Ontogeny of proteolytic immunity: IgM serine proteases

We report the chemical activity of immunoglobulin micro and kappa/lambda subunits expressed on the surface of B cells and in secreted IgM antibodies (Abs) found in the preimmune repertoire. Most of the nucleophilic reactivity of B cells measured by formation of covalent adducts of a hapten amidino phosphonate diester was attributed to micro and kappa/lambda subunits of the B cell receptor. Secreted IgM Abs displayed superior nucleophilic reactivity than IgG Abs. IgM Abs catalyzed the cleavage of model peptide substrates at rates up to 344-fold greater than IgG Abs. Catalytic activities were observed in polyclonal IgM Abs from immunologically na?ve mice and humans without immunological disease, as well as monoclonal IgM Abs to unrelated antigens. Comparison of several IgM Abs indicated divergent activity levels and substrate preferences, with the common requirement of a basic residue flanking the cleavage site. Fab fragments of a monoclonal IgM Ab expressed catalytic activity, confirming the V domain location of the catalytic site. The catalytic reaction was inhibited by the covalently reactive hapten probe and diisopropylfluorophosphate, suggesting a serine protease-like mechanism. These observations indicate the existence of serine protease-like BCRs and secreted IgM Abs as innate immunity components with potential roles in B cell development and Ab effector functions.     

Extramitochondrial localization of mortalin/mthsp70/PBP74/GRP75

Subcellular fractionation and immunofluorescence microscopy were used to identify the specific sites of intracellular residence of mortalin, also called a mitochondrial homologue of the hsp70 family, in immortal human cell lines previously assigned to four distinct complementation groups (A-D) for indefinite cell division. In addition to the mitochondria it was seen in the endoplasmic reticulum (ER) fractions of all the cell lines analyzed. Interestingly, three of the group A cells lines (EJ, GM639, and HT1080), in addition to the mitochondria and ER, exhibited cytosolically (extra-organelle) localized pool of mortalin. These findings demonstrate that mortalin is not present exclusively in mitochondria. Its residence in different organelles may be the basis of differential distribution observed previously in different human cell lines.     

Expression of the zinc-finger antiviral protein inhibits alphavirus replication

The rat zinc-finger antiviral protein (ZAP) was recently identified as a host protein conferring resistance to retroviral infection. We analyzed ZAP's ability to inhibit viruses from other families and found that ZAP potently inhibits the replication of multiple members of the Alphavirus genus within the Togaviridae, including Sindbis virus, Semliki Forest virus, Ross River virus, and Venezuelan equine encephalitis virus. However, expression of ZAP did not induce a broad-spectrum antiviral state as some viruses, including vesicular stomatitis virus, poliovirus, yellow fever virus, and herpes simplex virus type 1, replicated to normal levels in ZAP-expressing cells. We determined that ZAP expression inhibits Sindbis virus replication after virus penetration and entry, but before the amplification of newly synthesized plus strand genomic RNA. Using a temperature-sensitive Sindbis virus mutant expressing luciferase, we further showed that translation of incoming viral RNA is blocked by ZAP expression. Elucidation of the antiviral mechanism by which ZAP inhibits Sindbis virus translation may lead to the development of agents with broad activity against alphaviruses.     

Pubescine A,a cyclopeptide alkaloid from Discaria pubescens

From Discaria pubescens, a new peptide alkaloid, pubescine A (1), belonging to the frangulanine type has been isolated and its structure elucidated. Pubescine A seems to be isomeric to melonovine A. For the first time, d-leucine has been identified in a cyclopeptide alkaloid.     

Spontaneous transfer of monoacyl amphiphiles between lipid and protein surfaces.

The kinetics of transfer of natural and fluorescent nonesterified fatty acids (NEFA) and lysolecithins (lysoPC) from phospholipid and protein surfaces were measured. The kinetics of transfer of 12-(1-pyrenyl)dodecanoic acid, from liquid crystalline and gel phase single unilamellar phospholipid vesicles, very low, low, and high density lipoproteins, human serum albumin, and rat liver fatty acid-binding protein, were first-order and characterized by similar rate constants. The halftimes (t1/2) of NEFA transfer from lipids and proteins were dependent on the acyl chain structure according to log t1/2 = -0.62n + 0.59m + 12.0, where n and m, respectively, are the numbers of carbon atoms and double bonds. The structure of the donor surface had a measurable but smaller effect on transfer rates. The kinetics of NEFA and lysoPC transfer are slow relative to the lipolytic processes that liberate them. Therefore, one would predict a transient accumulation of NEFA and lysoPC during lipolysis and an attendant modulation of many metabolic processes within living cells and within the plasma compartment of blood. These data will be useful in the refinement of current models of membrane and lipoprotein function and in the selection of fluorescent NEFA analogs for studying transport in living cells.     

Fluorescence microscopy and 3D image reconstruction of cytokine initiated disruption of the Parkinson disease associated proteins α-synuclein, tau and ubiquitin in cultured glial cells

Human derived glioblastoma cells were cultured and treated with cytokines interleukin-6 (IL6), tumor necrosis factor alpha (TNF) and interferon-gamma (IFN) and imaged by fluorescence deconvolution microscopy to localize α-synuclein, tau and ubiquitin. Exposures were for short (2 h) and prolonged times (up to 96 h), with doses at both low (10 ng/ml), and high (100 ng/ml) concentrations. Further experiments used additive doses up to 200 ng/ml (2 × 100 ng), mimicking a super-infection state. Single, low doses of the cytokines initiated changes in levels of intracellular proteins, but these changes, be they increases or decreases, were not sustained, so we added higher doses of cytokine to the culture medium or fresh aliquots of cytokines over time. Finally, we treated cells with high, single doses of cytokine (200 ng/ml), to try to sustain perturbations of the proteins with cytokines. IFN caused a disruption and reduction of peripheral synuclein, TNF treatment resulted in increased levels of ubiquitin and IL6 disrupted and appeared to fragment tau. Of note, each of the proteins was found in a specific locale, tau being perinuclear, ubiquitin residing in the cytoplasm, and α-synuclein occupying the tips of cellular processes, exhibiting the characteristics of an adhesion protein/molecule [Word count = 198].