Multifaceted behavior of PEST sequence enriched nuclear proteins in cancer biology and role in gene therapy.

The amino acid sequence enriched with proline (P), glutamic acid (E), serine (S), and threonine (T) (PEST) is a signal-transducing agent providing unique features to its substrate nuclear proteins (PEST-NPs). The PEST motif is responsible for particular posttranslational modifications (PTMs). These PTMs impart distinct properties to PEST-NPs that are responsible for their activation/inhibition, intracellular localization, and stability/degradation. PEST-NPs participate in cancer metabolism, immunity, and protein transcription as oncogenes or as tumor suppressors. Gene-based therapeutics are getting the attention of researchers because of their cell specificity. PEST-NPs are good targets to explore as cancer therapeutics. Insights into PTMs of PEST-NPs demonstrate that these proteins not only interact with each other but also recruit other proteins to/from their active site to promote/inhibit tumors. Thus, the role of PEST-NPs in cancer biology is multivariate. It is hard to obtain therapeutic objectives with single gene therapy. An especially designed combination gene therapy might be a promising strategy in cancer treatment. This review highlights the multifaceted behavior of PEST-NPs in cancer biology. We have summarized a number of studies to address the influence of structure and PEST-mediated PTMs on activation, localization, stability, and protein-protein interactions of PEST-NPs. We also recommend researchers to adopt a pragmatic approach in gene-based cancer therapy.

The Pneumococcal Surface Proteins PspA and PspC Sequester Host C4-Binding Protein To Inactivate Complement C4b on the Bacterial Surface.

Complement is a critical component of antimicrobial immunity. Various complement regulatory proteins prevent host cells from being attacked. Many pathogens have acquired the ability to sequester complement regulators from host plasma to evade complement attack. We describe here how Streptococcus pneumoniae adopts a strategy to prevent the formation of the C3 convertase C4bC2a by the rapid conversion of surface bound C4b and iC4b into C4dg, which remains bound to the bacterial surface but no longer forms a convertase complex. Noncapsular virulence factors on the pneumococcus are thought to facilitate this process by sequestering C4b-binding protein (C4BP) from host plasma. When S. pneumoniae D39 was opsonized with human serum, the larger C4 activation products C4b and iC4b were undetectable, but the bacteria were liberally decorated with C4dg and C4BP. With targeted deletions of either PspA or PspC, C4BP deposition was markedly reduced, and there was a corresponding reduction in C4dg and an increase in the deposition of C4b and iC4b. The effect was greatest when PspA and PspC were both knocked out. Infection experiments in mice indicated that the deletion of PspA and/or PspC resulted in the loss of bacterial pathogenicity. Recombinant PspA and PspC both bound serum C4BP, and both led to increased C4b and reduced C4dg deposition on S. pneumoniae D39. We conclude that PspA and PspC help the pneumococcus to evade complement attack by binding C4BP and so inactivating C4b.

Age-related variations in the in vitro bactericidal activity of human sera against Pseudomonas aeruginosa.

The human serum is a vital component of the innate immunity of the host that acts as the first line of defence against invading pathogens. A key player in serum-mediated innate immune defence is a system of more than 35 proteins, collectively named as the complement system. After exposure of the pathogen, these proteins are activated in a cascade manner, ultimately forming a membrane attack complex (MAC) on the surface of the pathogen that directly lyses the bacterial cell. Formation of the MAC can be demonstrated in vitro by using serum bactericidal assay (SBA) that works in the absence of cellular components of blood after incubating the serum along with bacteria. Here, we describe the age-related differences in the bactericidal activity of human serum against Pseudomonas aeruginosa, an opportunistic human pathogen causing an array of hospital and community-acquired infections. We demonstrate that adult sera were highly effective in the in vitro killing of Pseudomonas aeruginosa as compared to children and the elderly (p < 0.0001). Sera from children were seriously compromised in the killing P. aeruginosa, whereas elderly sera showed a reduced level of killing. Data revealed a positive correlation between age and serum-killing with higher coefficient of determination values of 0.34, 0.27, and 0.58 and p values of < 0.0001, < 0.001, and < 0.0001, respectively, after 60, 90, and 120 minutes of incubation. Hence, our study highlights the age-related difference in the bactericidal activity of human sera. We conclude that sera of children are totally compromised, whereas elderly sera are only partially compromised, in the killing of P. aeruginosa.

Low-dose recombinant properdin provides substantial protection against Streptococcus pneumoniae and Neisseria meningitidis infection.

Modern medicine has established three central antimicrobial therapeutic concepts: vaccination, antibiotics, and, recently, the use of active immunotherapy to enhance the immune response toward specific pathogens. The efficacy of vaccination and antibiotics is limited by the emergence of new pathogen strains and the increased incidence of antibiotic resistance. To date, immunotherapy development has focused mainly on cytokines. Here we report the successful therapeutic application of a complement component, a recombinant form of properdin (Pn), with significantly higher activity than native properdin, which promotes complement activation via the alternative pathway, affording protection against N. menigitidis and S. pneumoniae. In a mouse model of infection, we challenged C57BL/6 WT mice with N. menigitidis B-MC58 6 h after i.p. administration of Pn (100 µg/mouse) or buffer alone. Twelve hours later, all control mice showed clear symptoms of infectious disease while the Pn treated group looked healthy. After 16 hours, all control mice developed sepsis and had to be culled, while only 10% of Pn treated mice presented with sepsis and recoverable levels of live Meningococci. In a parallel experiment, mice were challenged intranasally with a lethal dose of S. pneumoniae D39. Mice that received a single i.p. dose of Pn at the time of infection showed no signs of bacteremia at 12 h postinfection and had prolonged survival times compared with the saline-treated control group (P < 0.0001). Our findings show a significant therapeutic benefit of Pn administration and suggest that its antimicrobial activity could open new avenues for fighting infections caused by multidrug-resistant neisserial or streptococcal strains.

The lectin pathway of complement activation is a critical component of the innate immune response to pneumococcal infection.

The complement system plays a key role in host defense against pneumococcal infection. Three different pathways, the classical, alternative and lectin pathways, mediate complement activation. While there is limited information available on the roles of the classical and the alternative activation pathways of complement in fighting streptococcal infection, little is known about the role of the lectin pathway, mainly due to the lack of appropriate experimental models of lectin pathway deficiency. We have recently established a mouse strain deficient of the lectin pathway effector enzyme mannan-binding lectin associated serine protease-2 (MASP-2) and shown that this mouse strain is unable to form the lectin pathway specific C3 and C5 convertases. Here we report that MASP-2 deficient mice (which can still activate complement via the classical pathway and the alternative pathway) are highly susceptible to pneumococcal infection and fail to opsonize Streptococcus pneumoniae in the none-immune host. This defect in complement opsonisation severely compromises pathogen clearance in the lectin pathway deficient host. Using sera from mice and humans with defined complement deficiencies, we demonstrate that mouse ficolin A, human L-ficolin, and collectin 11 in both species, but not mannan-binding lectin (MBL), are the pattern recognition molecules that drive lectin pathway activation on the surface of S. pneumoniae. We further show that pneumococcal opsonisation via the lectin pathway can proceed in the absence of C4. This study corroborates the essential function of MASP-2 in the lectin pathway and highlights the importance of MBL-independent lectin pathway activation in the host defense against pneumococci.