Constitutive Secretory Pathway Research Articles

Alpha herpesvirus exocytosis from neuron cell bodies uses constitutive secretory mechanisms, and egress and spread from axons is independent of neuronal firing activity.

Alpha herpesviruses naturally infect the peripheral nervous system, and can spread to the central nervous system, causing severe debilitating or deadly disease. Because alpha herpesviruses spread along synaptic circuits, and infected neurons exhibit altered electrophysiology and increased spontaneous activity, we hypothesized that alpha herpesviruses use activity-dependent synaptic vesicle-like regulated secretory mechanisms for egress and spread from neurons. Using live-cell fluorescence microscopy, we show that Pseudorabies Virus (PRV) particles use the constitutive Rab6 post-Golgi secretory pathway to exit from the cell body of primary neurons, independent of local calcium signaling. Some PRV particles colocalize with Rab6 in the proximal axon, but we did not detect colocalization/co-transport in the distal axon. Thus, the specific secretory mechanisms used for viral egress from axons remains unclear. To address the role of neuronal activity more generally, we used a compartmentalized neuron culture system to measure the egress and spread of PRV from axons, and pharmacological and optogenetics approaches to modulate neuronal activity. Using tetrodotoxin to silence neuronal activity, we observed no inhibition, and using potassium chloride or optogenetics to elevate neuronal activity, we also show no increase in virus spread from axons. We conclude that PRV egress from neurons uses constitutive secretory mechanisms: generally, activity-independent mechanisms in axons, and specifically, the constitutive Rab6 post-Golgi secretory pathway in cell bodies.

The Regulated Secretion and Models of Intracellular Transport: The Goblet Cell as an Example.

Transport models are extremely important to map thousands of proteins and their interactions inside a cell. The transport pathways of luminal and at least initially soluble secretory proteins synthesized in the endoplasmic reticulum can be divided into two groups: the so-called constitutive secretory pathway and regulated secretion (RS) pathway, in which the RS proteins pass through the Golgi complex and are accumulated into storage/secretion granules (SGs). Their contents are released when stimuli trigger the fusion of SGs with the plasma membrane (PM). In specialized exocrine, endocrine, and nerve cells, the RS proteins pass through the baso-lateral plasmalemma. In polarized cells, the RS proteins secrete through the apical PM. This exocytosis of the RS proteins increases in response to external stimuli. Here, we analyze RS in goblet cells to try to understand the transport model that can be used for the explanation of the literature data related to the intracellular transport of their mucins.

The exocyst complex is an essential component of the mammalian constitutive secretory pathway.

Secreted proteins fulfill a vast array of functions, including immunity, signaling, and extracellular matrix remodeling. In the trans-Golgi network, proteins destined for constitutive secretion are sorted into post-Golgi carriers which fuse with the plasma membrane. The molecular machinery involved is poorly understood. Here, we have used kinetic trafficking assays and transient CRISPR-KO to study biosynthetic sorting from the Golgi to the plasma membrane. Depletion of all canonical exocyst subunits causes cargo accumulation in post-Golgi carriers. Exocyst subunits are recruited to and co-localize with carriers. Exocyst abrogation followed by kinetic trafficking assays of soluble cargoes results in intracellular cargo accumulation. Unbiased secretomics reveals impairment of soluble protein secretion after exocyst subunit knockout. Importantly, in specialized cell types, the loss of exocyst prevents constitutive secretion of antibodies in lymphocytes and of leptin in adipocytes. These data identify exocyst as the functional tether of secretory post-Golgi carriers at the plasma membrane and an essential component of the mammalian constitutive secretory pathway.

Germline gain-of-function MMP11 variant results in an aggressive form of colorectal cancer.

Matrix metalloproteinase-11 (MMP11) is an enzyme with proteolytic activity against matrix and nonmatrix proteins. Although most MMPs are secreted as inactive proenzymes and are later activated extracellularly, MMP11 is activated intracellularly by furin within the constitutive secretory pathway. It is a key factor in physiological tissue remodeling and its alteration may play an important role in the progression of epithelial malignancies and other diseases. TCGA colon and colorectal adenocarcinoma data showed that upregulation of MMP11 expression correlates with tumorigenesis and malignancy. Here, we provide evidence that a germline variant in the MMP11 gene (NM_005940: c.232C>T; p.(Pro78Ser)), identified by whole exome sequencing, can increase the tumorigenic properties of colorectal cancer (CRC) cells. P78S is located in the prodomain region, which is responsible for blocking MMP11's protease activity. This variant was detected in the proband and all the cancer-affected family members analyzed, while it was not detected in healthy relatives. In silico analyses predict that P78S could have an impact on the activation of the enzyme. Furthermore, our in vitro analyses show that the expression of P78S in HCT116 cells increases tumor cell invasion and proliferation. In summary, our results show that this variant could modify the structure of the MMP11 prodomain, producing a premature or uncontrolled activation of the enzyme that may contribute to an early CRC onset in these patients. The study of this gene in other CRC cases will provide further information about its role in CRC development, which might improve patient treatment in the future.

Expression of a Secretable, Cell-Penetrating CDKL5 Protein Enhances the Efficacy of Gene Therapy for CDKL5 Deficiency Disorder

Although delivery of a wild-type copy of the mutated gene to cells represents the most effective approach for a monogenic disease, proof-of-concept studies highlight significant efficacy caveats for treatment of brain disorders. Herein, we develop a cross-correction-based strategy to enhance the efficiency of a gene therapy for CDKL5 deficiency disorder, a severe neurodevelopmental disorder caused by CDKL5 gene mutations. We created a gene therapy vector that produces an Igk-TATk-CDKL5 fusion protein that can be secreted via constitutive secretory pathways and, due to the cell-penetration property of the TATk peptide, internalized by cells. We found that, although AAVPHP.B_Igk-TATk-CDKL5 and AAVPHP.B_CDKL5 vectors had similar brain infection efficiency, the AAVPHP.B_Igk-TATk-CDKL5 vector led to higher CDKL5 protein replacement due to secretion and penetration of the TATk-CDKL5 protein into the neighboring cells. Importantly, Cdkl5 KO mice treated with the AAVPHP.B_Igk-TATk-CDKL5 vector showed a behavioral and neuroanatomical improvement in comparison with vehicle or AAVPHP.B_CDKL5 vector-treated Cdkl5 KO mice. In conclusion, we provide the first evidence that a gene therapy based on a cross-correction approach is more effective at compensating Cdkl5-null brain defects than gene therapy based on the expression of the native CDKL5, opening avenues for the development of this innovative approach for other monogenic diseases.

A Feasible Alternative Strategy Targeting Furin Disrupts SARS-CoV-2 Infection Cycle.

ABSTRACTThe COVID-19 causing coronavirus (SARS-CoV-2) remains a public health threat worldwide. SARS-CoV-2 enters human lung cells via its spike glycoprotein binding to angiotensin-converting enzyme 2 (ACE2). Notably, the cleavage of the spike by the host cell protease furin in virus-producing cells is critical for subsequent spike-driven entry into lung cells. Thus, effective targeted therapies blocking the spike cleavage and activation in viral producing cells may provide an alternate strategy to break the viral transmission cycle and to overcome disease pathology. Here we engineered and described an antibody-based targeted strategy, which directly competes with the furin mediated proteolytic activation of the spike in virus-producing cells. The described approach involves engineering competitive furin substrate residues in the IgG1 Fc-extended flexible linker domain of SARS-CoV-2 spike targeting antibodies. Considering the site of spike furin cleavage and SARS-CoV-2 egress remains uncertain, the experimental strategy pursued here revealed novel mechanistic insights into proteolytic processing of the spike protein, which suggest that processing does not occur in the constitutive secretory pathway. Furthermore, our results show blockade of furin-mediated cleavage of the spike protein for membrane fusion activation and virus host-cell entry function. These findings provide an alternate insight of targeting applicability to SARS-CoV-2 and the future coronaviridae family members, exploiting the host protease system to gain cellular entry and subsequent chain of infections.IMPORTANCE Since its emergence in December 2019, COVID-19 has remained a global economic and health threat. Although RNA and DNA vector-based vaccines induced antibody response and immunological memory have proven highly effective against hospitalization and mortality, their long-term efficacy remains unknown against continuously evolving SARS-CoV-2 variants. As host cell-enriched furin-mediated cleavage of SARS-CoV-2 spike protein is critical for viral entry and chain of the infection cycle, the solution described here of an antibody Fc-conjugated furin competing peptide is significant. In a scenario where spike mutational drifts do not interfere with the Fc-conjugated antibody's epitope, the proposed furin competing strategy confers a broad-spectrum targeting design to impede the production of efficiently transmissible SARS-CoV-2 viral particles. In addition, the proposed approach is plug-and-play against other potentially deadly viruses that exploit secretory pathway independent host protease machinery to gain cellular entry and subsequent transmissions to host cells.

ALG-2 and peflin regulate COPII targeting and secretion in response to calcium signaling

ER-to-Golgi transport is the first step in the constitutive secretory pathway, which, unlike regulated secretion, is believed to proceed nonstop independent of Ca2+ flux. However, here we demonstrate that penta-EF hand (PEF) proteins ALG-2 and peflin constitute a hetero-bifunctional COPII regulator that responds to Ca2+ signaling by adopting one of several distinct activity states. Functionally, these states can adjust the rate of ER export of COPII-sorted cargos up or down by ∼50%. We found that at steady-state Ca2+, ALG-2/peflin hetero-complexes bind to ER exit sites (ERES) through the ALG-2 subunit to confer a low, buffered secretion rate, while peflin-lacking ALG-2 complexes markedly stimulate secretion. Upon Ca2+ signaling, ALG-2 complexes lacking peflin can either increase or decrease the secretion rate depending on signaling intensity and duration—phenomena that could contribute to cellular growth and intercellular communication following secretory increases or protection from excitotoxicity and infection following decreases. In epithelial normal rat kidney (NRK) cells, the Ca2+-mobilizing agonist ATP causes ALG-2 to depress ER export, while in neuroendocrine PC12 cells, Ca2+ mobilization by ATP results in ALG-2-dependent enhancement of secretion. Furthermore, distinct Ca2+ signaling patterns in NRK cells produce opposing ALG-2-dependent effects on secretion. Mechanistically, ALG-2-dependent depression of secretion involves decreased levels of the COPII outer shell and increased peflin targeting to ERES, while ALG-2-dependent enhancement of secretion involves increased COPII outer shell and decreased peflin at ERES. These data provide insights into how PEF protein dynamics affect secretion of important physiological cargoes such as collagen I and significantly impact ER stress.

Intracellular Trafficking of HBV Particles

The human hepatitis B virus (HBV), that is causative for more than 240 million cases of chronic liver inflammation (hepatitis), is an enveloped virus with a partially double-stranded DNA genome. After virion uptake by receptor-mediated endocytosis, the viral nucleocapsid is transported towards the nuclear pore complex. In the nuclear basket, the nucleocapsid disassembles. The viral genome that is covalently linked to the viral polymerase, which harbors a bipartite NLS, is imported into the nucleus. Here, the partially double-stranded DNA genome is converted in a minichromosome-like structure, the covalently closed circular DNA (cccDNA). The DNA virus HBV replicates via a pregenomic RNA (pgRNA)-intermediate that is reverse transcribed into DNA. HBV-infected cells release apart from the infectious viral parrticle two forms of non-infectious subviral particles (spheres and filaments), which are assembled by the surface proteins but lack any capsid and nucleic acid. In addition, naked capsids are released by HBV replicating cells. Infectious viral particles and filaments are released via multivesicular bodies; spheres are secreted by the classic constitutive secretory pathway. The release of naked capsids is still not fully understood, autophagosomal processes are discussed. This review describes intracellular trafficking pathways involved in virus entry, morphogenesis and release of (sub)viral particles.

Switching of cargo sorting from the constitutive to regulated secretory pathway by the addition of cystatin D sequence in salivary acinar cells.

The mechanism for segregation of cargo proteins into the regulated and constitutive secretory pathways in exocrine cells remains to be elucidated. We examined the transport of HaloTag proteins fused with full-length cystatin D (fCst5-Halo) or only its signal peptide (ssCst5-Halo) in parotid acinar cells. Although both fusion proteins were observed to be colocalized with amylase in the secretory granules, the coefficients for overlapping and correlation of fCst5-Halo with amylase were higher than those of ssCst5-Halo. The secretion of both the proteins was enhanced by the addition of the β-adrenergic receptor agonist isoproterenol as well as endogenous amylase. In contrast, unstimulated secretion of ssCst5-Halo without isoproterenol was significantly higher than that of fCst5-Halo and amylase. Simulation analysis using a mathematical model revealed that a large proportion of ssCst5-Halo was secreted through the constitutive pathway, whereas fCst5-Halo was transported into the secretory granules more efficiently. Precipitation of fCst5-Halo from cell lysates was increased at a low pH, which may mimic the milieu of the trans-Golgi networks. These data suggest that the addition of a full-length sequence of cystatin D facilitates efficient selective transport into the regulated pathway by aggregation at low pH in the trans-Golgi network.NEW & NOTEWORTHY The mechanism underlying the segregation of cargo proteins to the regulated and constitutive secretory pathways in exocrine cells remains to be solved. We analyzed unstimulated secretion in salivary acinar cells by performing double-labeling experiments using HaloTag technology and computer simulation. It revealed that the majority of HaloTag with only signal peptide sequence was secreted through the constitutive pathway and that the addition of a full-length cystatin D sequence changed its sorting to the regulated pathway.

The malaria parasite Plasmodium falciparum Sortilin is essential for merozoite formation and apical complex biogenesis.

The inner membrane complex and the apical secretory organelles are defining features of apicomplexan parasites. Despite their critical roles, the mechanisms behind the biogenesis of these structures in the malaria parasite Plasmodium falciparum are still poorly defined. We here show that decreasing expression of the P.falciparum homologue of the conserved endolysomal escorter Sortilin-VPS10 prevents the formation of the inner membrane complex and abrogates the generation of new merozoites. Moreover, protein trafficking to the rhoptries, the micronemes, and the dense granules is disrupted, which leads to the accumulation of apical complex proteins in the endoplasmic reticulum and the parasitophorous vacuole. We further show that protein export to the erythrocyte and transport through the constitutive secretory pathway are functional. Taken together, our results suggest that the malaria parasite P.falciparum Sortilin has potentially broader functions than most of its other eukaryotic counterparts.

Proprotein Convertase Selectivity in the Activation of the Human Papilloma Virus

The family of host cell serine proteinases known as proprotein convertases (PCs) is exploited by a variety of human pathogenic viruses to promote viral activation. The PCs, furin, PC5, PACE4, and PC7 reside in the trans‐Golgi network membranes of the constitutive secretion pathway and are expressed in most human tissues. We study PC selectivity for the activation of the human papilloma virus (HPV), and for that purpose we applied an approach that combines the use of recombinant PCs and an assay of HPV cell entry. We found differences in PC selectivity between HPV genotypes 16 and 18 for activation by furin, PACE4, and PC7, and showed that both viral genotypes are inactivated by PC5. These findings imply that proteolytic cleavage of the coat proteins in these two high cancer‐risk HPV genotypes involves multiple cleavage sites with different PC specificity, and challenge the current accepted model of HPV cell entry that postulates that only one PC cleavage site, located on the coat protein L2 at position Arg12, is needed for viral activation. The inactivation by mutagenesis of the six potential PC cleavage site motifs in HPV16, two in L1 and four in L2, demonstrated that additional PC cleavage sites in L1 and L2 are required for HPV16 infection. These results suggest that PC selectivity could be a determinant of viral tropism to anatomical sites of infection based on tissue PC expression profile.

Decreased proteinase A excretion by strengthening its vacuolar sorting and weakening its constitutive secretion in Saccharomyces cerevisiae.

Proteinase A (PrA), encoded by PEP4 gene, is detrimental to beer foam stability. There are two transport pathways for the new synthesized PrA in yeast, sorting to the vacuole normally, or excreting out of the cells under stress conditions. They were designated as the Golgi-to-vacuole pathway and the constitutive secretory pathway, respectively. To reduce PrA excretion in some new way instead of its coding gene deletion, which had a negative effect on cell metabolism and beer fermentation, we modified the PrA transport based on these above two pathways. In the Golgi-to-vacuole pathway, after the verification that Vps10p is the dominant sorting receptor for PrA Golgi-to-vacuolar transportation by VPS10 deletion, VPS10 was then overexpressed. Furthermore, SEC5, encoding exocyst complexes' central subunit (Sec5p) in the constitutive secretory pathway, was deleted. The results show that PrA activity in the broth fermented with WGV10 (VPS10 overexpressing strain) and W∆SEC5 (SEC5 deletion strain) was lowered by 76.96 and 32.39%, compared with the parental strain W303-1A, at the end of main fermentation. There are negligible changes in fermentation performance between W∆SEC5 and W303-1A, whereas, surprisingly, WGV10 had a significantly improved fermentation performance compared with W303-1A. WGV10 has an increased growth rate, resulting in higher biomass and faster fermentation speed; finally, wort fermentation is performed thoroughly. The results show that the biomass production of WGV10 is always higher than that of W∆SEC5 and W303-1A at all stages of fermentation, and that ethanol production of WGV10 is 1.41-fold higher than that of W303-1A. Obviously, VPS10 overexpression is beneficial for yeast and is a more promising method for reduction of PrA excretion.

The Intracellular Cholesterol Transport Inhibitor U18666A Inhibits the Exosome-Dependent Release of Mature Hepatitis C Virus.

There are still a variety of open questions regarding morphogenesis and release of hepatitis C virus. The HCV-infected cell produces significant more viral particles that are released, raising the question about the fate of the nonreleased particles. Moreover, the relevance of the endosomal pathway for the release of HCV is under debate. Use of the MVB (multivesicular body) inhibitor U18666A enabled a detailed analysis of the impact of MVBs for viral morphogenesis and release. It was revealed that infectious, fully assembled HCV particles are either MVB-dependently released or intracellularly degraded by the lysosome. Our data indicate that at least a fraction of HCV particles leaves the cell via the endosomal pathway independent from the constitutive secretory pathway. Our study describes a so-far-unprecedented cross talk between two pathways regulating on the one hand the release of infectious viral particles and on the other hand the intracellular degradation of nonreleased particles.

Subviral Hepatitis B Virus Filaments, like Infectious Viral Particles, Are Released via Multivesicular Bodies.

In addition to infectious viral particles, hepatitis B virus-replicating cells secrete large amounts of subviral particles assembled by the surface proteins, but lacking any capsid and genome. Subviral particles form spheres (22-nm particles) and filaments. Filaments contain a much larger amount of the large surface protein (LHBs) compared to spheres. Spheres are released via the constitutive secretory pathway, while viral particles are ESCRT-dependently released via multivesicular bodies (MVBs). The interaction of virions with the ESCRT machinery is mediated by α-taxilin that connects the viral surface protein LHBs with the ESCRT component tsg101. Since filaments in contrast to spheres contain a significant amount of LHBs, it is unclear whether filaments are released like spheres or like virions. To study the release of subviral particles in the absence of virion formation, a core-deficient HBV mutant was generated. Confocal microscopy, immune electron microscopy of ultrathin sections and isolation of MVBs revealed that filaments enter MVBs. Inhibition of MVB biogenesis by the small-molecule inhibitor U18666A or inhibition of ESCRT functionality by coexpression of transdominant negative mutants (Vps4A, Vps4B, and CHMP3) abolishes the release of filaments while the secretion of spheres is not affected. These data indicate that in contrast to spheres which are secreted via the secretory pathway, filaments are released via ESCRT/MVB pathway like infectious viral particles. This study revises the current model describing the release of subviral particles by showing that in contrast to spheres, which are secreted via the secretory pathway, filaments are released via the ESCRT/MVB pathway like infectious viral particles. These data significantly contribute to a better understanding of the viral morphogenesis and might be helpful for the design of novel antiviral strategies.

Chediak-Higashi syndrome: Lysosomal trafficking regulator domains regulate exocytosis of lytic granules but not cytokine secretion by natural killer cells

Mutations in lysosomal trafficking regulator (LYST) cause Chediak-Higashi syndrome (CHS), a rare immunodeficiency with impaired cytotoxic lymphocyte function, mainly that of natural killer (NK) cells. Our understanding of NK cell function deficiency in patients with CHS and how LYST regulates lytic granule exocytosis is very limited. We sought to delineate cellular defects associated with LYST mutations responsible for the impaired NK cell function seen in patients with CHS. We analyzed NK cells from patients with CHS with missense mutations in the LYST ARM/HEAT (armadillo/huntingtin, elongation factor 3, protein phosphatase 2A, and the yeast kinase TOR1) or BEACH (beige and Chediak-Higashi) domains. NK cells from patients with CHS displayed severely reduced cytotoxicity. Mutations in the ARM/HEAT domain led to a reduced number of perforin-containing granules, which were significantly increased in size but able to polarize to the immunologic synapse; however, they were unable to properly fuse with the plasma membrane. Mutations in the BEACH domain resulted in formation of normal or slightly enlarged granules that had markedly impaired polarization to the IS but could be exocytosed on reaching the immunologic synapse. Perforin-containing granules in NK cells from patients with CHS did not acquire certain lysosomal markers (lysosome-associated membrane protein 1/2) but were positive for markers of transport vesicles (cation-independent mannose 6-phosphate receptor), late endosomes (Ras-associated binding protein 27a), and, to some extent, early endosomes (early endosome antigen 1), indicating a lack of integrity in the endolysosomal compartments. NK cells from patients with CHS had normal cytokine compartments and cytokine secretion. LYST is involved in regulation of multiple aspects of NK cell lytic activity, ranging from governance of lytic granule size to control of their polarization and exocytosis, as well as regulation of endolysosomal compartment identity. LYST functions in the regulated exocytosis but not in the constitutive secretion pathway.

Editorial: secretion of cytokines and chemokines by innate immune cells.

Editorial: secretion of cytokines and chemokines by innate immune cells.

Serine/threonine kinase 16 and MAL2 regulate constitutive secretion of soluble cargo in hepatic cells.

MAL2 (myelin and lymphocyte protein 2) is thought to regulate at least two steps in the hepatic apical transcytotic pathway. As vesicle budding and delivery at each step are driven by complex machineries, we predicted that MAL2 participates in several large protein complexes with multiple binding partners. To identify novel MAL2 interactors, we performed split-ubiquitin yeast two-hybrid assays and identified STK16 (serine/threonine kinase 16) as a putative interactor which we verified morphologically and biochemically. As STK16 is a Golgi-associated constitutively active kinase implicated in regulating secretion and because of the massive constitutive secretory capacity of hepatic cells, we tested whether MAL2 and STK16 function in secretion. Expression of a dominant-negative kinase-dead STK16 mutant (E202A) or knockdown of MAL2 impaired secretion that correlated with decreased expression of albumin and haptoglobin. By using 19°C temperature blocks and lysosome deacidification, we determined that E202A expression or MAL2 knockdown did not interfere with albumin synthesis or processing, but led to albumin lysosomal degradation. We conclude that MAL2 and the constitutively active STK16 function to sort secretory soluble cargo into the constitutive secretory pathway at the TGN (trans-Golgi network) in polarized hepatocytes.

Repression of liver colorectal metastasis by the serpin Spn4A a naturally occurring inhibitor of the constitutive secretory proprotein convertases.

Liver is the most common site of metastasis from colorectal cancers, and liver of patients with liver colorectal metastasis have abnormal levels of the proprotein convertases (PCs). These proteases are involved in the activation and/or expression of various colon cancer-related mediators, making them promising targets in colorectal liver metastasis therapy. Here, we revealed that the serpin Spn4 from Drosophila melanogaster inhibits the activity of all the PCs found in the constitutive secretory pathway and represses the metastatic potential of the colon cancer cells HT-29 and CT-26. In these cells, Spn4A inhibited the processing of the PCs substrates IGF-1R and PDGF-A that associated their reduced anchorage-independent growth, invasiveness and survival in response to apoptotic agents. In vivo, Spn4A-expressing tumor cells showed repressed subcutaneous tumor development and liver metastases formation in response to their intrasplenic inoculation. In these cells Spn4A induced the expression of molecules with anti-metastatic functions and inhibited expression of pro-tumorigenic molecules. Taken together, our findings identify Spn4A as the only endogenous inhibitor of all the constitutive secretory pathway PCs, which is able to repress the metastatic potential of colon cancer cells. These results suggest the potential use of Spn4A and/or derivates as a useful adduct colorectal liver metastasis prevention.

Toward gene therapy for growth hormone deficiency via salivary gland expression of growth hormone

Salivary glands are useful targets for gene therapeutics. After gene transfer into salivary glands, regulated secretory pathway proteins, such as human growth hormone, are secreted into saliva, whereas constitutive secretory pathway proteins, such as erythropoietin, are secreted into the bloodstream. Secretion of human growth hormone (hGH) into the saliva is not therapeutically useful. In this study, we attempted to redirect the secretion of transgenic hGH from the saliva to the serum by site-directed mutagenesis. We tested hGH mutants first in vitro with AtT20 cells, a model endocrine cell line that exhibits polarized secretion of regulated secretory pathway proteins. Selected mutants were further studied in vivo using adenoviral-mediated gene transfer to rat submandibular glands. We identified two mutants with differences in secretion behavior compared to wild-type hGH. One mutant, ΔN1-6 , was detected in the serum of transduced rats, demonstrating that expression of this mutant in the salivary gland resulted in its secretion through the constitutive secretory pathway. This study demonstrates that mutagenesis of therapeutic proteins normally destined for the regulated secretory pathway may result in their secretion via the constitutive secretory pathway into the circulation for potential therapeutic benefit.

An Amphipathic Alpha-Helix in the Prodomain of Cocaine and Amphetamine Regulated Transcript Peptide Precursor Serves as Its Sorting Signal to the Regulated Secretory Pathway

Cocaine and Amphetamine Regulated Transcript (CART) peptides are anorexigenic neuropeptides. The L34F mutation in human CART peptide precursor (proCART) has been linked to obesity (Yanik et al. Endocrinology 147: 39, 2006). Decrease in CART peptide levels in individuals carrying the L34F mutation was attributed to proCART subcellular missorting. We studied proCART features required to enter the regulated secretory pathway. The subcellular localization and the secretion mode of monomeric EGFP fused to the full-length or truncated forms of human proCART transiently transfected in PC12 cells were analyzed. Our results showed that the N-terminal 1–41 fragment of proCART was necessary and sufficient to sort proCART to the regulated secretory pathway. In silico modeling predicted an alpha-helix structure located between residues 24–37 of proCART. Helical wheel projection of proCART alpha-helix showed an amphipathic configuration. The L34F mutation does not modify the amphipathicity of proCART alpha-helix and consistently proCARTL34F was efficiently sorted to the regulated secretory pathway. However, four additional mutations to proCARTL34F that reduced its alpha-helix amphipathicity resulted in the missorting of the mutated proCART toward the constitutive secretory pathway. These findings show that an amphipathic alpha-helix is a key cis-structure for the proCART sorting mechanism. In addition, our results indicate that the association between L34F mutation and obesity is not explained by proCART missorting.