Engineered resistance against proteinases

Exogenous proteinase inhibitors are valuable and economically interesting protective biotechnological tools. We examined whether small proteinase inhibitors when fused to a selected target protein can protect the target from proteolytic degradation without simultaneously affecting the function and activity of the target domain. Two proteinase inhibitors were studied: a Kazal-type silk proteinase inhibitor (SPI2) from Galleria mellonella, and the Cucurbita maxima trypsin inhibitor I (CMTI I). Both inhibitors target serine proteinases, are small proteins with a compact structure stabilized by a network of disulfide bridges, and are expressed as free polypeptides in their natural surroundings. Four constructs were prepared: the gene for either of the inhibitors was ligated to the 5' end of the DNA encoding one or the other of two selected target proteins, the coat protein (CP) of Potato potyvirus Y or the Escherichia coli beta-glucuronidase (GUS). CMTI I fused to the target proteins strongly hampered their functions. Moreover, the inhibitory activity of CMTI I was retained only when it was fused to the CP. In contrast, when fused to SPI2, specific features and functions of both target proteins were retained and the inhibitory activity of SPI2 was fully preserved. Measuring proteolysis in the presence or absence of either inhibitor, we demonstrated that proteinase inhibitors can protect target proteins used either free or as a fusion domain. Interestingly, their inhibitory efficiency was superior to that of a commercial inhibitor of serine proteinases, AEBSF.     

Glia Maturation Factor (GMF) Interacts with Arp2/3 Complex in a Nucleotide State-dependent Manner

Glia maturation factor (GMF) is a member of the actin-depolymerizing factor (ADF)/cofilin family. ADF/cofilin promotes disassembly of aged actin filaments, whereas GMF interacts specifically with Arp2/3 complex at branch junctions and promotes debranching. A distinguishing feature of ADF/cofilin is that it binds tighter to ADP-bound than to ATP-bound monomeric or filamentous actin. The interaction is also regulated by phosphorylation at Ser-3 of mammalian cofilin, which inhibits binding to actin. However, it is unknown whether these two factors play a role in the interaction of GMF with Arp2/3 complex. Here we show using isothermal titration calorimetry that mammalian GMF has very low affinity for ATP-bound Arp2/3 complex but binds ADP-bound Arp2/3 complex with 0.7 μm affinity. The phosphomimetic mutation S2E in GMF inhibits this interaction. GMF does not bind monomeric ATP- or ADP-actin, confirming its specificity for Arp2/3 complex. We further show that mammalian Arp2/3 complex nucleation activated by the WCA region of the nucleation-promoting factor N-WASP is not affected by GMF, whereas nucleation activated by the WCA region of WAVE2 is slightly inhibited at high GMF concentrations. Together, the results suggest that GMF functions by a mechanism similar to that of other ADF/cofilin family members, displaying a preference for ADP-Arp2/3 complex and undergoing inhibition by phosphorylation of a serine residue near the N terminus. Arp2/3 complex nucleation occurs in the ATP state, and nucleotide hydrolysis promotes debranching, suggesting that the higher affinity of GMF for ADP-Arp2/3 complex plays a physiological role by promoting debranching of aged branch junctions without interfering with Arp2/3 complex nucleation.     

Analysis of the rat hypothalamus proteome by data‐independent label‐free LC‐MS/MS

Studies of neuronal, endocrine, and metabolic disorders would be facilitated by characterization of the hypothalamus proteome. Protein extracts prepared from 16 whole rat hypothalami were measured by data‐independent label‐free nano LC‐MS/MS. Peptide features were detected, aligned, and searched against a rat Swiss‐Prot database using ProteinLynx Global Server v.2.5. The final combined dataset comprised 21 455 peptides, corresponding to 622 unique proteins, each identified by a minimum of two distinct peptides. The majority of the proteins (69%) were cytosolic, and 16% were membrane proteins. Important proteins involved in neurological and synaptic function were identified including several members of the Ras‐related protein family and proteins involved in glutamate biosynthesis.     

Target Cell Cyclophilins Facilitate Human Papillomavirus Type 16 Infection

Following attachment to primary receptor heparan sulfate proteoglycans (HSPG), human papillomavirus type 16 (HPV16) particles undergo conformational changes affecting the major and minor capsid proteins, L1 and L2, respectively. This results in exposure of the L2 N-terminus, transfer to uptake receptors, and infectious internalization. Here, we report that target cell cyclophilins, peptidyl-prolyl cis/trans isomerases, are required for efficient HPV16 infection. Cell surface cyclophilin B (CyPB) facilitates conformational changes in capsid proteins, resulting in exposure of the L2 N-terminus. Inhibition of CyPB blocked HPV16 infection by inducing noninfectious internalization. Mutation of a putative CyP binding site present in HPV16 L2 yielded exposed L2 N-terminus in the absence of active CyP and bypassed the need for cell surface CyPB. However, this mutant was still sensitive to CyP inhibition and required CyP for completion of infection, probably after internalization. Taken together, these data suggest that CyP is required during two distinct steps of HPV16 infection. Identification of cell surface CyPB will facilitate the study of the complex events preceding internalization and adds a putative drug target for prevention of HPV–induced diseases.     

Slipped Misalignment Mechanisms of Deletion Formation: In Vivo Susceptibility to Nucleases

Misalignment of repeated sequences during DNA replication can lead to deletions or duplications in genomic DNA. In Escherichia coli, such genetic rearrangements can occur at high frequencies, independent of the RecA-homologous recombination protein, and are sometimes associated with sister chromosome exchange (SCE). Two mechanisms for RecA-independent genetic rearrangements have been proposed: simple replication misalignment of the nascent strand and its template and SCE-associated misalignment involving both nascent strands. We examined the influence of the 3′ exonuclease of DNA polymerase III and exonuclease I on deletion via these mechanisms in vivo. Because mutations in these exonucleases stimulate tandem repeat deletion, we conclude that displaced 3′ ends are a common intermediate in both mechanisms of slipped misalignments. Our results also confirm the notion that two distinct mechanisms contribute to slipped misalignments: simple replication misalignment events are sensitive to DNA polymerase III exonuclease, whereas SCE-associated events are sensitive to exonuclease I. If heterologies are present between repeated sequences, the mismatch repair system dependent on MutS and MutH aborts potential deletion events via both mechanisms. Our results suggest that simple slipped misalignment and SCE-associated misalignment intermediates are similarly susceptible to destruction by the mismatch repair system.     

Morphometric classification of hairy cell leukemia in bone marrow trephine biopsy

OBJECTIVE: To analyze cytologic and histologic parameters in bone marrow trephine biopsy in an attempt to define heterogeneity of hairy cell leukemia cells. STUDY DESIGN: The study group consisted of 28 trephine biopsies. Immunohistochemistry for CD20 antigen was used. Image processing and measurements were performed with AnalySIS 3.0 image analysis system (Soft Imaging System GmbH, Germany) and custom built programs. For planimetric measurements of nuclei, automatic segmentation was implemented. The measured parameters were: surface area, perimeter, minimum, mean and maximum diameter, and a set of form factors. Relative volumes of bone trabeculae, adipose tissue, hematopoietic tissue and neoplastic infiltrate were assessed by the point counting method. Nuclear volume was measured by the point sampled intercept method. Bone marrow fibrosis was assessed using a curvilinear line test system. RESULTS: Significant variability of cell nuclei was found, and their classification into 3 types was possible. The relative frequency of those types was different in various cases and allowed subdivision of cases into 3 groups that differed in some clinical and histologic manifestations. CONCLUSION: The present study demonstrated the heterogeneity of cell populations of hairy cell leukemia.     

Cardiolipin clusters and membrane domain formation induced by mitochondrial proteins

We show in this study that mitochondrial creatine kinase promotes segregation and clustering of cardiolipin in mixed membranes, a phenomenon that has been proposed to occur at contact sites in the mitochondria. This property of mitochondrial creatine kinase is dependent on the native octameric structure of the protein and does not occur after heat-denaturation or with the native dimeric form of the protein. Cardiolipin segregation was demonstrated by differential scanning calorimetry using membranes containing cardiolipin and either dipalmitoylphosphatidylethanolamine or 1-palmitoyl-2-oleoylphosphatidylethanolamine. Addition of the ubiquitous form of mitochondrial creatine kinase leads to the formation of a phosphatidylethanolamine-rich domain as a result of the protein binding preferentially to the cardiolipin. Such phase separation does not occur if cardiolipin is replaced with dioleoyl phosphatidylglycerol. Lipid phase separation is observed with other cardiolipin-binding proteins, including cytochrome c and, to a very small extent, with truncated Bid (t-Bid), as well as with the cationic polypeptide poly-L-lysine, but among these proteins the octameric form of mitochondrial creatine kinase is by far the most effective in causing segregation and clustering of cardiolipin. The proteins included in this study are found at mitochondrial contact sites where they are known to associate with cardiolipin. Domains in mitochondria enriched in cardiolipin play an important role in apoptosis and in energy flux processes.     

Inhibition of PDGF-BB by Factor VII-activating protease (FSAP) is neutralized by protease nexin-1, and the FSAP-inhibitor complexes are internalized via LRP

FSAP (Factor VII-activating protease) can inhibit neointima formation and VSMC (vascular smooth-muscle cell) proliferation by cleavage of PDGF-BB (platelet-derived growth factor-BB). Negatively charged polyanions lead to autoactivation of the FSAP, but no information is available concerning the potential regulation of FSAP activity and its metabolism in the vessel wall. In the present study, we demonstrate that the enzymatic activity of FSAP can be inhibited by the serine protease inhibitor, PN-1 (protease nexin-1), that is found in the vasculature. This leads to the loss of the inhibitory effect of FSAP on PDGF-BB-mediated DNA synthesis and mitogen-activated protein kinase phosphorylation in VSMCs. The FSAP-PN-1 complexes bind to the LRP (low-density lipoprotein receptor-related protein) and are subsequently internalized. This binding is inhibited by receptor-associated protein, an antagonist of LRP, as well as heparin. While PDGFbetaR (PDGFbeta receptor) is internalized by an LRP-dependent mechanism after stimulation of cells by PDGF-BB, the FSAP-PN-1 complex neither influenced PDGF-BB-mediated phosphorylation of PDGFbetaR nor its internalization via LRP. Hence, PN-1 inhibits the enzymatic activity of FSAP and neutralizes its effect on PDGF-BB-mediated VSMC proliferation. The FSAP-inhibitor complexes are internalized via LRP without influencing the PDGF-BB signal transduction pathway.     

Disruption of the dynamic sub-cellular localization of the Xenopus tumorhead protein causes embryonic lethality at the early gastrula transition

Abstract The Xenopus laevis tumorhead (TH) protein, a positive regulator of cell proliferation during embryogenesis, shuttles from the cell periphery into the nucleus during embryogenesis. In these studies, we performed a detailed analysis of TH's subcellular localization pattern to characterize its dynamic behavior. We found that TH exhibits distinct patterns of localization in different germ layers. At the blastula stage, TH is present in the apical cell periphery of prospective mesodermal and ectodermal cells. At the gastrula stage, TH is distributed throughout the entire cytoplasm of prospective mesodermal and ectodermal cells, whereas it shows nuclear localization in presumptive endodermal cells. TH moves into the nucleus of mesodermal and ectodermal cells during the neurula and early tailbud stages. To understand if TH is regulated by changes in its subcellular localization, we used a TH mutant containing signals for farnesylation and palmitoylation to tether the protein to the plasma membrane. Ubiquitous overexpression of this mutant causes embryonic lethality at the early gastrula transition. Further examination using TUNEL assays indicated that wild-type TH overexpression induces apoptosis during gastrulation, and that this effect is exacerbated by the overexpression of the membrane-bound TH mutant. Taken together, our results suggest that changes in the sub-cellular localization of the TH protein are important for its function because blocking the nuclear translocation of overexpressed TH increases apoptosis and causes embryos to die. Our data also suggest that TH plays a role outside the nucleus when it is present at the cell periphery.     

Serious doubts over “Eggs forever?”

A recent commentary in this journal by Byskov et al. (2005) claims that, despite published results from numerous independent lines of investigation from our laboratory and others, there does not "exist any evidence for neo-folliculogenesis in the adult mammalian ovary." While we agree with Byskov et al. that our work represents a radical departure from the age-old dogma that mammalian females permanently lose the capacity for oocyte and follicle production during the perinatal period, careful examination of all of the available data leaves no doubt that adult female mammals retain the capacity for oogenesis and folliculogenesis. These findings do not change the fact that exhaustion of the oocyte pool occurs with advancing chronological age--a process responsible for driving the menopause in women--but rather question the basic mechanism underlying age-related ovarian failure. In this regard, studies of aging male mice have demonstrated that testicular atrophy is associated with a dramatic decline in the number, activity and quality of germline stem cells that maintain spermatogenesis during adulthood (Zhang et al., 2006). Therefore, to the contrary of the opinion of Byskov et al. that such a process would be "considered exceptional among stem cells," it is certainly reasonable to hypothesize that a similar deterioration of female germline stem cell function underlies the decline in oocyte quality and the onset of ovarian failure in aging females. Further, while we accept that a departure from conventional thinking can take years to gain widespread acceptance, we feel this resistance to change should not be construed as the sole means to voice opinions about the validity of our data or the maturity of our principal conclusion.