Ancient, independent evolution and distinct molecular features of the novel human T-lymphotropic virus type 4

Background

The human endogenous retrovirus HERV-K(HML-2) family is associated with testicular germ cell tumors (GCT). Various HML-2 proviruses encode viral proteins such as Env and Rec.

Results

We describe here that HML-2 Env gives rise to a 13 kDa signal peptide (SP) that harbors a different C-terminus compared to Rec. Subsequent to guiding Env to the endoplasmatic reticulum (ER), HML-2 SP is released into the cytosol. Biochemical analysis and confocal microscopy demonstrated that similar to Rec, SP efficiently translocates to the granular component of nucleoli. Unlike Rec, SP does not shuttle between nucleus and cytoplasm. SP is less stable than Rec as it is subjected to proteasomal degradation. Moreover, SP lacks export activity towards HML-2 genomic RNA, the main function of Rec in the original viral context, and SP does not interfere with Rec's RNA export activity.

Conclusion

SP is a previously unrecognized HML-2 protein that, besides targeting and translocation of Env into the ER lumen, may exert biological functions distinct from Rec. HML-2 SP represents another functional similarity with the closely related Mouse Mammary Tumor Virus that encodes an Env-derived SP named p14. Our findings furthermore support the emerging concept of bioactive SPs as a conserved retroviral strategy to modulate their host cell environment, evidenced here by a "retroviral fossil". While the specific role of HML-2 SP remains to be elucidated in the context of human biology, we speculate that it may be involved in immune evasion of GCT cells or tumorigenesis.     

Hsp90 at the crossroads of genetics and epigenetics

Hsp90 is a specialized molecular chaperone that is capable of buffering the expression of abnormal phenotypes.Inhi-bition of Hsp90 activity results in the expression of these phenotypes that are otherwise masked.Selection of offspringfrom the crossing of affected progenies results in inheritance and enrichment of these phenotypes,which can becomeindependent of their original stimuli.The current combined evidence favours a model involving the interplay betweengenetics and epigenetics.The recent proteomics efforts to characterize the Hsp90 interaction networks provide further cluesinto the molecular mechanisms behind this complex phenomenon.This review summarizes the most recent experimentalobservations and briefly discusses the genetic and epigenetic views used in explaining the different observations.     

The Clp chaperones and proteases of the human malaria parasite Plasmodium falciparum

The Clp chaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clp chaperones and proteases in the human malaria parasite Plasmodium falciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, were also identified. Expression of all Clp chaperones and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.     

Tamoxifen Enhances the Hsp90 Molecular Chaperone ATPase Activity

Background

Hsp90 is an essential molecular chaperone that is also a novel anti-cancer drug target. There is growing interest in developing new drugs that modulate Hsp90 activity.

Methodology/Principal Findings

Using a virtual screening approach, 4-hydroxytamoxifen, the active metabolite of the anti-estrogen drug tamoxifen, was identified as a putative Hsp90 ligand. Surprisingly, while all drugs targeting Hsp90 inhibit the chaperone ATPase activity, it was found experimentally that 4-hydroxytamoxifen and tamoxifen enhance rather than inhibit Hsp90 ATPase.

Conclusions/Significance

Hence, tamoxifen and its metabolite are the first members of a new pharmacological class of Hsp90 activators.     

Role and challenges of proteomics in pharma and biotech: technical, scientific and commercial perspective

Contemporary proteomics, currently in its exponential growth phase, is a bewildering array of tools. Proteomic methods are the result of a convergence of rapidly improving mass spectrometry technologies, protein chemistry and separation sciences, genomics and bioinformatics. Strides in improving proteomics technologies to map and measure proteomes and subproteomes are being made. However, no single proteomic platform appears ideally suited to address all research needs or accomplish ambitious goals satisfactorily. However, proteomics is in a unique position to contribute to protein discovery and to public health in terms of better biomarkers, diagnostics and treatment of disease. While the potential is great, many challenges and issues remain to be solved. Fundamental issues, such as biological variability, pre-analytic factors and analytical reproducibility, remain to be resolved. Neither an all-genetic approach nor an all-proteomic approach will solve biological complexity. Proteomics will be the foundation for constructing and extracting useful knowledge to pharma and biotech depicted in the following path: data --> structured data --> information --> information architecture --> knowledge --> useful knowledge.     

Major histocompatibility complex (MHC) class II: are lipid rafts the missing link?

Aside from their crucial roles in the presentation of nominal antigen to CD4+ T cells and susceptibility to autoimmune diseases, substantial evidences suggest that MHC class II molecules act as signal transducer receptors as well. The signals transmitted affect diverse biological functions. Paradoxically, the cytoplasmic and transmembrane domains of these molecules are devoid of classic signaling motifs. The recent discovery of the presence of membrane microdomains, also called lipid rafts, that are enriched in kinases and adaptor molecules, may contribute to the elucidation of the mechanisms by which MHC class II molecules transmit their signals.     

Mutation-Directed Chemical Cross-Linking of Human Immunodeficiency Virus Type 1 gp41 Oligomers

The human immunodeficiency virus type 1 transmembrane protein gp41 oligomer anchors the attachment protein, gp120, to the viral envelope and mediates viral envelope-cell membrane fusion following gp120-CD4 receptor-chemokine coreceptor binding. We have used mutation-directed chemical cross-linking with bis(sulfosuccinimidyl)suberate (BS³) to investigate the architecture of the gp41 oligomer. Treatment of gp41 with BS³ generates a ladder of four bands on sodium dodecyl sulfate-polyacrylamide gels, corresponding to monomers, dimers, trimers, and tetramers. By systematically replacing gp41 lysines with arginine and determining the mutant gp41 cross-linking pattern, we observed that gp41 N termini are cross-linked. Lysine 678, which is close to the transmembrane sequence, was readily cross-linked to Lys-678 on other monomers within the oligomeric structure. This arrangement appears to be facilitated by the close packing of membrane-anchoring sequences, since the efficiency of assembly of heterooligomers between wild-type and mutant Env proteins is improved more than twofold if the mutant contains the membrane-anchoring sequence. We also detected close contacts between Lys-596 and Lys-612 in the disulfide-bonded loop/glycan cluster of one monomer and lysines in the N-terminal amphipathic α-helical oligomerization domain (Lys-569 and Lys-583) and C-terminal α-helical sequence (Lys-650 and Lys-660) of adjacent monomers. Precursor-processing efficiency, gp120-gp41 association, soluble recombinant CD4-induced shedding of gp120 from cell surface gp41, and acquisition of gp41 ectodomain conformational antibody epitopes were unaffected by the substitutions. However, the syncytium-forming function was most dependent on the conserved Lys-569 in the N-terminal α-helix. These results indicate that gp160-derived gp41 expressed in mammalian cells is a tetramer and provide information about the juxtaposition of gp41 structural elements within the oligomer.     

The importance of an extra loop in the B-domain of an α-amylase from B. stearothermophilus US100

To provide insight into the potential role of a loop in domain B of several bacterial α-amylases, molecular and structural investigation of Bacillus stearothermophilus α-amylase (Amy US100) was used as a model. Combination deletion mutants of G₂₁₃, I₂₁₄ and G₂₁₅, described as a loop-forming on the surface bacterial amylases, were subjected to biochemical and structural investigation. Thermoactivity, thermostability as well calcium requirement were studied for each mutant.Thus, deletion of one residue differently affects only the thermostability. Shortening the loop by deletion of G₂₁₃-I₂₁₄ or I₂₁₄-G₂₁₅ improved the thermostability and reduces calcium requirement. However, the deletion of three residues has a negative effect on thermostability and reduces the optimal temperature by 17 °C.The structural investigation showed that stabilizing deletions contribute to reinforce the architecture of domain B and the active site conformation. The deletion of three residues reduces the flexibility of this region and abolishes a denser hydrogen bond network.