Myosin 1b and F-actin are involved in the control of secretory granule biogenesis.

Hormone secretion relies on secretory granules which store hormones in endocrine cells and release them upon cell stimulation. The molecular events leading to hormone sorting and secretory granule formation at the level of the TGN are still elusive. Our proteomic analysis of purified whole secretory granules or secretory granule membranes uncovered their association with the actomyosin components myosin 1b, actin and the actin nucleation complex Arp2/3. We found that myosin 1b controls the formation of secretory granules and the associated regulated secretion in both neuroendocrine cells and chromogranin A-expressing COS7 cells used as a simplified model of induced secretion. We show that F-actin is also involved in secretory granule biogenesis and that myosin 1b cooperates with Arp2/3 to recruit F-actin to the Golgi region where secretory granules bud. These results provide the first evidence that components of the actomyosin complex promote the biogenesis of secretory granules and thereby regulate hormone sorting and secretion.

Adipokines (Leptin, Adiponectin, Resistin) Differentially Regulate All Hormonal Cell Types in Primary Anterior Pituitary Cell Cultures from Two Primate Species.

Adipose-tissue (AT) is an endocrine organ that dynamically secretes multiple hormones, the adipokines, which regulate key physiological processes. However, adipokines and their receptors are also expressed and regulated in other tissues, including the pituitary, suggesting that locally- and AT-produced adipokines might comprise a regulatory circuit that relevantly modulate pituitary cell-function. Here, we used primary pituitary cell-cultures from two normal nonhuman-primate species [Papio-anubis/Macaca-fascicularis] to determine the impact of different adipokines on the functioning of all anterior-pituitary cell-types. Leptin and resistin stimulated GH-release, a response that was blocked by somatostatin. Conversely, adiponectin decreased GH-release, and inhibited GHRH-, but not ghrelin-stimulated GH-secretion. Furthermore: 1) Leptin stimulated PRL/ACTH/FSH- but not LH/TSH-release; 2) adiponectin stimulated PRL-, inhibited ACTH- and did not alter LH/FSH/TSH-release; and 3) resistin increased ACTH-release and did not alter PRL/LH/FSH/TSH-secretion. These effects were mediated through the activation of common (AC/PKA) and distinct (PLC/PKC, intra-/extra-cellular calcium, PI3K/MAPK/mTOR) signaling-pathways, and by the gene-expression regulation of key receptors/transcriptional-factors involved in the functioning of these pituitary cell-types (e.g. GHRH/ghrelin/somatostatin/insulin/IGF-I-receptors/Pit-1). Finally, we found that primate pituitaries expressed leptin/adiponectin/resistin. Altogether, these and previous data suggest that local-production of adipokines/receptors, in conjunction with circulating adipokine-levels, might comprise a relevant regulatory circuit that contribute to the fine-regulation of pituitary functions.

The Effects of Bariatric Surgery-Induced Weight Loss on Adipose Tissue in Morbidly Obese Women Depends on the Initial Metabolic Status.

BACKGROUND: Adipose tissue (AT) dysfunction in obesity is commonly linked to insulin resistance and promotes the development of metabolic disease. Bariatric surgery (BS) represents an effective strategy to reduce weight and to improve metabolic health in morbidly obese subjects. However, the mechanisms and pathways that are modified in AT in response to BS are not fully understood, and few information is still available as to whether these may vary depending on the metabolic status of obese subjects. METHODS: Abdominal subcutaneous adipose tissue (SAT) samples were obtained from morbidly obese women (n = 18) before and 13.3 ± 0.37 months after BS. Obese women were stratified into two groups: normoglycemic (NG; Glu < 100 mg/dl, HbA1c <5.7 %) or insulin resistant (IR; Glu 100-126 mg/dl, HbA1c 5.7-6.4 %) (n = 9/group). A multi-comparative proteomic analysis was employed to identify differentially regulated SAT proteins by BS and/or the degree of insulin sensitivity. Serum levels of metabolic, inflammatory, and anti-oxidant markers were also analyzed. RESULTS: Before surgery, NG and IR subjects exhibited differences in AT proteins related to inflammation, metabolic processes, the cytoskeleton, and mitochondria. BS caused comparable weight reductions and improved glucose homeostasis in both groups. However, BS caused dissimilar changes in metabolic enzymes, inflammatory markers, cytoskeletal components, mitochondrial proteins, and angiogenesis regulators in NG and IR women. CONCLUSIONS: BS evokes significant molecular rearrangements indicative of improved AT function in morbidly obese women at either low or high metabolic risk, though selective adaptive changes in key cellular processes occur depending on the initial individual's metabolic status.

Defective transport of the obesity mutant PC1/3 N222D contributes to loss of function.

Mutations in the PCSK1 gene encoding prohormone convertase 1/3 (PC1/3) are strongly associated with obesity in humans. The PC1/3(N222D) mutant mouse thus far represents the only mouse model that mimics the PC1/3 obesity phenotype in humans. The present investigation addresses the cell biology of the N222D mutation. Metabolic labeling experiments reveal a clear defect in the kinetics of insulin biosynthesis in islets from PC1/3(N222D) mutant mice, resulting in an increase in both proinsulin and its processing intermediates, predominantly lacking cleavage at the Arg-Arg site. Although the mutant PC1/3 zymogen is correctly processed to the 87-kDa form, pulse-chase immunoprecipitation experiments, labeling, and immunohistochemical experiments using uncleavable variants all demonstrate that the PC1/3-N222D protein is largely mislocalized compared with similar wild-type (WT) constructs, being predominantly retained in the endoplasmic reticulum. The PC1/3-N222D mutant also undergoes more efficient degradation via the ubiquitin-proteasome system than the WT enzyme. Lastly, the mutant PC1/3-N222D protein coimmunoprecipitates with WT PC1/3 and exerts a modest effect on intracellular retention of the WT enzyme. These profound alterations in the cell biology of PC1/3-N222D are likely to contribute to the defective insulin biosynthetic events observed in the mutant mice and may be relevant to the dramatic contributions of polymorphisms in this gene to human obesity.

Blockade of islet amyloid polypeptide fibrillation and cytotoxicity by the secretory chaperones 7B2 and proSAAS.

The deposition of fibrillated human islet ß-cell peptide islet amyloid polypeptide (hIAPP) into amyloid plaques is characteristic of the pathogenesis of islet cell death during type 2 diabetes. We investigated the effects of the neuroendocrine secretory proteins 7B2 and proSAAS on hIAPP fibrillation in vitro and on cytotoxicity. In vitro, 21-kDa 7B2 and proSAAS blocked hIAPP fibrillation. Structure-function studies showed that a central region within 21-kDa 7B2 is important in this effect and revealed the importance of the N-terminal region of proSAAS. Both chaperones blocked the cytotoxic effects of exogenous hIAPP on Rin5f cells; 7B2 generated by overexpression was also effective. ProSAAS and 7B2 may perform a chaperone role as secretory anti-aggregants in normal islet cell function and in type 2 diabetes.

Adiponectin receptors form homomers and heteromers exhibiting distinct ligand binding and intracellular signaling properties.

Adiponectin binds to two widely expressed receptors (AdipoR1 and AdipoR2) that contain seven transmembrane domains but, unlike G-protein coupled receptors, present an extracellular C terminus and a cytosolic N terminus. Recently, AdipoR1 was found to associate in high order complexes. However, it is still unknown whether AdipoR2 may also form homomers or heteromers with AdipoR1 or if such interactions may be functionally relevant. Herein, we have analyzed the oligomerization pattern of AdipoRs by FRET and immunoprecipitation and evaluated both the internalization of AdipoRs in response to various adiponectin isoforms and the effect of adiponectin binding to different AdipoR combinations on AMP-activated protein kinase phosphorylation and peroxisome proliferator-activated receptor α activation. Transfection of HEK293AD cells with AdipoR1 and AdipoR2 showed that both receptors colocalize at both the plasma membrane and the endoplasmic reticulum. Co-transfection with the different AdipoR pairs yielded high FRET efficiencies in non-stimulated cells, which indicates that AdipoR1 and AdipoR2 form homo- and heteromeric complexes under resting conditions. Live FRET imaging suggested that both homo- and heteromeric AdipoR complexes dissociate in response to adiponectin, but heteromers separate faster than homomers. Finally, phosphorylation of AMP-activated protein kinase in response to adiponectin was delayed in cells wherein heteromer formation was favored. In sum, our findings indicate that AdipoR1 and AdipoR2 form homo- and heteromers that present unique interaction behaviors and signaling properties. This raises the possibility that the pleiotropic, tissue-dependent functions of adiponectin depend on the expression levels of AdipoR1 and AdipoR2 and, therefore, on the steady-state proportion of homo- and heteromeric complexes.

The Golgi-associated long coiled-coil protein NECC1 participates in the control of the regulated secretory pathway in PC12 cells.

Golgi-associated long coiled-coil proteins, often referred to as golgins, are involved in the maintenance of the structural organization of the Golgi apparatus and the regulation of membrane traffic events occurring in this organelle. Little information is available on the contribution of golgins to Golgi function in cells specialized in secretion such as endocrine cells or neurons. In the present study, we characterize the intracellular distribution as well as the biochemical and functional properties of a novel long coiled-coil protein present in neuroendocrine tissues, NECC1 (neuroendocrine long coiled-coil protein 1). The present study shows that NECC1 is a peripheral membrane protein displaying high stability to detergent extraction, which distributes across the Golgi apparatus in neuroendocrine cells. In addition, NECC1 partially localizes to post-Golgi carriers containing secretory cargo in PC12 cells. Overexpression of NECC1 resulted in the formation of juxtanuclear aggregates together with a slight fragmentation of the Golgi and a decrease in K+-stimulated hormone release. In contrast, NECC1 silencing did not alter Golgi architecture, but enhanced K+-stimulated hormone secretion in PC12 cells. In all, the results of the present study identify NECC1 as a novel component of the Golgi matrix and support a role for this protein as a negative modulator of the regulated trafficking of secretory cargo in neuroendocrine cells.

Modulation of prohormone convertase 1/3 properties using site-directed mutagenesis.

Prohormone convertase (PC)1/3 and PC2 cleave active peptide hormones and neuropeptides from precursor proteins. Compared with PC2, recombinant PC1/3 exhibits a very low specific activity against both small fluorogenic peptides and recombinant precursors, even though the catalytic domains in mouse PC1/3 and PC2 share 56% amino acid sequence identity. In this report, we have designed PC2-specific mutations into the catalytic domain of PC1/3 in order to investigate the molecular contributions of these sequences to PC1/3-specific properties. The exchange of residues RQG(314) with the SY sequence present in the same location within PC2 paradoxically shifted the pH optimum of PC1/3 upward into the neutral range; other mutations in the catalytic domain had no effect. Although none of the full-length PC1/3 mutants examined exhibited increased specific activity, the 66-kDa form of the RQG(314)SY mutant was two to four times more active than the 66-kDa form of wild-type PC1/3. However, stable transfection of RQG(314)SY into PC12 cells did not result in greater activity against the endogenous substrate proneurotensin, implying unknown cellular controls of PC1/3 activity. Mutation of GIVTDA(243-248) to QPFMTDI, a molecular determinant of 7B2 binding, resulted in increased zymogen expression but no propeptide cleavage or secretion, suggesting that this mutant is trapped in the endoplasmic reticulum due to an inability to cleave its own propeptide. We conclude that many convertase-specific properties are attributable less to convertase-specific catalytic cleft residues than to convertase-specific domain interactions.

Rab18 inhibits secretory activity in neuroendocrine cells by interacting with secretory granules.

Rab proteins comprise a complex family of small GTPases involved in the regulation of intracellular membrane trafficking and reorganization. In this study, we identified Rab18 as a new inhibitory player of the secretory pathway in neuroendocrine cells. In adrenal chromaffin PC12 cells and pituitary AtT20 cells, Rab18 is located at the cytosol but associates with a subpopulation of secretory granules after stimulation of the regulated secretory pathway, strongly suggesting that induction of secretion provokes Rab18 activation and recruitment to these organelles. In support of this, a dominant-inactive Rab18 mutant was found to distribute diffusely in the cytosol, whereas a dominant-active Rab18 mutant was predominantly associated to secretory granules. Furthermore, interaction of Rab18 with secretory granules was associated to an inhibition in the secretory activity of PC12 and AtT20 cells in response to stimulatory challenges. Association of Rab18 with secretory granules was also observed by immunoelectron microscopy in normal, non-tumoral endocrine cells (pituitary melanotropes), wherein Rab18 protein content is inversely correlated to the level of secretory activity of cells. Taken together, these findings suggest that, in neuroendocrine cells, Rab18 acts as a negative regulator of secretory activity, likely by impairing secretory granule transport.

Identification and characterization of human polyserase-3, a novel protein with tandem serine-protease domains in the same polypeptide chain.

BACKGROUND: We have previously described the identification and characterization of polyserase-1 and polyserase-2, two human serine proteases containing three different catalytic domains within the same polypeptide chain. Polyserase-1 shows a complex organization and it is synthesized as a membrane-bound protein which can generate three independent serine protease domains as a consequence of post-translational processing events. The two first domains are enzymatically active. By contrast, polyserase-2 is an extracellular glycosylated protein whose three protease domains remain embedded in the same chain, and only the first domain possesses catalytic activity. RESULTS: Following our interest in the study of the human degradome, we have cloned a human liver cDNA encoding polyserase-3, a new protease with tandem serine protease domains in the same polypeptide chain. Comparative analysis of polyserase-3 with the two human polyserases described to date, revealed that this novel polyprotein is more closely related to polyserase-2 than to polyserase-1. Thus, polyserase-3 is a secreted protein such as polyserase-2, but lacks additional domains like the type II transmembrane motif and the low-density lipoprotein receptor module present in the membrane-anchored polyserase-1. Moreover, analysis of post-translational mechanisms operating in polyserase-3 maturation showed that its two protease domains remain as integral parts of the same polypeptide chain. This situation is similar to that observed in polyserase-2, but distinct from polyserase-1 whose protease domains are proteolytically released from the original chain to generate independent units. Immunolocalization studies indicated that polyserase-3 is secreted as a non-glycosylated protein, thus being also distinct from polyserase-2, which is a heavily glycosylated protein. Enzymatic assays indicated that recombinant polyserase-3 degrades the alpha-chain of fibrinogen as well as pro-urokinase-type plasminogen activator (pro-uPA). Northern blot analysis showed that polyserase-3 exhibits a unique expression pattern among human polyserases, being predominantly detected in testis, liver, heart and ovary, as well as in several tumor cell lines. CONCLUSION: These findings contribute to define the growing group of human polyserine proteases composed at present by three different proteins. All of them share a complex structural design with several catalytic units in a single polypeptide but also show specific features in terms of enzymatic properties, expression patterns and post-translational maturation mechanisms.

Identification and characterization of human archaemetzincin-1 and -2, two novel members of a family of metalloproteases widely distributed in Archaea.

Systematic analysis of degradomes, the complete protease repertoires of organisms, has demonstrated the large and growing complexity of proteolytic systems operating in all cells and tissues. We report here the identification of two new human metalloproteases that have been called archaemetzincin-1 (AMZ1) and archaemetzincin-2 (AMZ2) to emphasize their close relationship to putative proteases predicted by bioinformatic analysis of archaeal genomes. Both human proteins contain a catalytic domain with a core motif (HEXXHXXGX3CX4CXMX17CXXC) that includes an archetypal zinc-binding site, the methionine residue characteristic of metzincins, and four conserved cysteine residues that are not present at the equivalent positions of other human metalloproteases. Analysis of genome sequence databases revealed that AMZs are widely distributed in Archaea and vertebrates and contribute to the defining of a new metalloprotease family that has been called archaemetzincin. However, AMZ-like sequences are absent in a number of model organisms from bacteria to nematodes. Phylogenetic analysis showed that these enzymes have undergone a complex evolutionary process involving a series of lateral gene transfer, gene loss, and genetic duplication events that have shaped this novel family of metalloproteases. Northern blot analysis showed that AMZ1 and AMZ2 exhibit distinct expression patterns in human tissues. AMZ1 is mainly detected in liver and heart whereas AMZ2 is predominantly expressed in testis and heart, although both are also detectable at lower levels in other tissues. Both human enzymes were produced in Escherichia coli, and the purified recombinant proteins hydrolyzed synthetic substrates and bioactive peptides, demonstrating that they are functional proteases. Finally, these activities were abolished by inhibitors of metalloproteases, providing further evidence that AMZs belong to this catalytic class of proteolytic enzymes.

Cross-inhibition between furin and lethal factor inhibitors.

Bacillus anthracis synthesizes two toxins composed of the three proteins: protective antigen (PA), lethal factor (LF), and edema factor (EF). The cleavage of PA on the cell surface by the convertase furin leads to the translocation of LF and EF into the cytosol. We have investigated the cross-inhibitory activities of the furin inhibitors hexa-d-arginine amide (D6R) and nona-d-arginine amide (D9R), which block the proteolytic activation of PA; and of the LF inhibitor In-2-LF, a peptide hydroxamate. D6R and D9R inhibit LF with IC(50s) of 300 and 10microM, respectively; conversely, In-2-LF also inhibits furin (IC(50) 2microM). In-2-LF was efficiently cleaved by furin with the concomitant loss of inhibitory activity on both LF and furin. Incubation of In-2-LF with LF however generated a product that retained partial inhibitory activity against LF. Combined treatment of cells with D6R and In-2-LF enhanced protection against anthrax lethal toxin, indicating that combined administration of inhibitors could represent an effective therapeutic approach.

Inhibition of furin by polyarginine-containing peptides: nanomolar inhibition by nona-D-arginine.

Polyarginine-containing peptides represent potent inhibitors of furin, a mammalian endoprotease that plays an important role in metabolism, activation of pathogenic toxins, and viral proliferation. The therapeutic use of D-polyarginines is especially interesting because they are not cleaved by furin and possess inhibitory potency almost equal to L-polyarginines. In this study we attempted to determine the important elements within polyarginines that contribute to effective inhibition. Structure-function analyses of polyarginine peptides showed that inhibition by polyarginine-containing peptides appeared to depend on the total number of basic charges of the positively charged inhibitors bound to the negatively charged substrate binding pocket; peptide positioning did not appear to be rigorously determined. Screening of L- and D-decapeptide positional scanning combinatorial peptide libraries indicated a preference for basic residues in nearly all positions, similar to previous results with hexapeptide libraries. Length and terminal modification studies showed that the most potent D-polyarginine tested was nona-D-arginine (D9R) amide with a K(i) of 1.3 nm. D9R amide was shown to protect RAW264.7 cells against anthrax toxemia with an IC(50) of 3.7 microm. Because of its high stability, specificity, low toxicity, small molecular weight, and extremely low K(i) against furin, D9R amide or its derivatives may represent promising compounds for therapeutic use.

Protection against anthrax toxemia by hexa-D-arginine in vitro and in vivo.

The anthrax toxin protective antigen precursor is activated by proteolytic cleavage by furin or a furin-like protease. We present here data demonstrating that the small stable furin inhibitor hexa-D-arginine amide delays anthrax toxin-induced toxemia both in cells and in live animals, suggesting that furin inhibition may represent a reasonable avenue for therapeutic intervention in anthrax.