Keding Cheng

Mitochondrial translocation of protein kinase C delta in phorbolester-induced cytochrome C release and apoptosis

Apoptosis is induced by the release of cytochromec from mitochondria to the cytoplasm. The present studies demonstrate that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induces translocation of protein kinase C (PKC) δ from the cytoplasm to mitochondria. The results also show that translocation of PKCδ results in release of cytochrome c. The functional significance of this event is further supported by the demonstration that PKCδ translocation is required for TPA-induced apoptosis. These findings demonstrate that translocation of PKCδ to mitochondria is responsible, at least in part, for inducing cytochrome crelease and apoptosis.

Mass Spectrometric Characterization of Proteins from the SARS Virus A Preliminary Report

A new coronavirus has been implicated as the causative agent of severe acute respiratory syndrome (SARS). We have used convalescent sera from several SARS patients to detect proteins in the culture supernatants from cells exposed to lavage another SARS patient. The most prominent protein in the supernatant was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a ∼46-kDa species. This was found to be a novel nucleocapsid protein that matched almost exactly one predicted by an open reading frame in the recently published nucleotide sequence of the same virus isolate (>96% coverage). A second viral protein corresponding to the predicted ∼139-kDa spike glycoprotein has also been examined by MALDI-TOF MS (42% coverage). After peptide N-glycosidase F digestion, 12 glycosylation sites in this protein were confirmed. The sugars attached to four of the sites were also identified. These results suggest that the nucleocapsid protein is a major immunogen that may be useful for early diagnostics, and that the spike glycoprotein may present a particularly attractive target for prophylactic intervention in combating SARS.

Identification of differentially expressed proteins in the cervical mucosa of HIV-1-resistant sex workers.

Novel tools are necessary to understand mechanisms of altered susceptibility to HIV-1 infection in women of the Pumwani Sex Worker cohort, Kenya. In this cohort, more than 140 of the 2000 participants have been characterized to be relatively resistant to HIV-1 infection. Given that sexual transmission of HIV-1 occurs through mucosal surfaces such as that in the cervicovaginal environment, our hypothesis is that innate immune factors in the genital tract may play a role in HIV-1 infection resistance. Understanding this mechanism may help develop microbicides and/or vaccines against HIV-1. A quantitative proteomics technique (2D-DIGE: two-dimensional difference in-gel electrophoresis) was used to examine cervical mucosa of HIV-1 resistant women ( n = 10) for biomarkers of HIV-1 resistance. Over 15 proteins were found to be differentially expressed between HIV-1-resistant women and control groups ( n = 29), some which show a greater than 8-fold change. HIV-1-resistant women overexpressed several antiproteases, including those from the serpin B family, and also cystatin A, a known anti-HIV-1 factor. Immunoblotting for a selection of the identified proteins confirmed the DIGE volume differences. Validation of these results on a larger sample of individuals will provide further evidence these biomarkers are associated with HIV-1 resistance and could help aid in the development of effective microbicides against HIV-1.

Recent development of mass spectrometry and proteomics applications in identification and typing of bacteria

Identification and typing of bacteria occupy a large fraction of time and work in clinical microbiology laboratories. With the certification of some MS platforms in recent years, more applications and tests of MS‐based diagnosis methods for bacteria identification and typing have been created, not only on well‐accepted MALDI‐TOF‐MS‐based fingerprint matches, but also on solving the insufficiencies of MALDI‐TOF‐MS‐based platforms and advancing the technology to areas such as targeted MS identification and typing of bacteria, bacterial toxin identification, antibiotics susceptibility/resistance tests, and MS‐based diagnostic method development on unique bacteria such as Clostridium and Mycobacteria. This review summarizes the recent development in MS platforms and applications in bacteria identification and typing of common pathogenic bacteria.

Reduced focal adhesion kinase and paxillin phosphorylation in BCR‐ABL‐transfected cells

BCR‐ABL formation is critical to oncogenic transformation in chronic myelogenous leukemia and has been implicated as a key event leading to alterations in cytoskeletal structures and adhesion in the leukemic cells. The authors therefore investigated the effect of p210BCR‐ABL on actin polymerization as well as on the expression and phosphorylation state of the adhesion proteins paxillin and focal adhesion kinase (FAK).

Bcl-xL Blocks Activation of Related Adhesion Focal Tyrosine Kinase/Proline-rich Tyrosine Kinase 2 and Stress-activated Protein Kinase/c-Jun N-terminal Protein Kinase in the Cellular Response to Methylmethane Sulfonate

The stress-activated protein kinase/c-Jun N-terminal protein kinase (JNK) is induced in response to ionizing radiation and other DNA-damaging agents. Recent studies indicate that activation of JNK is necessary for induction of apoptosis in response to diverse agents. Here we demonstrate that methylmethane sulfonate (MMS)-induced activation of JNK is inhibited by overexpression of the anti-apoptotic protein Bcl-xL, but not by caspase inhibitors CrmA and p35. By contrast, UV-induced JNK activity is insensitive to Bcl-xL. The results demonstrate that treatment with MMS is associated with an increase in tyrosine phosphorylation of related adhesion focal tyrosine kinase (RAFTK)/proline-rich tyrosine kinase 2 (PYK2), an upstream effector of JNK and that this phosphorylation is inhibited by overexpression of Bcl-xL. Furthermore, overexpression of a dominant-negative mutant of RAFTK (RAFTK K-M) inhibits MMS-induced JNK activation. The results indicate that inhibition of RAFTK phosphorylation by MMS in Bcl-xL cells is attributed to an increase in tyrosine phosphatase activity in these cells. Hence, treatment of Bcl-xL cells with sodium vanadate, a tyrosine phosphatase inhibitor, restores MMS-induced activation of RAFTK and JNK. These findings indicate that RAFTK-dependent induction of JNK in response to MMS is sensitive to Bcl-xL, but not to CrmA and p35, by a mechanism that inhibits tyrosine phosphorylation and thereby activation of RAFTK. Taken together, these findings support a novel role for Bcl-xL that is independent of the caspase cascade.

Rapid, Sensitive, and Specific Escherichia coli H Antigen Typing by Matrix-Assisted Laser Desorption Ionization–Time of Flight-Based Peptide Mass Fingerprinting

ABSTRACT Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has gained popularity in recent years for rapid bacterial identification, mostly at the genus or species level. In this study, a rapid method to identify the Escherichia coli flagellar antigen (H antigen) at the subspecies level was developed using a MALDI-TOF MS platform with high specificity and sensitivity. Flagella were trapped on a filter membrane, and on-filter trypsin digestion was performed. The tryptic digests of each flagellin then were collected and analyzed by MALDI-TOF MS through peptide mass fingerprinting. Sixty-one reference strains containing all 53 H types and 85 clinical strains were tested and compared to serotyping designations. Whole-genome sequencing was used to resolve conflicting results between the two methods. It was found that DHB (2,5-dihydroxybenzoic acid) worked better than CHCA (α-cyano-4-hydroxycinnamic acid) as the matrix for MALDI-TOF MS, with higher confidence during protein identification. After method optimization, reference strains representing all 53 E. coli H types were identified correctly by MALDI-TOF MS. A custom E. coli flagellar/H antigen database was crucial for clearly identifying the E. coli H antigens. Of 85 clinical isolates tested by MALDI-TOF MS-H, 75 identified MS-H types (88.2%) matched results obtained from traditional serotyping. Among 10 isolates where the results of MALDI-TOF MS-H and serotyping did not agree, 60% of H types characterized by whole-genome sequencing agreed with those identified by MALDI-TOF MS-H, compared to only 20% by serotyping. This MALDI-TOF MS-H platform can be used for rapid and cost-effective E. coli H antigen identification, especially during E. coli outbreaks.

A proteomics-based approach for monoclonal antibody characterization

The determination of monoclonal antibody specificity is dependent upon the availability of purified antigen. Such material is not always available and this has proven to be one of the rate-limiting steps in monoclonal antibody production. The aim of the present study was to develop a generic approach to defining antibody specificity that bypassed the need for pure antigens through the use of proteomics. The scheme and its application to several biological mixtures are described. The results demonstrate the ability of the approach to identify antibodies against both the major components and the minor contaminants of a protein mixture. This approach should markedly enhance the characterization of antibodies to complex antigen mixtures.

MS-H: a novel proteomic approach to isolate and type the E. coli H antigen using membrane filtration and liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Serotyping is the long-standing gold standard method to determine E. coli H antigens; however, this method requires a panel of H-antigen specific antibodies and often culture-based induction of the H-antigen flagellar motility. In this study, a rapid and accurate method to isolate and identify the Escherichia coli (E. coli) H flagellar antigen was developed using membrane filtration and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Flagella were isolated from pure culture, digested with trypsin, and then subjected to LC-MS/MS using one of two systems (Agilent-nano-LC-QSTAR XL or Proxeon-nano-LC-LTQ-Orbitrap XL). The resulting peptide sequence data were searched against a custom E. coli flagella/H antigen database. This approach was evaluated using flagella isolated from reference E. coli strains representing all 53 known H antigen types and 41 clinical E. coli strains. The resulting LC-MS/MS classifications of H antigen types (MS-H) were concordant with the known H serogroup for all 53 reference types, and of 41 clinical isolates tested, 38 (92.7%) were concordant with the known H serogroup. MS-H clearly also identified two clinical isolates (4.9%) that were untypeable by serotyping. Notably, successful detection and classification of flagellar antigens with MS-H did not generally require induction of motility, establishing this proteomic approach as more rapid and cost-effective than traditional methods, while providing equitable specificity for typing E. coli H antigens.

Evidence for the presence of a low-mass β1 integrin on the cell surface

Although the cell line K562 reportedly expresses a single species of β1 integrin, α5β1, surface staining with monoclonal antibodies JB1A, 12G10 and B3B11 to the β1 chain clearly demonstrated differences in the expression levels of the epitopes detected by these antibodies. The present studies were initiated to determine the basis for this molecular heterogeneity in the integrins. Cross-linking of surface integrins with B3B11 caused their selective aggregation. This distribution was similar to that observed for the α5 chain. In contrast, cross-linking the β1 chains with 12G10 did not cause codistribution of α5, suggesting that these two species were not associated on the cell surface. Immunoprecipitates of the surface integrins of K562 cells indicated the presence of 120 and 140 kDa forms of the β1 chain which were detected by 12G10 and B3B11, respectively. Immunological, biochemical and mass spectrometric analysis of K562 surface integrins also failed to demonstrate the presence of any α chain in association with the 120 kDa species of β1 of K562 cells. Treatment of the two forms of β1 with PGNase reduced their masses to ∼90 kDa, suggesting that N-glycosylation was responsible for the mass differences. Collectively, these results provide evidence for a novel species of β1 on the cell surface, which does not appear to be associated with any α chain. The data also suggest that differences in glycosylation may be involved in defining the association between the integrin α and β chains and the functional properties of these integrins.