Sonic Hedgehog as a mediator of long-range signaling

The ability of Hedgehog (Hh) proteins to exert their biological effects is regulated by a series of post-translational processes. These processes include an intramolecular cleavage, covalent addition of cholesterol and/or palmitate, and conversion into a multimeric freely diffusible form. The processing of Hh proteins affects their trafficking, potency, and ability to signal over many cell diameters. Accordingly, the loss of gene products required for these processes abrogates the Hh proteins' abilities to exert their effects, which can be long range, short range, or both. We review here recent evidence demonstrating that Hh proteins are directly responsible for their long-range biological effects. Additionally, we integrate both genetic and biochemical data to delineate a model illustrating how the unusual biochemistry of Hh family members may allow them to act as morphogens, signaling over both short and long distances.     

HIV-1 enhancing effect of prostatic acid phosphatase peptides is reduced in human seminal plasma

We recently reported that HIV-1 infection can be inhibited by innate antimicrobial components of human seminal plasma (SP). Conversely, naturally occurring peptidic fragments from the SP-derived prostatic acid phosphatase (PAP) have been reported to form amyloid fibrils called "SEVI" and enhance HIV-1 infection in vitro. In order to understand the biological consequence of this proviral effect, we extended these studies in the presence of human SP. PAP-derived peptides were agitated to form SEVI and incubated in the presence or absence of SP. While PAP-derived peptides and SEVI alone were proviral, the presence of 1% SP ablated their proviral activity in several different anti-HIV-1 assays. The anti-HIV-1 activity of SP was concentration dependent and was reduced following filtration. Supraphysiological concentrations of PAP peptides and SEVI incubated with diluted SP were degraded within hours, with SP exhibiting proteolytic activity at dilutions as high as 1:200. Sub-physiological concentrations of two prominent proteases of SP, prostate-specific antigen (PSA) and matriptase, could degrade physiological and supraphysiological concentrations of PAP peptides and SEVI. While human SP is a complex biological fluid, containing both antiviral and proviral factors, our results suggest that PAP peptides and SEVI may be subject to naturally occurring proteolytic components capable of reducing their proviral activity.     

Regulation of C-type Lectin Antimicrobial Activity by a Flexible N-terminal Prosegment

Members of the RegIII family of intestinal C-type lectins are directly antibacterial proteins that play a vital role in maintaining host-bacterial homeostasis in the mammalian gut, yet little is known about the mechanisms that regulate their biological activity. Here we show that the antibacterial activities of mouse RegIIIγ and its human ortholog, HIP/PAP, are tightly controlled by an inhibitory N-terminal prosegment that is removed by trypsin in vivo. NMR spectroscopy revealed a high degree of conformational flexibility in the HIP/PAP inhibitory prosegment, and mutation of either acidic prosegment residues or basic core protein residues disrupted prosegment inhibitory activity. NMR analyses of pro-HIP/PAP variants revealed distinctive colinear backbone amide chemical shift changes that correlated with antibacterial activity, suggesting that prosegment-HIP/PAP interactions are linked to a two-state conformational switch between biologically active and inactive protein states. These findings reveal a novel regulatory mechanism governing C-type lectin biological function and yield new insight into the control of intestinal innate immunity.The gastrointestinal tracts of mammals are heavily colonized with vast symbiotic microbial communities and are also a major portal of entry for bacterial pathogens. To cope with these complex microbial challenges, intestinal epithelial cells produce a diverse repertoire of protein antibiotics from multiple distinct protein families (1). These proteins are secreted apically into the luminal environment of the intestine where they play a pivotal role in protecting against enteric infections (2, 3) and may also function to limit opportunistic invasion by symbiotic bacteria (4).We previously identified lectins as a novel class of secreted antibacterial proteins in the mammalian intestine. RegIIIγ is a member of the RegIII subgroup of the C-type lectin family and is expressed in the small intestine in response to microbial cues (5), stored in epithelial cell secretory granules, and released into the small intestinal lumen (5). Similarly, HIP/PAP (hepatointestinal pancreatic/pancreatitis-associated protein; the human ortholog of RegIIIγ)⁶ is expressed in the human intestine (6) and is up-regulated in patients with inflammatory bowel disease (7). These proteins are produced in multiple epithelial lineages, including enterocytes and Paneth cells (5, 6). Both RegIIIγ and HIP/PAP are directly bactericidal at low micromolar concentrations for Gram-positive bacteria (5), revealing a previously unappreciated biological function for mammalian lectins. The antibacterial functions of RegIIIγ and HIP/PAP are dependent upon binding bacterial targets through interactions with peptidoglycan (5). As peptidoglycan is localized on surfaces of Gram-positive bacteria but is buried in the periplasmic space of Gram-negative bacteria, this binding activity provides a molecular explanation for the Gram-positive specific bactericidal effects of these lectins. Although the mechanism of lectin-mediated antibacterial activity remains unclear, RegIIIγ and HIP/PAP have been shown to elicit extensive damage to the cell surfaces of targeted bacteria (5).In this study, we show that C-type lectin bactericidal activity is under stringent post-translational control. RegIIIγ and HIP/PAP each undergo in vivo proteolytic removal of a flexible anionic N-terminal prosegment that maintains the proteins in a biologically inactive state. NMR spectroscopy suggests that the prosegment functions by controlling a two-state conformational switch between the biologically active and inactive states of the protein. We propose that this regulatory mechanism allows the host to restrict expression of RegIII lectin antibacterial activity to the intestinal lumen. Together, our findings represent a unique example of post-translational control of C-type lectin biological activity, and provide novel insight into the regulation of lectin-mediated innate immunity in the mammalian intestine.     

The concept of the CCN protein family revisited: a centralized coordination network

The wide array of biological properties attributed to the CCN family of proteins (Perbal in Lancet 363(9402):62–64, 2004) led me to reconsider the possible relationship and roles that these proteins may play as a team, instead of acting on their own as individual regulators in various signaling pathways. The dynamic model which I present in this review stems from the contribution of the biological properties that we established for CCN3, one of the three founding members of the CCN family, which was identified by our group as the first CCN protein showing growth inhibitory properties (1992), expressed mainly in quiescent cells (1996), and showing anti-tumor activities in several cellular models both ex vivo and in vivo. At the present time CCN3 is the only member of the family that has been reported to negatively act on the progression of the cell cycle. The unique dual localisation of CCN3 in the nucleus and outside cells, either at the membrane or in the extracellular matrix, that I first established in 1999, and that now appears to be shared by several other CCN proteins, is a unique essential feature which can no longer be ignored. Based on the structural and functional properties of CCN3, shared by most of the CCN family members, I propose an « all in one » concept in which CCN proteins are team members with specific functions that are aimed at the same goal. This model accounts both for the functional specificity of the various CCN proteins, their sequential and opposite or complementary effects in various biological context, and for the biological consequences of their physical interaction and biological cross-regulation.     

HIP/PAP, a C-type lectin overexpressed in hepatocellular carcinoma, binds the RII alpha regulatory subunit of cAMP-dependent protein kinase and alters the cAMP-dependent protein kinase signalling.

HIP/PAP is a C-type lectin overexpressed in hepatocellular carcinoma (HCC). Pleiotropic biological activities have been ascribed to this protein, but little is known about the function of HIP/PAP in the liver. In this study, therefore, we searched for proteins interacting with HIP/PAP by screening a HCC cDNA expression library. We have identified the RII alpha regulatory subunit of cAMP-dependent protein kinase (PKA) as a partner of HIP/PAP. HIP/PAP and RII alpha were coimmunoprecipitated in HIP/PAP expressing cells. The biological relevance of the interaction between these proteins was established by demonstrating, using fractionation methods, that they are located in a same subcellular compartment. Indeed, though HIP/PAP is a protein secreted via the Golgi apparatus we showed that a fraction of HIP/PAP escaped the secretory apparatus and was recovered in the cytosol. Basal PKA activity was increased in HIP/PAP expressing cells, suggesting that HIP/PAP may alter PKA signalling. Indeed, we showed, using a thymidine kinase-luciferase reporter plasmid in which a cAMP responsive element was inserted upstream of the thymidine kinase promoter, that luciferase activity was enhanced in HIP/PAP expressing cells. Thus our findings suggest a novel mechanism for the biological activity of the HIP/PAP lectin.     

Secretory apparatus assessed by analysis of pancreatic secretory stress protein expression in a rat model of chronic pancreatitis

Secretory stress proteins (SSP) are a family of proteins including isoforms of pancreatitis-associated protein (PAP) and pancreatic stone protein (PSP/reg). In vitro exposure to trypsin results in the formation of insoluble fibrillar structures. SSP are constitutively secreted into pancreatic juice at low levels. The WBN/Kob rat is a model for chronic pancreatitis, displaying focal inflammation, destruction of the parenchyma and changes in the architecture of the acinar cell; the synthesis and secretion of SSP are also increased. We have investigated the secretory apparatus by SSP immunohistochemistry at the light- and electron-microscopical (EM) levels. Immunocytochemistry of PSP/reg in Wistar control rats reveals low levels, with individual acinar cells exhibiting high immunoreactivity in zymogen granules. PAP is not detectable. In the WBN/Kob rat, PSP/reg and PAP immunoreactivity is markedly increased. Double immunofluorescence for PSP/reg and PAP I or II demonstrates that these proteins colocalize to the same cell. Acinar cells change their secretory architecture by fusion of zymogen granules and elongation of the fused organelles. The immunogold technique has demonstrated an increase of SSP in zymogen granules in WBN/Kob rats. PSP/reg-positive zymogen granules fuse to form elongated structures with fibrillar contents. An extensive PSP/reg-positive fibrillar network is established in the cytosol. Extracellular fibrils have been observed in several ductules. Thus, SSP-derived fibrils form concomitantly with acinar damage in the WBN/Kob rat. Based on the known tryptic cleavage site of SSP, the in vivo generation of fibrils is presumably the result of premature trypsin activation.     

Expression of Reg/PAP family members during motor nerve regeneration in rat

In this study, we examined the expression of mRNAs for Regenerating gene (Reg)/pancreatitis-associated protein (PAP) family members following hypoglossal nerve injury in rats. In addition to four rat family members (RegI, Reg-2/PAP I, PAP II, and PAP III) that had been identified, we newly cloned and sequenced a type-IV Reg gene in rats. Among these five family members, the expression of Reg-2/PAP I mRNA was predominantly enhanced in injured motor neurons after axotomy. Furthermore, a marked induction of PAP III mRNA was observed in the distal part of the injured nerve. A polyclonal antibody was raised against PAP III, and a Western blotting analysis using this antibody confirmed an increased level of PAP III protein in the injured nerve. These results suggest that Reg family members would be new mediators among injured neurons and glial cells, and may play pivotal roles during nerve regeneration.     

Characterization of p51/52, a cell-growth regulated protein of WI-38 cells.

A number of proteins have been identified whose expression or activity is regulated by cell growth. We have produced a monoclonal antibody against a new cell-growth regulated protein found in normal human fibroblasts. We have shown that this antibody recognizes a 51/52-kDa doublet (p51/52) found mainly in normal cells. This doublet is sensitive to degradation by the calcium-activated protease, calpain, breaking down to a 37/38-kDa doublet. The relative amount of the two members of the 51/52-kDa doublet changes when serum-starved cells reenter the cell cycle. Quiescent cells express mainly the 51-kDa form; the 52-kDa form becomes more abundant upon refeeding serum-starved cells. Transformed cells express either very small amounts of this doublet, and then predominantly the 52-kDa form, or no detectable amount of either form. These characteristics distinguish this molecule from several other known growth-regulated proteins such as statin and the anti-oncogene p53.     

Identification of a carboxyl-terminal diaphanous-related formin homology protein autoregulatory domain

Mammalian and fungal Diaphanous-related formin homology (DRF) proteins contain several regions of conserved sequence homology. These include an amino-terminal GTPase binding domain (GBD) that interacts with activated Rho family members and formin homology domains that mediate targeting or interactions with signaling kinases and actin-binding proteins. DRFs also contain a conserved Dia-autoregulatory domain (DAD) in their carboxyl termini that binds the GBD. The GBD is a bifunctional autoinhibitory domain that is regulated by activated Rho. Expression of the isolated DAD in cells causes actin fiber formation and stimulates serum response factor-regulated gene expression. Inhibitor experiments show that the effects of exogenous DAD expression are dependent upon cellular Dia proteins. Alanine substitution of DAD consensus residues that disrupt GBD binding also eliminate DAD biological activity. Thus, DAD expression activates nuclear signaling and actin remodeling by mimicking activated Rho and unlatching the autoinhibited state of the cellular complement of Dia proteins.     

Apoptosis: a mitochondrial perspective on cell death

Mitochondria play an important role in both the life and death of cells. The past 7-8 years have seen an intense surge in research devoted toward understanding the critical role of mitochondria in the regulation of cell death. Mitochondria have, next to their function in respiration, an important role in apoptotic signaling pathway. Apoptosis is a form of programmed cell death important in the development and tissue homeostasis of multicellular organisms. Apoptosis can be initiated by a wide array of stimuli, including multiple signaling pathways that, for the most part, converge at the mitochondria. Although classically considered the powerhouses of the cell, it is now understood that mitochondria are also "gatekeepers" that ultimately determine the fate of the cell. Malfunctioning at any level of the cell is eventually translated in the release of apoptogenic factors from the mitochondrial intermembrane space resulting in the organized demise of the cell. These mitochondrial factors may contribute to both caspase-dependent and caspase-independent processes in apoptotic cell death. In addition, several Bcl-2 family members and other upstream proteins also contribute to and regulate the apoptosis. In this review, we attempt to summarize our current view of the mechanism that leads to the influx and efflux of many proteins from/to mitochondria during apoptosis.     

The LOV domain: a chromophore module servicing multiple photoreceptors

Three different families of blue-light receptors have been characterized from higher plants: three cryptochromes, two phototropins, and the three members of the ZTL/ADO family. Phototropins and the ZTL/ADO proteins have chromophore modules, designated LOV domains, that bind flavin mononucleotide and undergo formation of a C(4a) flavin-cysteinyl adduct. All contain the highly conserved amino acid motif GXNCRFLQ. Over 90 prokaryote proteins also contain LOV domains with this motif upstream from one of several different functional groups. All of these that have been investigated to date act as photoreceptors in vitro and form the adduct upon irradiation. Four members of the class LOV-histidine kinase, one from a plant pathogen (Pseudomonas syringae), one from an animal pathogen Brucella melitensis), and two from a marine bacterium (Erythrobacter litoralis) respectively, mediate light-activated histidine phosphorylation. Decay of the adduct in darkness after a blue light pulse coincides with loss of the capacity for phosphorylation upon addition of ATP. At present, the biological role(s) of these light-sensitive proteins is under investigation.     

A family of 16-kDa pancreatic secretory stress proteins form highly organized fibrillar structures upon tryptic activation

A group of 16-kDa proteins, synthesized and secreted by rat pancreatic acinar cells and composed of pancreatic stone protein (PSP/reg) and isoforms of pancreatitis-associated protein (PAP), show structural homologies, including conserved amino acid sequences, cysteine residues, and highly sensitive N-terminal trypsin cleavage sites, as well as conserved functional responses in conditions of pancreatic stress. Trypsin activation of recombinant stress proteins or counterparts contained in rat pancreatic juice (PSP/reg, PAP I and PAP III) resulted in conversion of 16-kDa soluble proteins into 14-kDa soluble isoforms (pancreatic thread protein and pancreatitis-associated thread protein, respectively) that rapidly polymerize into insoluble sedimenting structures. Activated thread proteins show long lived resistance to a wide spectrum of proteases contained in pancreatic juice, including serine proteases and metalloproteinases. In contrast, PAP II, following activation with trypsin or pancreatic juice, does not form insoluble structures and is rapidly digested by pancreatic proteases. Scanning and transmission electron microscopy indicate that activated thread proteins polymerize into highly organized fibrillar structures with helical configurations. Through bundling, branching, and extension processes, these fibrillar structures form dense matrices that span large topological surfaces. These findings suggest that PSP/reg and PAP I and III isoforms consist of a family of highly regulated soluble secretory stress proteins, which, upon trypsin activation, convert into a family of insoluble helical thread proteins. Dense extracellular matrices, composed of helical thread proteins organized into higher ordered matrix structures, may serve physiological functions within luminal compartments in the exocrine pancreas.     

Recent progress in the study of Hedgehog signaling

The Hedgehog (Hh) family of secreted signaling proteins plays a critical role in regulating the development of several tissues and organ systems.The ability of Hh proteins to exert their biological effects is regulated by a series of post-translational processes.These processes include an intramolecular cleavage,covalent addition of cholesterol and/or palmitate,and conversion into a multimeric freely diffusible form.The processing of Hh proteins affects their trafficking,potency,and ability to signal over several cell diameters.Here we review the current understanding of the Hh signaling mechanisms that govern the establishment of the Hh gradient and the transduction of the Hh signal in the light of recent data.     

Caulollins from Caulobacter crescentus, a pair of partially unstructured proteins of betagamma-crystallin superfamily, gain structure upon binding calcium

The betagamma-crystallin superfamily comprises members from various taxa and species, which have similar domain topologies as that of lens beta- and gamma-crystallins. We have studied new microbial members of this understudied betagamma-crystallin superfamily from the bacterium Caulobacter crescentus. These proteins, which we named "caulollins", are paralogues with a single betagamma-crystallin domain, made up of two Greek key motifs with AB-type arrangement seen in gamma-crystallin. The second Greek key motif has Cys in place of a generally conserved Phe/Tyr residue, and the Tyr corner, considered important for the proper betagamma-crystallin fold, is missing, making this a sequentially diverse atypical betagamma-crystallin domain. This atypical domain binds two Ca2+ with moderate affinity (0.8-20 microM). In apo form, caulollins are partially unstructured proteins and gain structure upon binding Ca2+. Unlike many other microbial betagamma-crystallin domains, this domain is monomeric, though in the presence of Ca2+ it becomes more compact. Ca2+ binding increases the intrinsic stability of proteins, suggesting the role of Ca2+ as an extrinsic stabilizer. N-Terminal extension does not play any role in modulating Ca2+ binding, intrinsic stability, or oligomerization. We noted that there are several such variant domains in the genomes of unrelated species. It appears that caulollins along with these members form a subfamily in the betagamma-crystallin superfamily that would be partially unstructured in apo form, unlike many other domains from lens or microbial crystallins. This work further suggests that Ca2+ binding is a widespread feature of the betagamma-crystallin superfamily.     

Bovine Chromaffin Cells Release a Transforming Growth Factor-β-Like Molecule Contained Within Chromaffin Granules

Abstract: Bovine chromaffin cells contain within their storage vesicles and release upon cholinergic stimulation a complex mixture of proteins and peptides. We present data suggesting that one of these proteins resembles transforming growth factor (TGF)-β in terms of its biological activity. The assay used to assess the activity of TGF-β is based on cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct. The assay is highly specific in detecting TGF-β1, -β2, and -β3 but does not detect several cytokines and growth factors, such as fibroblast growth factor-2, transforming growth factor-α, platelet-derived growth factor-AB, insulin-like growth factor-I, or neurotrophin-3 or -4. Moreover, we show that this assay does not detect a wide range of TGF-β superfamily members (activin A, bone morphogenetic protein-2, -4, -6, and -7, growth/differentiation factor-5, and glial cell line-derived neurotrophic factor). Chromaffin granules contain ∼1 ng of TGF-β/10 mg of protein. The biological activity elicited by the chromaffin granule component can be neutralized by using an antibody against TGF-β1/β2/β3. TGF-β is releasable from cultured chromaffin cells stimulated with the cholinergic agonist carbachol (10⁻⁵ M ). These data suggest that TGF-β is stored in chromaffin granules and can be released by exocytosis.