced suppressive activity and with significantly increased CD8⁺ T cells and CD4⁺ T cells producing interferon-γ and/or interleukin-2. We showed that Fas–Fas-L–mediated apoptosis of CD4⁺FOXP3^high cells and rapid cell-cycling of CD8⁺ T cells were collectively responsible for the reduced proportion of CD4⁺FOXP3^high cells in expanded cultures. The depletion of CD4⁺FOXP3^high cells and activation of Caspase 8 in CD4⁺FOXP3^high cells was attenuated by Fas antagonist antibody, ZB4, in short-term culture. However, the loss of CD4⁺FOXP3^high cells during expansion was not prevented by either Fas or Fas-L antagonist antibodies.ConclusionsTaken together, the data show that Fas–Fas-L–mediated apoptosis may limit the expansion of anti-CD25 bead-isolated cells in vitro.     

La teneur en proline et la résistance des plantes aux sels

Application of sodium chloride to crop plants in the culture medium leads to an increase in their endogenous content of free proline. There is a certain concentration of sodium chloride above which the proline content of the plants strongly rises (critical point). A relationship between endogenous sodium and proline contents could be found. In salt-sensitive plants (wheat) the critical point lies below that of salt-tolerant plants (barley). The critical concentration is not changed by certain alterations of the culture medium. The determination of the critical point by means of measurement of the proline concentration served as a basis for the analysis of the salt tolerance of crop plants.      

Effects of ring contraction on the conformational preferences of α-substituted proline analogs

The structural consequences derived from the incorporation of either a methyl or a phenyl group at the α carbon of proline were recently investigated by quantum mechanical calculations (J Org Chem 2008, 73, 3418). In this work, the effect produced by contraction of the pyrrolidine ring on such α-substituted proline analogs has been explored using the same computational methods. Specifically, the intrinsic conformational preferences of the N-acetyl-N'-methylamide derivatives of the lower proline homolog L-azetidine-2-carboxylic acid (Aze), characterized by a four- instead of a five-membered ring, and its α-methyl (αMeAze) and α-phenyl (αPhAze) derivatives have been determined using quantum mechanical calculations and compared to those observed before for the proline counterparts. Replacement of the pyrrolidine ring by an azetidine cycle leads to a reduction of the conformational flexibility, especially for the Aze and αMeAze derivatives, which should be attributed to the quasi-planar geometry of the four-membered ring. Furthermore, the azetidine nitrogen shows pyramidalization, which depending on the peptide backbone conformation favors the formation of an attractive N-H···N interaction or alleviates a severe steric hindrance. Calculations on different environments predict that the tendency of αMeAze to adopt γ-turns is higher than that of unsubstituted Aze and α-methylproline, this feature being in full agreement with the experimental observations available.     

Hyoscyamine and proline accumulation in water-stressed Hyoscyamus muticus ‘hairy root’ cultures

Summary The patterns of hyoscyamine and proline accumulation were studied in Agrobacterium-transformed ‘hairy root’ cultures of Hyoscyamus muticus to determine if proline is a metabolic precursor of hyoscyamine. Root cultures were stressed osmotically with mannitol and the subsequent growth, hyoscyamine levels, and proline levels were measured after each transfer to fresh experimental medium for a total of four transfers. H. muticus ‘hairy roots’ were also treated with [U-¹⁴C] proline or [1,4-¹⁴C] putrescine and analyzed for radioactive hyoscyamine. Growth of ‘hairy root’ cultures was reduced by up to 90% in 0.4 M mannitol, and this inhibition persisted for at least four transfers. ‘Hairy root’ cultures of H. muticus accumulated hyoscyamine and free proline (up to 6-fold and 25-fold, respectively) when osmotically stressed with mannitol, and this effect also persisted for four transfers when grown in the same mannitol concentration. Because the total production of hyoscyamine was also increased by twofold, we conclude that the elevated hyoscyamine concentration results from increased hyoscyamine synthesis and not from reduced growth. H. muticus ‘hairy roots’ incorporated radioactivity from [1,4-¹⁴C] putrescine efficiently into hyoscyamine in both treatments, but failed to convert [U-¹⁴C] proline into hyoscyamine. We thus conclude that accumulated proline does not serve as a precursor for hyoscyamine.     

Alzheimer's disease‐causing proline substitutions lead to presenilin 1 aggregation and malfunction

Do different neurodegenerative maladies emanate from the failure of a mutual protein folding mechanism? We have addressed this question by comparing mutational patterns that are linked to the manifestation of distinct neurodegenerative disorders and identified similar neurodegeneration‐linked proline substitutions in the prion protein and in presenilin 1 that underlie the development of a prion disorder and of familial Alzheimer's disease (fAD), respectively. These substitutions were found to prevent the endoplasmic reticulum (ER)‐resident chaperone, cyclophilin B, from assisting presenilin 1 to fold properly, leading to its aggregation, deposition in the ER, reduction of γ‐secretase activity, and impaired mitochondrial distribution and function. Similarly, reduced quantities of the processed, active presenilin 1 were observed in brains of cyclophilin B knockout mice. These discoveries imply that reduced cyclophilin activity contributes to the development of distinct neurodegenerative disorders, propose a novel mechanism for the development of certain fAD cases, and support the emerging theme that this disorder can stem from aberrant presenilin 1 function. This study also points at ER chaperones as targets for the development of counter‐neurodegeneration therapies.