Low Molecular Mass Form Research Articles

Overoxidation and Oligomerization of Trypanosoma cruzi Cytosolic and Mitochondrial Peroxiredoxins.

Peroxiredoxins (Prxs) have been shown to be important enzymes for trypanosomatids, counteracting oxidative stress and promoting cell infection and intracellular survival. In this work, we investigate the in vitro sensitivity to overoxidation and the overoxidation dynamics of Trypanosoma cruzi Prxs in parasites in culture and in the infection context. We showed that recombinant m-TXNPx, in contrast to what was observed for c-TXNPx, exists as low molecular mass forms in the overoxidized state. We observed that T. cruzi Prxs were overoxidized in epimastigotes treated with oxidants, and a significant proportion of the overoxidized forms were still present at least 24 h after treatment suggesting that these forms are not actively reversed. In in vitro infection experiments, we observed that Prxs are overoxidized in amastigotes residing in infected macrophages, demonstrating that inactivation of at least part of the Prxs by overoxidation occurs in a physiological context. We have shown that m-TXNPx has a redox-state-dependent chaperone activity. This function may be related to the increased thermotolerance observed in m-TXNPx-overexpressing parasites. This study suggests that despite the similarity between protozoan and mammalian Prxs, T. cruzi Prxs have different oligomerization dynamics and sensitivities to overoxidation, which may have implications for their function in the parasite life cycle and infection process.

Different Molecular Forms of TFF3 in the Human Respiratory Tract: Heterodimerization with IgG Fc Binding Protein (FCGBP) and Proteolytic Cleavage in Bronchial Secretions

The polypeptide TFF3 belongs to the trefoil factor family (TFF) of lectins. TFF3 is typically secreted from mucous epithelia together with mucins. Both intestinal and salivary TFF3 mainly exist as disulfide-linked heterodimers with IgG Fc binding protein (FCGBP). Here, we investigated bronchial tissue specimens, bronchial secretions, and bronchoalveolar lavage (BAL) fluid from patients with a chronic obstructive pulmonary disease (COPD) background by fast protein liquid chromatography and proteomics. For the first time, we identified different molecular forms of TFF3 in the lung. The high-molecular mass form represents TFF3-FCGBP oligomers, whereas the low-molecular mass forms are homodimeric and monomeric TFF3 with possibly anti-apoptotic activities. In addition, disulfide-linked TFF3 heterodimers with an Mr of about 60k and 30k were detected in both bronchial secretions and BAL fluid. In these liquids, TFF3 is partly N-terminally truncated probably by neutrophil elastase cleavage. TFF3-FCGBP is likely involved in the mucosal innate immune defense against microbial infections. We discuss a hypothetical model how TFF3 might control FCGBP oligomerization. Furthermore, we did not find indications for interactions of TFF3-FCGBP with DMBT1gp340 or the mucin MUC5AC, glycoproteins involved in mucosal innate immunity. Surprisingly, bronchial MUC5AC appeared to be degraded when compared with gastric MUC5AC.

Hyaluronan primes the oxidative burst in human neutrophils.

Hyaluronan (HA) is a glycosaminoglycan that in its natural, high molecular mass (HMM) form, promotes tissue repair and homeostasis. With inflammation, HA metabolism and HMM HA fragmentation to low molecular mass (LMM) forms is greatly enhanced. Considerable evidence suggests that LMM HA may act as a damage-associated molecular pattern to initiate innate immune responses. However, the responsiveness of myeloid cells to LMM HA is controversial and largely unknown for neutrophils. Peripheral blood cells from healthy donors were incubated ex vivo with pharmaceutical grade HA of different molecular mass (HMM, LMM, and HA fragments<10kDa). Key innate immune functions were assessed, namely production of cytokines and reactive oxygen species release (ROS), granule mobilization, and apoptosis. None of the tested sizes of HA altered cytokine production by PBMC and neutrophils. Also, HA had no effect on neutrophil granule mobilization and apoptosis. In contrast, HA primed neutrophils for rapid and robust release of ROS in response to a secondary stimulus (N-formyl-methionyl-leucyl phenylalanine). Priming occurred within 20min of exposure to HA and was similar for all tested molecular mass. The observed effect was independent of granule mobilization and associated with the activation of intracellular signaling pathways involving Src family kinases, glycogen synthase kinase-3, and the proline-rich Akt substrate of 40kDa. Our findings provide new evidence that HA, irrespective of molecular mass, is a specific priming agent of the neutrophil oxidative burst, which is a critical, early component of an innate immune response.

Molecular profile of mannan-binding lectin in hepatitis C patients with MBL gene polymorphisms by a modified mannan-coated nitrocellulose assay

The aim of this study was to develop an assay to analyze the serum profile of Mannose-binding lectin (MBL) through a simple and “in-house” method (called “dot-N-man”). Furthermore, the study attempted to associate molecular masses of MBL to the profile of MBL gene polymorphisms in patients with hepatitis C. Heterogeneity in molecular masses of MBL is due to the impairment of oligomers formation, which is linked to genetic polymorphisms in the MBL gene. Individuals with AA genotype (wild-type) produce high-molecular-mass proteins, whereas AO and OO individuals produce intermediate and low-molecular-mass proteins, respectively. Sera of thirty patients carrying the hepatitis C virus (HCV) were investigated using MBL binding assay with mannan-coated nitrocellulose (dot-N-man). Purified MBL was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Dot-N-Man assay yielded MBL with molecular masses ranging between 55 and 320 kDa, comparable to low and high molecular mass forms of MBL. Nonreducing SDS-PAGE showed high molecular mass bands in all AA individuals while bands of 270 and 205 kDa were observed in sera for a number of patients with AO and OO genotypes, respectively. Immunoblotting confirmed the MBL samples obtained from the dot-N-man. These results provide new insights to understand the MBL molecular forms profile in patients infected with HCV- which could be useful in future investigations on the influence of the MBL structure/genotype on both the progression of infection and the response to hepatitis C therapy.

The where, when, how, and why of hyaluronan binding by immune cells.

Hyaluronan is made and extruded from cells to form a pericellular or extracellular matrix (ECM) and is present in virtually all tissues in the body. The size and form of hyaluronan present in tissues are indicative of a healthy or inflamed tissue, and the interactions of hyaluronan with immune cells can influence their response. Thus, in order to understand how inflammation is regulated, it is necessary to understand these interactions and their consequences. Although there is a large turnover of hyaluronan in our bodies, the large molecular mass form of hyaluronan predominates in healthy tissues. Upon tissue damage and/or infection, the ECM and hyaluronan are broken down and an inflammatory response ensues. As inflammation is resolved, the ECM is restored, and high molecular mass hyaluronan predominates again. Immune cells encounter hyaluronan in the tissues and lymphoid organs and respond differently to high and low molecular mass forms. Immune cells differ in their ability to bind hyaluronan and this can vary with the cell type and their activation state. For example, peritoneal macrophages do not bind soluble hyaluronan but can be induced to bind after exposure to inflammatory stimuli. Likewise, naïve T cells, which typically express low levels of the hyaluronan receptor, CD44, do not bind hyaluronan until they undergo antigen-stimulated T cell proliferation and upregulate CD44. Despite substantial knowledge of where and when immune cells bind hyaluronan, why immune cells bind hyaluronan remains a major outstanding question. Here, we review what is currently known about the interactions of hyaluronan with immune cells in both healthy and inflamed tissues and discuss how hyaluronan binding by immune cells influences the inflammatory response.

Subcellular Trafficking and Activity of Hyal‐1 and Its Processed Forms in Murine Macrophages

The hyaluronidase Hyal-1 is an acid hydrolase that degrades hyaluronic acid (HA), a component of the extracellular matrix. It is often designated as a lysosomal protein. Yet few data are available on its intracellular localization and trafficking. We demonstrate here that in RAW264.7 murine macrophages, Hyal-1 is synthesized as a glycosylated precursor that is only weakly mannose 6-phosphorylated. Nevertheless, this precursor traffics to endosomes, via a mannose 6-phosphate-independent secretion/recapture mechanism that involves the mannose receptor. Once in endosomes, it is processed into a lower molecular mass form that is transported to lysosomes, where its activity could be detected using native gel zymography. Indeed, this activity co-distributed with lysosomal hydrolases in the densest fraction of a self-forming Percoll(TM) density gradient. Moreover, it shifted toward the lower density region, in parallel with those hydrolases, when a decrease of lysosomal density was induced by the endocytosis of sucrose. Interestingly, the activity of the processed form of Hyal-1 was largely underestimated when assayed by zymography after SDS-PAGE and subsequent renaturation of the proteins, by contrast to the full-length protein that could efficiently degrade HA in those conditions. These results suggest that noncovalent associations support the lysosomal activity of Hyal-1.

The Roles of APOBEC3G Complexes in the Incorporation of APOBEC3G into HIV-1

BackgroundThe incorporation of human APOBEC3G (hA3G) into HIV is required for exerting its antiviral activity, therefore the mechanism underlying hA3G virion encapsidation has been investigated extensively. hA3G was shown to form low-molecular-mass (LMM) and high-molecular-mass (HMM) complexes. The function of different forms of hA3G in its viral incorporation remains unclear.Methodology/Principal FindingsIn this study, we investigated the subcellular distribution and lipid raft association of hA3G using subcellular fractionation, membrane floatation assay and pulse-chase radiolabeling experiments respectively, and studied the correlation between the ability of hA3G to form the different complex and its viral incorporation. Our work herein provides evidence that the majority of newly-synthesized hA3G interacts with membrane lipid raft domains to form Lipid raft-associated hA3G (RA hA3G), which serve as the precursor of mature HMM hA3G complex, while a minority of newly-synthesized hA3G remains in the cytoplasm as a soluble LMM form. The distribution of hA3G among the soluble LMM form, the RA LMM form and the mature forms of HMM is regulated by a mechanism involving the N-terminal part of the linker region and the C-terminus of hA3G. Mutagenesis studies reveal a direct correlation between the ability of hA3G to form the RA LMM complex and its viral incorporation.Conclusions/SignificanceTogether these data suggest that the Lipid raft-associated LMM A3G complex functions as the cellular source of viral hA3G.

Extracellular aspartic protease SAP2 of Candida albicans yeast cleaves human kininogens and releases proinflammatory peptides, Met-Lys-bradykinin and des-Arg9-Met-Lys-bradykinin

Bradykinin-related peptides, universal mediators of inflammation collectively referred to as the kinins, are often produced in excessive amounts during microbial infections. We have recently shown that the yeast Candida albicans, the major fungal pathogen to humans, can exploit two mechanisms to enhance kinin levels at the sites of candidial infection, one depending on adsorption and activation of the endogenous kinin-generating system of the host on the fungal cell wall and the other relying on cleavage of kinin precursors, the kininogens, by pathogen-secreted proteases. This work aimed at assigning this kininogenase activity to the major secreted aspartic protease of C. albicans (SAP2). The purified SAP2 was shown to cleave human kininogens, preferably the low molecular mass form (LK) and optimally in an acidic environment (pH 3.5-4.0), and to produce two kinins, Met-Lys-bradykinin and its derivative, [Hydroxyproline(3)]-Met-Lys-bradykinin, both of which are capable of interacting with cellular bradykinin receptors of the B2 subtype. Additionally, albeit with a lower yield, des-Arg(9)-Met-Lys-bradykinin, an effective agonist of B1-subtype receptors, was released. The pathophysiological potential of these kinins and des-Arg-kinin was also proven by presenting their ability to stimulate human promonocytic cells U937 to release proinflammatory interleukin 1β (IL-1β) and IL-6.

Heparan Sulfate Chains of Syndecan-1 Regulate Ectodomain Shedding

Matrix metalloproteinases release intact syndecan-1 ectodomains from the cell surface giving rise to a soluble, shed form of the proteoglycan. Although it is known that shed syndecan-1 controls diverse pathophysiological responses in cancer, wound healing, inflammation, infection, and immunity, the mechanisms regulating shedding remain unclear. We have discovered that the heparan sulfate chains present on syndecan core proteins suppress shedding of the proteoglycan. Syndecan shedding is dramatically enhanced when the heparan sulfate chains are enzymatically degraded or absent from the core protein. Exogenous heparan sulfate or heparin does not inhibit shedding, indicating that heparan sulfate must be attached to the core protein to suppress shedding. Regulation of shedding by heparan sulfate occurs in multiple cell types, for both syndecan-1 and syndecan-4 and in murine and human syndecans. Mechanistically, the loss of heparan sulfate enhances the susceptibility of the core protein to proteolytic cleavage by matrix metalloproteinases. Enhanced shedding of syndecan-1 following loss of heparan sulfate is accompanied by a dramatic increase in core protein synthesis. This suggests that in response to an increase in the rate of shedding, cells attempt to maintain a significant level of syndecan-1 on the cell surface. Together these data indicate that the amount of heparan sulfate present on syndecan core proteins regulates both the rate of syndecan shedding and core protein synthesis. These findings assign new functions to heparan sulfate chains, thereby broadening our understanding of their physiological importance and implying that therapeutic inhibition of heparan sulfate degradation could impact the progression of some diseases.

1058 POSTER Adaptor Protein Ruk/CIN85 Expression in Human Tumours

Background: The characterization of molecular alterations in each single tumour is at the basis of personalized anticancer approaches aimed to give each patient the most appropriate therapy. Adaptor/scaffold proteins are the key components of signalling networks involved in the control of cell physiology. In particular, by binding to numerous effector proteins adaptor/scaffold protein Ruk/CIN85 assembles multimeric complexes implicated in control of receptor tyrosine kinase signalling, neuronal and T cells apoptosis, adhesion and invasion. Material and Methods: Using Western-blot analysis, samples of uterine cervix, stomach, skin, thyroid, brain tumours and adjacent normal tissues were analyzed. Results: Increase of Ruk/CIN85 full-length form (p85) expression was revealed in uterine cervix, stomach and skin cancer samples in comparison with corresponding control samples. Down-regulation of p85 was revealed in the majority of thyroid tumour samples in comparison with adjacent normal tissue samples. Using anti-RukS Western-blot analysis, multiple molecular forms of CIN85/Ruk with molecular weight of 140, 130, 85, 70, 56, 50, 40 and 38 kDa were detected. Changes in expression level of some Ruk/CIN85 multiple molecular forms as well as up-regulation of low-molecular mass forms (40 and 34 kDa) were detected in skin cancer samples. Expression level up-regulation of high-molecular (140 and 130 kDa) and low-molecular (40 and 34 kDa) Ruk/CIN85 multiple molecular forms of thyroid tumour, renal tumour and glioblastoma samples in comparison with conditionally normal tissue were revealed. The additional feature of Ruk/CIN85 forms expression patterns in uterine as well as stomach malignancies was the high content of p70, p40 and p34 forms while control tissue samples were characterized by the predominant increase of high molecular forms (p140, p130, p100). Conclusions: The obtained results suggest that changes in the expression level of multiple molecular forms of CIN85/Ruk in tumour samples can lead to the loss of coordinated control of apoptosis and proliferation in the transformed cells. These data will offer new opportunities for the identification and validation of key molecular tumour targets to be exploited for novel therapeutic approaches.

1059 POSTER Sensitizing Hemopoietic Malignant Cells to Glucocorticoid Induced Apoptosis by PK Inhibitors

Background: The characterization of molecular alterations in each single tumour is at the basis of personalized anticancer approaches aimed to give each patient the most appropriate therapy. Adaptor/scaffold proteins are the key components of signalling networks involved in the control of cell physiology. In particular, by binding to numerous effector proteins adaptor/scaffold protein Ruk/CIN85 assembles multimeric complexes implicated in control of receptor tyrosine kinase signalling, neuronal and T cells apoptosis, adhesion and invasion. Material and Methods: Using Western-blot analysis, samples of uterine cervix, stomach, skin, thyroid, brain tumours and adjacent normal tissues were analyzed. Results: Increase of Ruk/CIN85 full-length form (p85) expression was revealed in uterine cervix, stomach and skin cancer samples in comparison with corresponding control samples. Down-regulation of p85 was revealed in the majority of thyroid tumour samples in comparison with adjacent normal tissue samples. Using anti-RukS Western-blot analysis, multiple molecular forms of CIN85/Ruk with molecular weight of 140, 130, 85, 70, 56, 50, 40 and 38 kDa were detected. Changes in expression level of some Ruk/CIN85 multiple molecular forms as well as up-regulation of low-molecular mass forms (40 and 34 kDa) were detected in skin cancer samples. Expression level up-regulation of high-molecular (140 and 130 kDa) and low-molecular (40 and 34 kDa) Ruk/CIN85 multiple molecular forms of thyroid tumour, renal tumour and glioblastoma samples in comparison with conditionally normal tissue were revealed. The additional feature of Ruk/CIN85 forms expression patterns in uterine as well as stomach malignancies was the high content of p70, p40 and p34 forms while control tissue samples were characterized by the predominant increase of high molecular forms (p140, p130, p100). Conclusions: The obtained results suggest that changes in the expression level of multiple molecular forms of CIN85/Ruk in tumour samples can lead to the loss of coordinated control of apoptosis and proliferation in the transformed cells. These data will offer new opportunities for the identification and validation of key molecular tumour targets to be exploited for novel therapeutic approaches.

The cellular source for APOBEC3G's incorporation into HIV-1

BackgroundHuman APOBEC3G (hA3G) has been identified as a cellular inhibitor of HIV-1 infectivity. Viral incorporation of hA3G is an essential step for its antiviral activity. Although the mechanism underlying hA3G virion encapsidation has been investigated extensively, the cellular source of viral hA3G remains unclear.ResultsPrevious studies have shown that hA3G forms low-molecular-mass (LMM) and high-molecular-mass (HMM) complexes. Our work herein provides evidence that the majority of newly-synthesized hA3G interacts with membrane lipid raft domains to form Lipid raft-associated hA3G (RA hA3G), which serve as the precursor of the mature HMM hA3G complex, while a minority of newly-synthesized hA3G remains in the cytoplasm as a soluble LMM form. The distribution of hA3G among the soluble LMM form, the RA LMM form and the mature forms of HMM is regulated by a mechanism involving the N-terminal part of the linker region and the C-terminus of hA3G. Mutagenesis studies reveal a direct correlation between the ability of hA3G to form the RA LMM complex and its viral incorporation.ConclusionsTogether these data suggest that the Lipid raft-associated LMM A3G complex functions as the cellular source of viral hA3G.

Bioavailability of sediment-associated and low-molecular-mass species of radionuclides/trace metals to the mussel Mytilus edulis

Sediments can act as a sink for contaminants in effluents from industrial and nuclear installations or when released from dumped waste. However, contaminated sediments may also act as a potential source of radionuclides and trace metals to the water phase due to remobilisation of metals as dissolved species and resuspension of particles. The marine mussel Mytilus edulis is a filter-feeding organism that via the gills is subjected to contaminants in dissolved form and from contaminants associated to suspended particles via the digestive system. In this paper the bioavailability of sediment-associated and seawater diluted Cs, Co, Cd and Zn radioactive tracers to the filtering bivalve M. edulis has been examined. The mussels were exposed to tracers diluted in ultrafiltered (<10 kDa) seawater (Low Molecular Mass form) or to tracers associated with sediment particles from the Stepovogo Fjord at Novaya Zemlya in short-term uptake experiments, followed by 1-month depuration experiments in flow-through tanks. A toxicokinetic model was fitted to the uptake and depuration data, and the obtained parameters were used to simulate the significance of the two uptake pathways at different suspended sediment loads and sediment-seawater distribution coefficients. The results of the model simulations, assuming steady state conditions, suggest that resuspended particles from contaminated sediments can be a highly significant pathway for mussels in the order 109Cd ≌ 65Zn < 134Cs < 60Co. The significance increases with higher suspended sediment load and with higher K d. Furthermore, the experimental depuration data suggest that Cs is retained longer and Co, Cd and Zn shorter by the mussels when associated with ingested sediments, than if the metals are taken up from the low molecular mass (LMM) phase.

Molecular heterogeneity of urinary albumin in glomerulonephritis: Comparison of cardiovascular disease with albuminuria

Background Despite the unstable structure of urinary albumin in kidney diseases, urinary albumin fragments have been identified by denaturing methods such as two-dimensional electrophoresis. This study examined the relationship between the structural heterogeneity of urinary albumin and protease effects. Methods Urine samples from patients with glomerulonephritis (GN), cardiovascular diseases (CVD), and healthy subjects were analyzed by non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), Western blot, diagonal 2-dimensional non-reducing/reducing (d2D) SDS PAGE, and albumin zymography. Results The major band was monomer albumin in CVD and healthy subjects; however, 13 urinary albumin bands ranging from 55 to 172 kDa were identified by non-reducing SDS PAGE in GN. The results from d2D SDS PAGE showed urinary albumin polymerization between disulfide bridges, interactions with other proteins, and reduction induced degradation in GN patients. The results from albumin zymography showed that low-molecular mass forms of albumin did not necessarily correspond to high protease activity. Furthermore, concentrated healthy urine showed similar protease digestion as in GN without low-molecular mass of albumin. Conclusions The molecular alterations observed cannot be explained only by urinary proteases. The specific alteration of urinary albumin molecules in GN can be attributed to different mechanisms to CVD.

APOBEC3G: an intracellular centurion

The intrinsic antiretroviral factor APOBEC3G (A3G) is highly active against HIV-1 and other retroviruses. In different cell types, A3G is expressed in high-molecular-mass (HMM) RNA- protein complexes or low-molecular-mass (LMM) forms displaying different biological activities. In resting CD4 T cells, a LMM form of A3G potently restricts HIV-1 infection soon after virion entry. However, when T cells are activated, LMM A3G is recruited into HMM complexes that include Staufen-containing RNA granules. These complexes are probably nucleated by the induced expression of Alu/hY retroelement RNAs that accompany T-cell activation. HMM A3G sequesters these retroelement RNAs away from the nuclear long interspersed nuclear element-derived enzymes required for Alu/hY retrotransposition. Human immunodeficiency virus (HIV) exploits this 'window of opportunity' provided by the loss of LMM A3G in activated CD4 T cells to productively infect these cells. During HIV virion formation, newly synthesized LMM A3G is preferentially encapsidated but only under conditions where Vif is absent and thus not able to target A3G for proteasome-mediated degradation. Together, these findings highlight the discrete functions of the different forms of A3G. LMM A3G opposes the external threat posed by exogenous retroviruses, while HMM A3G complexes oppose the internal threat posed by the retrotransposition of select types of retroelements.

APOBEC3G and HIV-1: Strike and counterstrike

APOBEC3G (A3G), a deoxycytidine deaminase, is a powerful host antiretroviral factor that can restrict HIV-1 infection. This restriction is counteracted by the HIV-1 virion infectivity factor (Vif) protein, whose activity culminates in depletion of A3G from infected cells. In the absence of Vif, viruses encapsidate A3G, which acts in part to mutate viral DNA formed during reverse transcription upon subsequent infection of a new cell. Cellular A3G also functions as a post-entry restriction factor for HIV in resting CD4 T cells, where it resides in a low molecular mass form. Unfortunately, this barrier is forfeited when CD4 T cells are activated because A3G is recruited into inactive high molecular mass ribonucleoprotein complexes. In addition to restricting HIV, A3G and related deaminases may counter other retroviruses and protect the cell from endogenous mobile retroelements. Understanding A3G complex assembly and its interplay with HIV Vif may make possible future development of a new class of HIV therapeutic agents.

APOBEC3G and HIV-1: Strike and counterstrike

APOBEC3G (A3G), a deoxycytidine deaminase, is a powerful host antiretroviral factor that can restrict HIV-1 infection. This restriction is counteracted by the HIV-1 virion infectivity factor (Vif) protein, whose activity culminates in depletion of A3G from infected cells. In the absence of Vif, viruses encapsidate A3G, which acts in part to mutate viral DNA formed during reverse transcription upon subsequent infection of a new cell. Cellular A3G also functions as a post-entry restriction factor for HIV in resting CD4 T cells, where it resides in a low molecular mass form. Unfortunately, this barrier is forfeited when CD4 T cells are activated because A3G is recruited into inactive high molecular mass ribonucleoprotein complexes. In addition to restricting HIV, A3G and related deaminases may counter other retroviruses and protect the cell from endogenous mobile retroelements. Understanding A3G complex assembly and its interplay with HIV Vif may make possible future development of a new class of HIV therapeutic agents.

Mitochondrial Protein Sorting: DIFFERENTIATION OF β-BARREL ASSEMBLY BY Tom7-MEDIATED SEGREGATION OF Mdm10

The mitochondrial outer membrane contains two distinct machineries for protein import and protein sorting that function in a sequential manner: the general translocase of the outer membrane (TOM complex) and the sorting and assembly machinery (SAM complex), which is dedicated to beta-barrel proteins. The SAM(core) complex consists of three subunits, Sam35, Sam37, and Sam50, that can associate with a fourth subunit, the morphology component Mdm10, to form the SAM(holo) complex. Whereas the SAM(core) complex is required for the biogenesis of all beta-barrel proteins, Mdm10 and the SAM(holo) complex play a selective role in beta-barrel biogenesis by promoting assembly of Tom40 but not of porin. We report that Tom7, a conserved subunit of the TOM complex, functions in an antagonistic manner to Mdm10 in biogenesis of Tom40 and porin. We show that Tom7 promotes segregation of Mdm10 from the SAM(holo) complex into a low molecular mass form. Upon deletion of Tom7, the fraction of Mdm10 in the SAM(holo) complex is significantly increased, explaining the opposing functions of Tom7 and Mdm10 in beta-barrel sorting. Thus the role of Tom7 is not limited to the TOM complex. Tom7 functions in mitochondrial protein biogenesis by a new mechanism, segregation of a sorting component, leading to a differentiation of beta-barrel assembly.

P3-239: Heparin activates βeta-secretase(BACE1) of Alzheimer’s disease and increases autocatalysis of the enzyme

The beta–site APP cleaving enzyme 1 (BACE1) is an aspartic protease that generates the N–terminus of the beta–amyloid protein from the beta–amyloid precursor protein (APP). BACE1 is a key target for Alzheimer drug development. However, little is known about the physiological regulation of the enzyme. Heparin can promote beta–secretase cleavage of endogenous APP in neuroblastoma cells. However, heparin has also been reported to directly inhibit BACE1 activity in vitro. To clarify the role of heparin in regulating BACE1. We examined the effect of heparin on the activity of recombinant human BACE1 (rBACE1) in vitro. Low concentrations (1 microg/mL) of heparin were found to stimulate rBACE1, whereas higher concentrations (10 or 100 microg/mL) were inhibitory. Heparin affinity chromatography demonstrated that heparin interacted strongly with the partially active pro–enzyme form of rBACE1 and bound to a peptide homologous to the N–terminal pro–sequence of BACE1. Low concentrations of heparin promoted autocatalytic cleavage of rBACE1, resulting in a decrease in the concentration of the pro–enzyme form and an increase in lower molecular mass forms. Furthermore, mature (pro–sequence cleaved) enzyme appeared to lack the capacity to be stimulated by heparin providing support for the view that activation by heparin was through pro–domain cleavage. The results indicate that high affinity binding of heparin to the pro–domain of BACE1 activates the enzyme and increases autocatalysis of BACE1 in vitro. Our study provides evidence that heparan sulfate proteoglycans could regulate the rate of Abeta production in vivo.

Endogenous factors enhance HIV infection of tissue naive CD4 T cells by stimulating high molecular mass APOBEC3G complex formation

Human immunodeficiency virus (HIV) can infect resting CD4 T cells residing in lymphoid tissues but not those circulating in peripheral blood. The molecular mechanisms producing this difference remain unknown. We explored the potential role of the tissue microenvironment and its influence on the action of the antiviral factor APOBEC3G (A3G) in regulating permissivity to HIV infection. We found that endogenous IL-2 and -15 play a key role in rendering resident naive CD4 T cells susceptible to HIV infection. Infection of memory CD4 T cells also requires endogenous soluble factors, but not IL-2 or -15. A3G is found in a high molecular mass complex in HIV infection–permissive, tissue-resident naive CD4 T cells but resides in a low molecular mass form in nonpermissive, blood-derived naive CD4 T cells. Upon treatment with endogenous soluble factors, these cells become permissive for HIV infection, as low molecular mass A3G is induced to assemble into high molecular mass complexes. These findings suggest that in lymphoid tissues, endogenous soluble factors, likely including IL-2 and -15 and others, stimulate the formation of high molecular mass A3G complexes in tissue-resident naive CD4 T cells, thereby relieving the potent postentry restriction block for HIV infection conferred by low molecular mass A3G.