In 1993, the WHO declared tuberculosis a global emergency on the basis that there are 8 million new cases per year. The complete genome of the strain H37Rv of the causative microorganism, Mycobacterium tuberculosis, comprising 3924 genes has been sequenced. We compared the proteomes of two non‐virulent vaccine strains of M. bovis BCG (Chicago and Copenhagen) with two virulent strains of M. tuberculosis (H37Rv and Erdman) to identify protein candidates of value for the development of vaccines, diagnostics and therapeutics. The mycobacterial strains were analysed by two‐dimensional electrophoresis (2‐DE) combining non‐equilibrium pH gradient electrophoresis (NEPHGE) with SDS–PAGE. Distinct and characteristic proteins were identified by mass spectrometry and introduced into a dynamic 2‐DE database (http://www.mpiib‐berlin.mpg.de/2D‐PAGE). Silver‐stained 2‐DE patterns of mycobacterial cell proteins or culture supernatants contained 1800 or 800 spots, respectively, from which 263 were identified. Of these, 54 belong to the culture supernatant. Sixteen and 25 proteins differing in intensity or position between M. tuberculosis H37Rv and Erdman, and H37Rv and M. bovis BCG Chicago, respectively, were identified and categorized into protein classes. It is to be hoped that the availability of the mycobacterial proteome will facilitate the design of novel measures for prevention and therapy of one of the great health threats, tuberculosis.

Heat shock proteins Hsp90 and Hsp70 facilitate protein folding but can also direct proteins for ubiquitin-mediated degradation. The mechanisms regulating these opposite activities involve Hsp binding to co-chaperones including CHIP and HOP at their C-termini. We demonstrated that the extreme C-termini of Hsp70 and Hsp90 contain phosphorylation sites targeted by kinases including CK1, CK2 and GSK3-β in vitro. The phosphorylation of Hsp90 and Hsp70 prevents binding to CHIP and thus enhances binding to HOP. Highly proliferative cells contain phosphorylated chaperones in complex with HOP and phospho-mimetic and non-phosphorylable Hsp mutant proteins show that phosphorylation is directly associated with increased proliferation rate. We also demonstrate that primary human cancers contain high levels of phosphorylated chaperones and show increased levels of HOP protein and mRNA. These data identify C-terminal phosphorylation of Hsp70 and Hsp90 as a switch for regulating co-chaperone binding and indicate that cancer cells possess an elevated protein folding environment by the concerted action of co-chaperone expression and chaperone modifications. In addition to identifying the pathway responsible for regulating chaperone-mediated protein folding/degradation balances in normal cells, the data provide novel mechanisms to account for the aberrant chaperone activities observed in human cancer cells and have implications for the application of anti-chaperone therapies in cancer treatment.

Efficient assembly and repair of the oxygen-evolving photosystem II (PSII) complex is vital for maintaining photosynthetic activity in plants, algae, and cyanobacteria. How chlorophyll is delivered to PSII during assembly and how vulnerable assembly complexes are protected from photodamage are unknown. Here, we identify a chlorophyll and β-carotene binding protein complex in the cyanobacterium Synechocystis PCC 6803 important for formation of the D1/D2 reaction center assembly complex. It is composed of putative short-chain dehydrogenase/reductase Ycf39, encoded by the slr0399 gene, and two members of the high-light-inducible protein (Hlip) family, HliC and HliD, which are small membrane proteins related to the light-harvesting chlorophyll binding complexes found in plants. Perturbed chlorophyll recycling in a Ycf39-null mutant and copurification of chlorophyll synthase and unassembled D1 with the Ycf39-Hlip complex indicate a role in the delivery of chlorophyll to newly synthesized D1. Sequence similarities suggest the presence of a related complex in chloroplasts.

We have investigated the location of the Psb27 protein and its role in photosystem (PS) II biogenesis in the cyanobacterium Synechocystis sp. PCC 6803. Native gel electrophoresis revealed that Psb27 was present mainly in monomeric PSII core complexes but also in smaller amounts in dimeric PSII core complexes, in large PSII supercomplexes, and in the unassembled protein fraction. We conclude from analysis of assembly mutants and isolated histidine-tagged PSII subcomplexes that Psb27 associates with the “unassembled” CP43 complex, as well as with larger complexes containing CP43, possibly in the vicinity of the large lumenal loop connecting transmembrane helices 5 and 6 of CP43. A functional role for Psb27 in the biogenesis of CP43 is supported by the decreased accumulation and enhanced fragmentation of unassembled CP43 after inactivation of the psb27 gene in a mutant lacking CP47. Unexpectedly, in strains unable to assemble PSII, a small amount of Psb27 comigrated with monomeric and trimeric PSI complexes upon native gel electrophoresis, and Psb27 could be copurified with histidine-tagged PSI isolated from the wild type. Yeast two-hybrid assays suggested an interaction of Psb27 with the PsaB protein of PSI. Pull-down experiments also supported an interaction between CP43 and PSI. Deletion of psb27 did not have drastic effects on PSII assembly and repair but did compromise short-term acclimation to high light. The tentative interaction of Psb27 and CP43 with PSI raises the possibility that PSI might play a previously unrecognized role in the biogenesis/repair of PSII.

Hepcidin is a major regulator of iron metabolism. Hepcidin-based therapeutics/diagnostics could play roles in hematology in the future, and thus, hepcidin transport is crucial to understand. In this study, we identify α₂-macroglobulin (α₂-M) as the specific hepcidin-binding molecule in blood. Interaction of ¹²⁵I-hepcidin with α₂-M was identified using fractionation of plasma proteins followed by native gradient polyacrylamide gel electrophoresis and mass spectrometry. Hepcidin binding to nonactivated α₂-M displays high affinity (K_d 177 ± 27 nM), whereas hepcidin binding to albumin was nonspecific and displayed nonsaturable kinetics. Surprisingly, the interaction of hepcidin with activated α₂-M exhibited a classical sigmoidal binding curve demonstrating cooperative binding of 4 high-affinity (K_d 0.3 μM) hepcidin-binding sites. This property probably enables efficient sequestration of hepcidin and its subsequent release or inactivation that may be important for its effector functions. Because α₂-M rapidly targets ligands to cells via receptor-mediated endocytosis, the binding of hepcidin to α₂-M may influence its functions. In fact, the α₂-M–hepcidin complex decreased ferroportin expression in J774 cells more effectively than hepcidin alone. The demonstration that α₂-M is the hepcidin transporter could lead to better understanding of hepcidin physiology, methods for its sensitive measurement and the development of novel drugs for the treatment of iron-related diseases.

Phlebotomine sand flies are incriminated in the transmission of several human and veterinary pathogens. To elucidate their role as vectors, proper species identification is crucial. Since traditional morphological determination is based on minute and often dubious characteristics on their head and genitalia, which require certain expertise and may be damaged in the field-collected material, there is a demand for rapid, simple and cost-effective molecular approaches.