Roles of adroxazine, a new heterocyclic hormone of the adrenals, and leucogenenol, a thymothyroid hormone, in the formation of blood cells

Treatment of normal rats with the thymothyroid hormone leucogenenol accelerates the rate at which the 'functional' cells, neutrophils, red blood cells and lymphocytes, develop in the bone marrow from their corresponding committed precursor cells. Following the initial treatment of normal rats with leucogenenol, there is a temporary elevation in the bone marrow of the relative concentrations of myeloblasts, rubriblasts and lymphoblasts. It has now been found that bilaterally adrenalectomized rats have a different response from normal rats to treatment with leucogenenol. Although treatment of bilaterally adrenalectomized rats with leucogenenol accelerates the rate of development of their functional cells, the initial injection of leucogenenol is followed by a temporary decrease in the relative concentrations of myeloblasts, rubriblasts and lymphoblasts in their bone marrow. Additionally, adrenalectomized rats treated concurrently with tritiated thymidine and leucogenenol show a significant lower percentage of labeled cells in their bone marrow than do correspondingly treated normal rats. These results indicate that adrenalectomized rats have a lower than normal concentration of stem cells in their bone marrow that can be committed to become functional cells. Treatment with adroxazine, a new recently isolated heterocyclic hormone of the adrenals, causes adrenalectomized rats to respond as normal rats to the injection of leucogenenol. There is a temporary elevation of myeloblasts, rubriblasts and lymphoblasts in their bone marrow as well as a normal increase in the percentage of cells that are labeled following concurrent treatment with tritiated thymidine and leucogenenol. It may be concluded that treatment with adroxazine increases the rate of replication of uncommitted bone marrow stem cells while treatment with leucogenenol increases the rate at which committed cells develop into their respective functional cells. A scheme is presented to show the suggested roles that leucogenenol and adroxazine play in regulating the formation of blood cells.     

Human milk-fat globule membrane derived mucin is a disulfide-linked heteromer

The human milk-fat globule membrane (HMFG) contains a number of antigens also expressed on breast tumors. The dominant antigens are a mucin of MW greater than 400,000 and a group of antigens of MW 67,000-70,000. The mucin was separated with monoclonal antibodies against HMFG and a MW 70,000 doublet was found to be associated with the mucin under non-reducing conditions, the latter identifiable by another set of monoclonal antibodies. Immunoprecipitation of the mucin and analysis of the precipitated material using Western blots and identification of transferred material with monoclonal antibodies demonstrated that the MW 70,000 protein and the mucin were co-precipitated and linked by reducible disulfide bonds. Treatment with detergents, protein-dissociating agents and glycosidases did not release the component from the mucin. Mucins in HMFG membrane and breast tumor cells may be associated therefore to disulfide-linked linker proteins similar to those described for intestinal and gastric mucins, that would co-purify with the mucin under non-reducing conditions.     

Adroxazine, a hormone of the adrenals that stimulates the rate of replication of bone marrow stem cells

Treatment of normal animals with the thymothyroid hormone, leucogenenol, increases the rate at which appropriate committed precursor cells of the bone marrow develop sequentially into their corresponding functional cells: neutrophils, lymphocytes and red blood cells. Hence following treatment with leucogenenol, at a time that is dependent on the quantity of leucogenenol injected, there is a temporary increase in the bone marrow of the relative concentrations of early forms of committed cells such as myeloblasts. However, following treatment of bilaterally adrenalectomized rats with leucogenenol the early forms of committed cells in the bone marrow, such as myeloblasts, instead of showing a temporary relative increase show a temporary significant decrease in concentration. A new compound, adroxazine, C34H65NO2, was isolated from the methanol extract of bovine adrenal tissue. This compound, on injection into bilaterally adrenalectomized rats, causes leucogenenol to have the same effect on the development of their bone marrow cells as it does in normal rats. Adroxazine is present in the cortex of the adrenals and in blood serum. It is suggested that the population of primitive replicating cells is controlled by the concentration of adroxazine in the serum.     

Effect of leucogenenol,a thymothyroid hormone,on the enzymes and several other constituents of the serum

It has been found that rats treated with the thymothyroid hormone, leucogenenol, have significantly elevated levels of alkaline phosphatase and lactate dehydrogenase in their serum 8 hrs. later. These levels remain elevated for 24 hrs. and 12 hrs., respectively. The concentrations of creatine phosphokinase and aspartate aminotransferase as well as total protein, albumin, calcium, inorganic phosphorus, glucose, urea nitrogen, uric acid and creatinine in the serum are not affected by treatment with leucogenenol. These results are in agreement with the previously reported findings that treatment with leucogenenol increases the rate of development of blood cells of the bone marrow, increases the rate of recovery from immunosuppression and enhances the immune response.     

Tumor antigens of human ad5 in transformed cells and in cells infected with transformation-defective host-range mutants

We have studied the polypeptides associated with the expression of the transforming region of the Ad5 genome by immunoprecipitating antigens (using the double antibody and protein A-Sepharose techniques) from cells infected with wild-type (wt) Ad5 or transformation-defective host range (hr) mutants and from cells transformed by Ad5. Three different antisera were used: P antiserum specific for early viral products (Russell et al., 1967) and two different hamster tumor antisera. Immunoprecipitation of antigens from wt-infected KB cells followed by SDS-polyacrylamide gel electrophoresis of precipitated proteins revealed that a major polypeptide having a molecular weight of approximately 58,000 was detected with all three antisera and with both the double antibody and the protein A-Sepharose techniques, while P antiserum also precipitated polypeptides of molecular weights 72,000, 67,000 and 44,000, which probably represent the DNA binding protein and related polypeptides, respectively. With the double antibody technique, in addition to the proteins mentioned above, P antiserum and the hamster tumor antisera precipitated a 10,500 dalton polypeptide which was not detected when the protein A-Sepharose procedure was used. Using either the double antibody or the protein A-Sepharose technique, we found that hr mutants from complementation group II failed to induce the synthesis of the 58,000 dalton protein, whereas mutants from complementation group I produced normal or near normal amounts. Using the double antibody technique, we found that the 10,500 dalton protein was absent or made in reduced amounts by group I mutants. A 58,000 dalton protein was detected in a number of different Ad5-transformed cell lines, including the 293 human line, the 14b hamster line and several transformed rat cell lines. This observation and the fact that transformation negative group II mutants fail to induce the synthesis of a 58,000 dalton polypeptide suggest that this protein is one of the Ad5-specific products necessary for cell transformation.     

Studies of the action of leucogenenol on the myeloid and lymphoid tissues of the sublethally irradiated mouse

Studies of the action of leucogenenol on the peripheral leukocytes, the myeloid cells found in bone marrow, and the lymphoid cells found in the spleen of sublethally irradiated mice strongly suggest that leucogenenol stimulates the maturation and/or cellular division of cells of both the myeloid and lymphoid series. Accordingly, as indicated by the increase in the number of peripheral leukocytes, as well as the increase in the number of myeloid and lymphoid cells found in the bone marrow and spleen, mice treated with leucogenenol appear to recover more rapidly than untreated controls.     

Cold acclimation and oxygen consumption in the thymus

Mitochondrial uncoupling protein 1 is usually associated with brown adipose tissue but has recently been discovered in rat and mouse thymus. We wished to establish whether there was a thermogenic role for UCP 1 in thymus and thus examined the effect of 5 weeks cold-acclimation on rat thymus tissue abundance, thymocyte oxygen consumption, thymus mitochondrial abundance, uncoupling protein 1 expression and function. We found that thymocytes from cold-acclimated rats had oxygen consumption rates 8 times less than those from rats held at room temperature and that thymocytes from cold-acclimated rats or rats kept at room temperature were noradrenaline insensitive. In addition, we found that thymus tissue or mitochondrial abundance was not increased after cold-acclimation. However uncoupling protein 1 expression per unit mass of mitochondria was increased after cold-acclimation, as determined by immunoblotting (approximately 1.7-fold) and GDP binding (approximately 1.5-fold). Consistent with our protein expression data, we also observed an increased, state 4 (approximately 1.5-fold), GDP-inhibitable (approximately 1.3-fold) and palmitate activatable (approximately 1.6-fold) oxygen consumption rates in isolated thymus mitochondria. However, extrapolation of our data showed that cold-acclimation only increased the amount of UCP 1 per gram of thymus tissue approximately 1.2-fold. Taken together, we conclude that UCP 1 does not have a thermogenic role in thymus.     

Interactions of Cbl with Grb2 and phosphatidylinositol 3'-kinase in activated Jurkat cells.

T-cell receptor (TCR) cross-linking increases tyrosine phosphorylation of multiple proteins, only a few of which have been identified. One of the most rapidly tyrosine-phosphorylated polypeptides is the 120-kDa product of the proto-oncogene c-cbl, a cytosolic and cytoskeletal protein containing multiple proline-rich motifs that are potential binding sites for proteins containing Src homology 3 (SH3) domains. We report here that in cultured Jurkat T cells, Cbl is coprecipitated with antibody against the adapter protein Grb2. Upon activation of Jurkat T cells via the TCR-CD3 complex, we find that high-affinity binding of Cbl requires the N-terminal SH3 domain of GST-Grb2 fusion protein but after cross-linking of the TCR-CD3 and CD4 receptors, Cbl binds equally to its SH2 domain. Grb2 antisera also precipitated p85 from serum-starved cells, while TCR activation increased p85 and tyrosine-phosphorylated Cbl but not Cbl protein in Grb2 immunocomplexes. Phosphatidylinositol (PI) 3-kinase activity was immunoprecipitated from serum-starved cells with Cbl and to a lesser extent with Grb2 antisera, and TCR cross-linking increased this activity severalfold. The PI 3-kinase activity associated with Cbl amounted to 5 to 10% of the total cellular activity that could be precipitated by p85 antisera. The Ras exchange factor Son-of-sevenless 1 (Sos-1) was not found in anti-Cbl immunoprecipitates from activated cells, and Cbl was not detectable in anti-Sos-1 precipitates, supporting the likelihood that Sos-Grb2 and Cbl-Grb2 are present as distinct complexes. Taken together, these data suggest that Cbl function in Jurkat T cells involves its constitutive association with Grb2 and its recruitment of PI 3-kinase in response to TCR activation.     

Identification and characterization of thymus LIM protein: targeted disruption reduces thymus cellularity.

We have identified a novel LIM gene encoding the thymus LIM protein (TLP), expressed specifically in the thymus in a subset of cortical epithelial cells. TLP was identified as a gene product which is upregulated in a thymus in which selection of T cells is occurring (Rag(-/-) OT-1) compared to its expression in a thymus in which selection is blocked at the CD4+ CD8+ stage of T-cell development (Rag(-/-) Tap(-/-) OT-1). TLP has an apparent molecular mass of 23 kDa and exists as two isomers (TLP-A and TLP-B), which are generated by alternative splicing of the message. The sequences of TLP-A and TLP-B are identical except for the C-terminal 19 or 20 amino acids. Based on protein sequence alignment, TLP is most closely related to the cysteine-rich proteins, a subclass of the family of LIM-only proteins. In both medullary and cortical thymic epithelial cell lines transduced with TLP, the protein localizes to the cytoplasm but does not appear to be strongly associated with actin. In immunohistochemical studies, TLP seems to be localized in a subset of epithelial cells in the cortex and is most abundant near the corticomedullary junction. We generated mice with a targeted disruption of the Tlp locus. In the absence of TLP, thymocyte development and thymus architecture appear to be normal but thymocyte cellularity is reduced by approximately 30%, with a proportional reduction in each subpopulation.     

RGS-GAIP, a GTPase-activating Protein for Gαi Heterotrimeric G Proteins, Is Located on Clathrin-coated Vesicles

RGS-GAIP (Gα-interacting protein) is a member of the RGS (regulator of G protein signaling) family of proteins that functions to down-regulate Gα_i/Gα_q-linked signaling. GAIP is a GAP or guanosine triphosphatase-activating protein that was initially discovered by virtue of its ability to bind to the heterotrimeric G protein Gα_i3, which is found on both the plasma membrane (PM) and Golgi membranes. Previously, we demonstrated that, in contrast to most other GAPs, GAIP is membrane anchored and palmitoylated. In this work we used cell fractionation and immunocytochemistry to determine with what particular membranes GAIP is associated. In pituitary cells we found that GAIP fractionated with intracellular membranes, not the PM; by immunogold labeling GAIP was found on clathrin-coated buds or vesicles (CCVs) in the Golgi region. In rat liver GAIP was concentrated in vesicular carrier fractions; it was not found in either Golgi- or PM-enriched fractions. By immunogold labeling it was detected on clathrin-coated pits or CCVs located near the sinusoidal PM. These results suggest that GAIP may be associated with both TGN-derived and PM-derived CCVs. GAIP represents the first GAP found on CCVs or any other intracellular membranes. The presence of GAIP on CCVs suggests a model whereby a GAP is separated in space from its target G protein with the two coming into contact at the time of vesicle fusion.     

Biosynthesis of rat insulin-like growth factor II. I. Immunochemical demonstration of a approximately 20-kilodalton biosynthetic precursor of rat insulin-like growth factor II in metabolically labeled BRL-3A rat liver cells

BRL-3A rat liver cells synthesize mature 7484-dalton rat insulin-like growth factor II (rIGF-II) as a approximately 22-kDa precursor, presumably prepro-rIGF-II. In the present study, we have biosynthetically labeled intact BRL-3A cells with [35S]cysteine and immunoprecipitated cell lysates and media with antisera to rIGF-II. A approximately 20-kDa protein was identified in immunoprecipitates of cell lysates having properties consistent with pro-rIGF-II. The approximately 20-kDa protein is precipitated by immune sera but not by nonimmune serum. Its immunoprecipitation is specifically inhibited by unlabeled rIGF-II but not by insulin. It is not precipitated from labeled lysates of a subclone of BRL-3A cells (BRL-3A2) that does not synthesize rIGF-II. The approximately 20-kDa protein is rapidly labeled intracellularly (10 min) but is not detected in BRL-3A media. In pulse-chase experiments, radioactivity in the approximately 20-kDa protein disappears during the chase and appears, at later times, in specifically immunoprecipitated approximately 19-, approximately 10-, approximately 8-, and approximately 7-kDa proteins in media and, to a limited extent, intracellularly. A protein with electrophoretic mobility identical to that of the approximately 20-kDa protein observed in cell lysates is immunoprecipitated from 35S-proteins whose synthesis is directed by BRL-3A RNA in a reticulocyte lysate cell-free translation system supplemented with microsomal membranes, and presumably arises by cotranslational removal of the signal peptide from approximately 22-kDa prepro-rIGF-II. Processing of the approximately 20-kDa protein in intact BRL-3A cells to intermediate and mature rIGF-II species appears to occur at the time of secretion and/or shortly thereafter, with the different forms appearing at approximately the same time.     

Relationship of bursal anti-steroidogenic peptide (BASP) and histone H1

Previous in vitro research from our laboratory has demonstrated the existence of a protein purified from the chicken bursa of Fabricius, with potent antisteroidogenic and antiproliferative action on granulose cells and lymphocytes, respectively called Bursal anti-steroidogenic peptide (BASP). This protein is heat-labile, basic, and amino- and carboxy-terminus blocked. In highly purified form, the protein presents as a doublet on SDS-PAGE electrophoresis with an apparent MW of approximately 29 and approximately 32 kDa. Recently, Nanoflow Q-TOF Mass Spectrometry amino acid sequencing allowed determination of a convincing partial amino acid sequence, strongly suggesting a probable relationship of BASP with histone H1. Bursal cDNA expression library screening, using an antibody produced against BASP, also identified a clone with a sequence matching histone H1. Presently, we have demonstrated that SDS-PAGE electrophoresis of highly purified and bioactive BASP, and commercially-available calf thymus derived histone H1, produced similar doublets at approximately the same apparent MW, and that the electrophoretic profile of these 2 preparations were strikingly similar following 2 dimensional gel electrophoresis. The BASP doublet produced on SDS-PAGE was recognized by a commercially available monoclonal antibody recognizing a highly conserved region of histone H1. Furthermore, calf thymus histone H1 was found to suppress mitogen-stimulated chicken B-cell proliferation in a concentration-related manner, similar to the action of BASP. These data indicate that BASP shares substantial structural homology with, and may be identical to, histone H1.     

Cloning,expression, characterization,and interaction of two components of a human mitochondrial fatty acid synthase. Malonyltransferase and acyl carrier protein

The possibility that human cells contain, in addition to the cytosolic type I fatty acid synthase complex, a mitochondrial type II malonyl-CoA-dependent system for the biosynthesis of fatty acids has been examined by cloning, expressing, and characterizing two putative components. Candidate coding sequences for a malonyl-CoA:acyl carrier protein transacylase (malonyltransferase) and its acyl carrier protein substrate, identified by BLAST searches of the human sequence data base, were located on nuclear chromosomes 22 and 16, respectively. The encoded proteins localized exclusively in mitochondria only when the putative N-terminal mitochondrial targeting sequences were present as revealed by confocal microscopy of HeLa cells infected with appropriate green fluorescent protein fusion constructs. The mature, processed forms of the mitochondrial proteins were expressed in Sf9 cells and purified, the acyl carrier protein was converted to the holoform in vitro using purified human phosphopantetheinyltransferase, and the functional interaction of the two proteins was studied. Compared with the dual specificity malonyl/acetyltransferase component of the cytosolic type I fatty acid synthase, the type II mitochondrial counterpart exhibits a relatively narrow substrate specificity for both the acyl donor and acyl carrier protein acceptor. Thus, it forms a covalent acyl-enzyme complex only when incubated with malonyl-CoA and transfers exclusively malonyl moieties to the mitochondrial holoacyl carrier protein. The type II acyl carrier protein from Bacillus subtilis, but not the acyl carrier protein derived from the human cytosolic type I fatty acid synthase, can also function as an acceptor for the mitochondrial transferase. These data provide compelling evidence that human mitochondria contain a malonyl-CoA/acyl carrier protein-dependent fatty acid synthase system, distinct from the type I cytosolic fatty acid synthase, that resembles the type II system present in prokaryotes and plastids. The final products of this system, yet to be identified, may play an important role in mitochondrial function.     

Proteomic identification of interactions between histones and plasma proteins: Implications for cytoprotection

Extracellular histones released from cells during acute inflammation contribute to organ failure and death in a mouse model of sepsis, and histones are known to exert in vitro cytotoxicity in the absence of serum. Since addition of histones to serum and plasma is known to induce protein aggregation, we reasoned that plasma proteins may afford protection from cytotoxicity. We found that MODE‐K mouse small intestinal epithelial cells were protected from histone‐induced toxicity in the presence of 10% FCS. Therefore, the main aim of this study was to identify histone‐interacting plasma proteins that might be involved in cytoprotection. The precipitate formed following addition of calf thymus histones to human EDTA plasma was characterised by shotgun proteomics, identifying a total of 36 protein subunits, including complement components, coagulation factors, protease inhibitors and apolipoproteins. The highly sulphated glycosaminoglycan heparin inhibited histone‐induced plasma protein aggregation. Moreover, histones bound to heparin agarose were capable of pulling down plasma proteins from solution, indicating their effective cross‐linking properties. It was particularly notable that inter‐α‐trypsin inhibitor was prominent among the histone‐precipitated proteins, since it contains a chondroitin sulphate glycan chain, and suggests a potential role for this protein in histone sequestration during acute inflammation in vivo.     

Precipitation of sword bean proteins by heating and addition of magnesium chloride in a crude extract

Sword bean (Canavalia gladiata) seeds are a traditional food in Asian countries. In this study, we aimed to determine the optimal methods for the precipitation of sword bean proteins useful for the food development. The soaking time for sword beans was determined by comparing it with that for soybeans. Sword bean proteins were extracted from dried seeds in distilled water using novel methods. We found that most proteins could be precipitated by heating the extract at more than 90 °C. Interestingly, adding magnesium chloride to the extract at lower temperatures induced specific precipitation of a single protein with a molecular weight of approximately 48 kDa. The molecular weight and N-terminal sequence of the precipitated protein was identical to that of canavalin. These data suggested that canavalin was precipitated by the addition of magnesium chloride to the extract. Our results provide important insights into the production of processed foods from sword bean.     

Functional switching of a novel prokaryotic 2-Cys peroxiredoxin (PpPrx) under oxidative stress

Many proteins have been isolated from eukaryotes as redox-sensitive proteins, but whether these proteins are present in prokaryotes is not clear. Redox-sensitive proteins contain disulfide bonds, and their enzymatic activity is modulated by redox in vivo. In the present study, we used thiol affinity purification and mass spectrometry to isolate and identify 19 disulfide-bond-containing proteins in Pseudomonas putida exposed to potential oxidative damages. Among these proteins, we found that a typical 2-Cys Prx-like protein (designated PpPrx) displays diversity in structure and apparent molecular weight (MW) and can act as both a peroxidase and a molecular chaperone. We also identified a regulatory factor involved in this structural and functional switching. Exposure of pseudomonads to hydrogen peroxide (H₂O₂) caused the protein structures of PpPrx to convert from high MW complexes to low MW forms, triggering a chaperone-to-peroxidase functional switch. This structural switching was primarily guided by the thioredoxin system. Thus, the peroxidase efficiency of PpPrx is clearly associated with its ability to form distinct protein structures in response to stress.     

Heparin modulates the secretion of a major excreted protein-like molecule by vascular smooth muscle cells

Previous work from our laboratory has shown that heparin specifically induces the release of a pair of proteins of approximately 35,000 and 37,000 Da into the culture medium of vascular smooth muscle cells (SMC). In this report, we demonstrate that the previously identified 37,000-Da smooth muscle protein is composed of two protein species with very similar molecular weights based on migration patterns in SDS-polyacrylamide gels. The larger molecular weight species in this doublet has a similar molecular weight and shares antigenic determinants with major excreted protein (MEP), a lysosomal proteinase previously shown to be secreted by normal and transformed fibroblasts and epidermal cells. Antisera to MEP precipitated the higher molecular weight band from the doublet; preimmune serum was not reactive with the smooth muscle protein. Exposure of smooth muscle cells to heparin resulted in decreased amounts of immunoprecipitable protein released into the medium. Thus, it now appears that three proteins in the 35,000-38,000 molecular weight range are modulated by heparin, and that the largest of the heparin-modulated vascular SMC proteins has a similar molecular weight and is immunologically related to MEP. The release of MEP-like protein from SMC is decreased by heparin, while the remaining two heparin-modulated proteins are increased in the presence of heparin.     

Chemical determination of leucogenenol and its production by Penicillium gilmanii

A method for the quantitative determination of leucogenenol, a leucocytosis-inducing compound produced by Penicillium gilmanii, has been developed. The formation of leucogenenol in P. gilmanii and its diffusion into the culture fluid has been followed over a period of 12 weeks. The highest concentration of leucogenenol was found in the mycelium of rapidly growing cultures. The greatest concentration occurred in the culture fluid, however, after cessation of rapid growth. The concentration of leucogenenol in the mycelium of cultures over 4 weeks old fluctuated widely. This fluctuation was apparently due to the formation and rapid growth of daughter colonies.     

Detection of an interaction between the HN and F proteins in Newcastle disease virus-infected cells.

For many paramyxoviruses, including Newcastle disease virus (NDV), syncytium formation requires the expression of both surface glycoproteins (HN and F) in the same cell, and evidence suggests that fusion involves a specific interaction between the HN and F proteins. Because a potential interaction in paramyxovirus-infected cells has never been demonstrated, such as interaction was explored by using coimmunoprecipitation and cross-linking. Both HN and F proteins could be precipitated with heterologous antisera after a 5-min radioactive pulse as well as after a 2-h chase in nonradioactive medium, but at low levels. Chemical cross-linking increased detection of complexes containing HN and F proteins at the cell surface. After cross-linking, intermediate- as well as high-molecular-weight species containing both proteins were precipitated with monospecific antisera. Precipitation of proteins with anti-HN after cross-linking resulted in the detection of complexes which electrophresed in the stacker region of the gel, from 160 to 300 kDa, at 150 kDa, and at 74 kDa. Precipitates obtained with anti-F after cross-linking contained species which migrated in the stacker region of the gel, between 160 and 300 kDa, at 120 kDa, and at 66 kDa. The three to four discrete complexes ranging in size from 160 to 300 kDa contained both HN and F proteins when precipitated with either HN or F antisera. That cross-linking of complexes containing both HN and F proteins was not simply a function of overexpression of viral glycoproteins at the cell surface was addressed by demonstrating cross-linking at early time points postinfection, when levels of viral surface glycoproteins are low. Use of cells infected with an avirulent strain of NDV showed that chemically cross-linked HN and F proteins were precipitated independent of cleavage of F0. Furthermore, under conditions that maximized HN protein binding to its receptor, there was no change in the percentages of HN and F0 proteins precipitated with heterologous antisera, but a decrease in F1 protein precipitated was observed upon attachment. These data argue that the HN and F proteins interact in the rough endoplasmic reticulum. Upon attachment of the HN protein to its receptor, the HN protein undergoes a conformational change which causes a conformational change in the associated F protein, releasing the hydrophobic fusion peptide into the target membrane and initiating fusion.