C-terminal Basic Residues Research Articles

Neuropeptidomics of Genetically Defined Cell Types in Mouse Brain.

Peptidomic techniques are powerful tools to identify peptides in a biological sample. In the case of brain, which contains a complex mixture of cell types, standard peptidomics procedures reveal the major peptides in a dissected brain region. It is difficult to obtain information on peptides within a specific cell type using standard approaches, unless that cell type can be isolated. This protocol describes a targeted peptidomic approach that uses affinity chromatography to purify peptides that are substrates of carboxypeptidase E (CPE), an enzyme present in the secretory pathway of neuroendocrine cells. Many CPE products function as neuropeptides and/or peptide hormones, and therefore represent an important subset of the peptidome. Because CPE removes C-terminal Lys and Arg residues from peptide processing intermediates, organisms lacking CPE show a large decrease in the levels of the mature forms of most neuropeptides and peptide hormones, and a very large increase in the levels of the processing intermediates that contain C-terminal Lys and/or Arg (i.e., the CPE substrates). These CPE substrates can be purified on an anhydrotrypsin-agarose affinity resin, which specifically binds peptides with C-terminal basic residues. When this method is used with mice lacking CPE activity in genetically defined cell types, it allows the detection of peptides specifically produced in that cell type.

Brain Expression of CPB2 and Effects of Cpb2 Deficiency in Mouse Models of Behavior.

Procarboxypeptidase B2 (proCPB2 or TAFI) is a zymogen that after activation cleaves C-terminal basic residues from peptides or proteins with many identified targets. A splice variant of CPB2 has been found in the brain lacking essential residues for its carboxypeptidase function. The aim was to determine CPB2 expression in the brain and effects of CPB2 deficiency (Cpb2 -/-) on behavior. Behavioral effects were tested by comparing Cpb2 -/- mice in short-term (open field and elevated zero maze tests) and long-term (Phenotyper) observations with wild-type (WT) controls. Long-term observation compared day 1 (acclimatizing to novel environment) to day 4 (fully acclimatized) with the inactive (day) and active (night) periods analyzed separately. Brain expression of CPB2 mRNA and protein was interrogated in publicly available databases. Long-term observation demonstrated differences between WT and Cpb2 -/- mice in several parameters. For example, Cpb2 -/- mice moved more frequently on both days 1 and 4, especially in the normally inactive periods. Cpb2 -/- mice spent more time on the shelter and less time in it. Differences were more pronounced on day 4 after the mice had fully acclimatized. In short-term observations, no differences were observed between Cpb2 -/- mice and WT mice. Brain expression of CBP2 was not detectable in the human protein atlas. Databases of single-cell RNAseq did not show expression of CPB2 mRNA in either human or mouse brain. Continuous observation of home-cage behavior suggests that Cpb2 -/- mice are more active than WT mice, show different day-night activity levels, and might have a different way of processing information.

In vivo characterization of the maturation steps of a pigment dispersing factor neuropeptide precursor in the Drosophila circadian pacemaker neurons.

Pigment dispersing factor (PDF) is a key signaling molecule coordinating the neuronal network associated with the circadian rhythms in Drosophila. The precursor (proPDF) of the mature PDF (mPDF) consists of 2 motifs, a larger PDF-associated peptide (PAP) and PDF. Through cleavage and amidation, the proPDF is predicted to produce cleaved-PAP (cPAP) and mPDF. To delve into the in vivo mechanisms underlying proPDF maturation, we generated various mutations that eliminate putative processing sites and then analyzed the effect of each mutation on the production of cPAP and mPDF by 4 different antibodies in both ectopic and endogenous conditions. We also assessed the knockdown effects of processing enzymes on the proPDF maturation. At the functional level, circadian phenotypes were measured for all mutants and knockdown lines. As results, we confirm the roles of key enzymes and their target residues: Amontillado (Amon) for the cleavage at the consensus dibasic KR site, Silver (Svr) for the removal of C-terminal basic residues from the intermediates, PAP-KR and PDF-GK, derived from proPDF, and PHM (peptidylglycine-α-hydroxylating monooxygenase) for the amidation of PDF. Our results suggest that the C-terminal amidation occurs independently of proPDF cleavage. Moreover, the PAP domain is important for the proPDF trafficking into the secretory vesicles and a close association between cPAP and mPDF following cleavage seems required for their stability within the vesicles. These studies highlight the biological significance of individual processing steps and the roles of the PAP for the stability and function of mPDF which is essential for the circadian clockworks.

C-Terminal Basic Residues of Troponin T Facilitate Troponin I Transitions during Activation

During activation of striated muscle, the inhibitory region of TnI moves from actin toward TnC allowing tropomyosin to adopt an activating position on actin. Basic amino acid residues within the C-terminal 14 residues of human cardiac TnT limit the extent of activation and are required for full relaxation (Johnson et al., 2018, Biophys J). FRET measurements show that those basic residues alter Ca2+-induced structural changes. With wild type TnT the distance between Cys 143 of TnI (within the inhibitory region) and Cys 190 of tropomyosin increased upon Ca2+ activation while the distance between Cys 143 of TnI and Cys 84 of TnC decreased.

BMP6 binding to heparin and heparan sulfate is mediated by N-terminal and C-terminal clustered basic residues

BackgroundThe bone morphogenetic protein 6 (BMP6) is a crucial inducer of hepcidin, the peptide hormone that regulates the iron availability in our body. Hepcidin expression is influenced by hepatic heparan sulfate (HS) and by heparin administration, suggesting BMP6 interaction with heparin/HS. The BMP2/4 subfamily has been deeply characterized to have a N-terminal heparin/HS binding domain (HBD), whose basic residues contact the sulfate groups on heparin and HS. Such detailed characterization is still required for other, structurally different BMPs, including BMP6. MethodsBMP6 peptides encompassing potential HBDs were analysed on heparin-functionalized plates and microcantilevers, and on membrane HS expressing CHO-K1 cells. Monomeric wild-type BMP6 and mutants were produced, substituting the basic residues with non-charged ones, and their affinity to the heparin-column was measured. The BMP6-heparin interaction was also predicted at atomic level by in silico molecular dynamics. ResultsN-terminal and C-terminal BMP6 peptides showed high heparin affinity in solid-phase assays. The mutation of the two sites (R5L, R6S, R7L and K126N, K127N, R129S) abolished the heparin-binding activity of the recombinant monomeric BMP6. Monomeric BMP6 and peptides specifically bound to membrane HS of CHO-K1 cells through the same domains. Molecular dynamic studies supported the role of the two HBDs, suggesting a cooperative behaviour. ConclusionsIn BMP6, N-terminal (R5, R6, R7) and C-terminal (K126, K127, R129) domains mediate the interaction with heparin and HS. General significanceThis study provides the molecular mechanism supporting the use of heparin to sequester BMP6 and inhibit hepcidin expression, a novel clinical approach for high-hepcidin iron disorders.

Neuropeptidomic Analysis of a Genetically Defined Cell Type in Mouse Brain and Pituitary

Neuropeptides and peptide hormones are important cell-cell signaling molecules that mediate many physiological processes. Unlike classic neurotransmitters, peptides undergo cell-type-specific post-translational modifications that affect their biological activity. To enable the identification of the peptide repertoire of a genetically defined cell type, we generated mice with a conditional disruption of the gene for carboxypeptidase E (Cpe), an essential neuropeptide-processing enzyme. The loss of Cpe leads to accumulation of neuropeptide precursors containing C-terminal basic residues, which serve as tags for affinity purification. The purified peptides are subsequently identified using quantitative peptidomics, thereby revealing the specific forms of neuropeptides in cells with the disrupted Cpe gene. To validate the method, we used mice expressing Cre recombinase under the proopiomelanocortin (Pomc) promoter and analyzed hypothalamic and pituitary extracts, detecting peptides derived from proopiomelanocortin (as expected) and also proSAAS in POMC neurons. This technique enables the analyses of specific forms of peptides in any Cre-expressing cell type.

The E. coli transcription factor GrlA is regulated by subcellular compartmentalization and activated in response to mechanical stimuli

Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen that colonizes the gastrointestinal tract and has evolved intricate mechanisms to sense and respond to the host environment. Upon the sensation of chemical and physical cues specific to the host's intestinal environment, locus of enterocyte effacement (LEE)-encoded virulence genes are activated and promote intestinal colonization. The LEE transcriptional activator GrlA mediates EHEC's response to mechanical cues characteristic of the intestinal niche, including adhesive force that results from bacterial adherence to epithelial cells and fluid shear that results from intestinal motility and transit. GrlA expression and release from its inhibitor GrlR was not sufficient to induce virulence gene transcription; mechanical stimuli were required for GrlA activation. The exact mechanism of GrlA activation, however, remained unknown. We isolated GrlA mutants that activate LEE transcription, independent of applied mechanical stimuli. In nonstimulated EHEC, wild-type GrlA associates with cardiolipin membrane domains via a patch of basic C-terminal residues, and this membrane sequestration is disrupted in EHEC that expresses constitutively active GrlA mutants. GrlA transitions from an inactive, membrane-associated state and relocalizes to the cytoplasm in response to mechanical stimuli, allowing GrlA to bind and activate the LEE1 promoter. GrlA expression and its relocalization in response to mechanical stimuli are required for optimal virulence regulation and colonization of the host intestinal tract during infection. These data suggest a posttranslational regulatory mechanism of the mechanosensor GrlA, whereby virulence gene expression can be rapidly fine-tuned in response to the highly dynamic spatiotemporal mechanical profile of the gastrointestinal tract.

Basic residues within the cardiac troponin T C terminus are required for full inhibition of muscle contraction and limit activation by calcium

Striated muscle is activated by myosin- and actin-linked processes, with the latter being regulated through changes in the position of tropomyosin relative to the actin surface. The C-terminal region of cardiac troponin T (TnT), a tropomyosin-associated protein, is required for full TnT inactivation at low Ca2+ and for limiting its activation at saturating Ca2+ Here, we investigated whether basic residues in this TnT region are involved in these activities, whether the TnT C terminus undergoes Ca2+-dependent conformational changes, and whether these residues affect cardiac muscle contraction. We generated a human cardiac TnT variant in which we replaced seven C-terminal Lys and Arg residues with Ala and added a Cys residue at either position 289 or 275 to affix a fluorescent probe. At pCa 3.7, actin filaments containing high-alanine TnT had an elevated ATPase rate like that obtained when the last TnT 14 residues were deleted. Acrylodan-tropomyosin fluorescence changes and S1-actin binding kinetics revealed that at pCa 8, the high-alanine TnT-containing filaments did not enter the first inactive state. FRET analyses indicated that the C-terminal TnT region approached Cys-190 of tropomyosin as actin filaments transitioned to the inactive B state; that transition was abolished with high-alanine TnT. High-alanine TnT-containing cardiac muscle preparations had increased Ca2+ sensitivity of both steady-state isometric force and sinusoidal stiffness as well as increased maximum steady-state isometric force and sinusoidal stiffness. We conclude that C-terminal basic residues in cardiac TnT are critical for the regulation of cardiac muscle contraction.

Affinity Purification of Neuropeptide Precursors from Mice Lacking Carboxypeptidase E Activity.

Peptidomic techniques are powerful tools to identify peptides in a biological sample. This protocol describes a targeted peptidomic approach that uses affinity chromatography to purify peptides that are substrates of carboxypeptidase E (CPE), an enzyme present in the secretory pathway of neuroendocrine cells. Many CPE products function as neuropeptides and/or peptide hormones, and therefore represent an important subset of the peptidome. Because CPE removes C-terminal Lys and Arg residues from peptide-processing intermediates, organisms lacking CPE show a large decrease in the levels of the mature forms of most neuropeptides and peptide hormones, and a very large increase in the levels of the processing intermediates that contain C-terminal Lys and/or Arg (i.e., the CPE substrates). These CPE substrates can be purified on an anhydrotrypsin-agarose affinity resin, which specifically binds peptides with C-terminal basic residues. Not all peptides with basic C-terminal residues within a cell are CPE substrates, and these other peptides will also be purified on the anhydrotrypsin affinity column. However, a comparison of peptides purified from wild-type mice and from mice lacking CPE allows for the rapid identification of CPE substrates based on their large increase in the absence of CPE.

Abstract 1967: A novel 40kDa CPE-ΔN isoform promotes proliferation and invasion in pancreatic cancer cells

Abstract Carboxypeptidase E (CPE) is a prohormone processing exopeptidase that cleaves C-terminal basic residues from peptide hormones liberated endoproteolytically from prohormones. It is a 476- amino acid protein with a signal peptide in its N-terminus and is mainly expressed in neuropeptide-rich areas of brain and endocrine tissues. As a multifunctional protein, CPE plays many non-enzymatic roles such as prohormone sorting, vesicle transport, and secretion in addition to its carboxypeptidase activity. Moreover, high expression of CPE is found in metastatic tumor tissues, including liver cancer, renal clear cell carcinoma, colorectal cancer, cervical cancer and melanoma, suggesting CPE may be involved in tumor metastasis and progression. In this study, a novel CPE splice isoform mRNA was identified by Northern blotting, which is ~1.83kb in size. RACE assay and sequence analysis confirmed existence of this CPE isoform mRNA, which partially lacks a N-terminus region, and has a shorter 3’-UTR compared to WT-CPE mRNA, by removal of 188 nucleotides within the first exon and 589 nucleotides out of the 3’-UTR, respectively. Bioinformatics analysis revealed that this CPE isoform mRNA has a shortened ORF, which starts coding from the 3rd ATG relative to wild-type (WT) CPE mRNA and encodes a 40kDa CPE-αN protein with the N-terminus truncated. Western blot analysis showed that 40kD CPE-αN is expressed in multiple cancer cell lines and tumor tissues. In Panc-1pancreatic cancer cells, knockdown of endogenous CPE(s) inhibited cell growth and invasion; in contrast, ectopic expression of 40kD CPE isoform significantly promoted proliferation, colony formation, invasion and migration, whereas overexpression of WT-CPE had only a small effect. These results suggest that the 40kD CPE-αN isoform plays a role in cell growth and metastasis. Note: This abstract was not presented at the meeting. Citation Format: XuYu Yang, Cong Ling, Hong Lou, Loh Yoke Peng. A novel 40kDa CPE-ΔN isoform promotes proliferation and invasion in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1967. doi:10.1158/1538-7445.AM2017-1967

Dissecting carboxypeptidase E: properties, functions and pathophysiological roles in disease

Since discovery in 1982, carboxypeptidase E (CPE) has been shown to be involved in the biosynthesis of a wide range of neuropeptides and peptide hormones in endocrine tissues, and in the nervous system. This protein is produced from pro-CPE and exists in soluble and membrane forms. Membrane CPE mediates the targeting of prohormones to the regulated secretory pathway, while soluble CPE acts as an exopeptidase and cleaves C-terminal basic residues from peptide intermediates to generate bioactive peptides. CPE also participates in protein internalization, vesicle transport and regulation of signaling pathways. Therefore, in two types of CPE mutant mice, Cpefat/Cpefat and Cpe knockout, loss of normal CPE leads to a lot of disorders, including diabetes, hyperproinsulinemia, low bone mineral density and deficits in learning and memory. In addition, the potential roles of CPE and ΔN-CPE, an N-terminal truncated form, in tumorigenesis and diagnosis were also addressed. Herein, we focus on dissecting the pathophysiological roles of CPE in the endocrine and nervous systems, and related diseases.

The importance of evolutionarily conserved C-terminal basic residues for the stability of proapoptotic Bax protein.

Bax is a protein that promotes apoptosis (a form of cell death). The atomistic details of the mechanism by which Bax is activated during apoptosis remain a subject of debate. C‐terminal basic residues in the sequence of Bax show remarkable conservation across a variety of species. The role of these charged residues in the stability of Bax was investigated by submitting substituted mutants to molecular dynamics simulations at high temperatures. Mutation of either or both K189 and K190 led to dramatic changes in helical content, radius of gyration, proximity of the C terminus to the core of the protein, exposure of the BH3 domain, and bundling of the core. These results suggest a critical role of positively charged residues close to the C terminus in the structural stability of Bax.

Abstract 2507: Circulating proteolytic products of carboxypeptidase N for early detection of breast cancer

Abstract Background: Carboxypeptidase N (CPN) plays an important role in regulating vasoactive peptide hormones, growth factors, and cytokines by specifically cleaving their C-terminal basic residues. Herein we demonstrated that the circulating peptides specifically cleaved by CPN in the tumor microenvironment can indeed be stage-specific indicators of breast cancer. Methods: The activity of CPN was evaluated using an ex-vivo peptide cleavage assay, in which synthesized C3f peptide (His6-C3f_S1304-R1320-His6) is incubated in interstitial fluids of breast tumor and adjacent normal breast tissues in mice with orthotopic implantation of the human cell line MDA-MB-231. Fragment profiling, by an approach combining nanopore fractionation and mass spectrometry, revealed the nature and extent of cleavage by CPN. These results correlated with the level of CPN-catalyzed peptides in blood specimens taken from the tumor-bearing mice, healthy women and breast cancer patients. CPN expression in the same set of samples was further examined by immunohistochemistry and immunoblotting. Results: We showed that generation of C3f_R1310-L1319 specifically correlated with the CPN expression level. In both the mouse and clinical patient samples, the amount of CPN was clearly increased in tumor tissues compared to that seen in normal breast tissue, while its counterpart in blood remained constant. The amount of 6 CPN-catalyzed peptides predominantly increased in sera taken from the mice (n=8) at 2 weeks after orthotropic implantation. Six homologous peptides displayed the significantly higher expression in the patients' plasma as early as the first pathologic stage of breast cancer. Conclusions: The amount of circulating CPN-catalyzed peptides reported here reflects the extent of this enzyme's activity in tumors, and our results clearly indicate their strong potential as biomarkers for non-invasive and early diagnosis of breast cancer. It would certainly be naïve to think that CPN-catalyzed peptides and the enzyme itself are the only biomarkers specific for breast cancer diagnosis. We do however argue that they can be used as a complementary measure for very early diagnosis of tumor growth, particularly because CPN activity within the tumor at this point is already detectable by blood sampling. Such sensitivity together with all of the features mentioned above lend well to their translation to the clinic, for breast cancer and perhaps other pathologies. Citation Format: Ye Hu. Circulating proteolytic products of carboxypeptidase N for early detection of breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2507. doi:10.1158/1538-7445.AM2014-2507

Abstract LB-247: Circulating proteolytic products of tumor-resident enzyme as potential biomarkers for early detection of breast cancer

Abstract In theory, any or all of tumor-secreted proteins can serve as cancer biomarkers. However, the reality is challenging to monitor because of the large degree of fluctuation in abundance and localization of these tumor-secreted proteins, especially in the early stage of tumor development and/or metastasis. As such, it seems feasible that we might take advantage of the fact that secreted proteases/peptidases in the tumor microenvironment generate proteolytic products, also referred to as “circulating peptides”, which are detectable in bloodstream and provide ample information about the body, “coded” in the patterns and quantity of these peptides. Herein we clearly link the catalytic activity of Carboxypeptidase N (CPN) to its proteolytic products during breast tumor progression in mouse model and clinical samples. CPN plays important roles in regulating vasoactive peptide hormones, growth factors, and cytokines by specifically cleaving their C-terminal basic residues. Circulating fragment profiling, by an approach combining nanopore fractionation and mass spectrometry, revealed the nature and extent of cleavage by CPN. These results correlated with the level of CPN-catalyzed peptides in blood taken from the tumor-bearing mice, healthy women and breast cancer patients. We showed that generation of C3f_R1310-L1319 specifically correlated with the CPN expression level. In both mouse and clinical patient samples, the amount of CPN was increased in tumor tissues compared to that seen in normal breast tissue, while its counterpart in blood remained constant. The amount of 6 CPN-catalyzed peptides predominantly increased in sera taken from both the mice at 2 weeks after orthotropic implantation and the patients’ plasma as early as the first pathologic stage of breast cancer. In conclusion, the circulating level of the selected 6 CPN-catalyzed proteolytic products reflects the extent of this enzyme's activity in tumors, and our results clearly indicate their strong potential as biomarkers for non-invasive early diagnosis of breast cancer. Citation Format: Ye Hu. Circulating proteolytic products of tumor-resident enzyme as potential biomarkers for early detection of breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-247. doi:10.1158/1538-7445.AM2014-LB-247

Circulating Proteolytic Products of Carboxypeptidase N for Early Detection of Breast Cancer

Carboxypeptidase N (CPN) is important in regulating vasoactive peptide hormones, growth factors, and cytokines by specifically cleaving their C-terminal basic residues. We investigated whether circulating peptides specifically cleaved by CPN in the tumor microenvironment can be stage-specific indicators of breast cancer. CPN activity was measured using an ex vivo peptide cleavage assay by incubating synthesized C3f peptide (His6-C3f_S1304-R1320-His6) in interstitial fluids of breast tumors and adjacent normal breast tissues in mice with orthotopic implantation of the human cell line MDA-MB-231. The nature and extent of peptide cleavage by CPN was investigated by fragment profiling using nanopore fractionation and mass spectrometry. The fragment profiles in interstitial fluid correlated with concentrations of CPN-catalyzed peptides in blood samples taken from the tumor-bearing mice, healthy women, and breast cancer patients. CPN expression in the same set of samples was further examined by immunohistochemistry and immunoblotting. We showed that generation of C3f_R1310-L1319 specifically correlated with the CPN expression level. In both the mouse and clinical patient samples, CPN was clearly increased in tumor tissues compared with normal breast tissue, whereas corresponding CPN abundance in blood remained constant. Concentrations of 6 CPN-catalyzed peptides predominantly increased in sera taken from the mice (n = 8) at 2 weeks after orthotopic implantation. Six homologous peptides displayed significantly higher expression in the patients' plasma as early as the first pathologic stage of breast cancer. Circulating CPN-catalyzed peptide concentrations reflect the CPN activity in tumors. These biomarkers show strong potential for the noninvasive and early diagnosis of breast cancer.

The Molecular Basis for Species-specific Activation of Human TRPA1 Protein by Protons Involves Poorly Conserved Residues within Transmembrane Domains 5 and 6

The surveillance of acid-base homeostasis is concerted by diverse mechanisms, including an activation of sensory afferents. Proton-evoked activation of rodent sensory neurons is mainly mediated by the capsaicin receptor TRPV1 and acid-sensing ion channels. In this study, we demonstrate that extracellular acidosis activates and sensitizes the human irritant receptor TRPA1 (hTRPA1). Proton-evoked membrane currents and calcium influx through hTRPA1 occurred at physiological acidic pH values, were concentration-dependent, and were blocked by the selective TRPA1 antagonist HC030031. Both rodent and rhesus monkey TRPA1 failed to respond to extracellular acidosis, and protons even inhibited rodent TRPA1. Accordingly, mouse dorsal root ganglion neurons lacking TRPV1 only responded to protons when hTRPA1 was expressed heterologously. This species-specific activation of hTRPA1 by protons was reversed in both mouse and rhesus monkey TRPA1 by exchange of distinct residues within transmembrane domains 5 and 6. Furthermore, protons seem to interact with an extracellular interaction site to gate TRPA1 and not via a modification of intracellular N-terminal cysteines known as important interaction sites for electrophilic TRPA1 agonists. Our data suggest that hTRPA1 acts as a sensor for extracellular acidosis in human sensory neurons and should thus be taken into account as a yet unrecognized transduction molecule for proton-evoked pain and inflammation. The species specificity of this property is unique among known endogenous TRPA1 agonists, possibly indicating that evolutionary pressure enforced TRPA1 to inherit the role as an acid sensor in human sensory neurons.

A specific cell-penetrating peptide induces apoptosis in SKOV3 cells by down-regulation of Bcl-2

Peptides are emerging as pharmaceutical agents in cancer therapy. The peptide, TLSGAFELSRDK (TLS) is a targeting ligand that can specifically triggers cellular uptake by binding to SKOV3 cells. Cell surface proteins and the C-terminal basic residues of the TLS are required for effective cell penetration, and the uptake process is energy-dependent. It inhibited the proliferation of SKOV3 cells and induced early-stage apoptosis by down-regulation of Bcl-2 expression mediated through a caspase-dependent pathway. The synergistic anti-proliferative effects of the peptide TLS and doxorubicin on SKOV3 cells were further investigated. Taken together, TLS, acting as a combination of a targeted ligand and a therapeutic agent, was a promising candidate for the development of peptide-based therapies in ovarian cancer.

Role of C-terminal Membrane-proximal Basic Residues in Cell Surface Trafficking of HIV Coreceptor GPR15 Protein

Cell surface density of G protein-coupled receptors (GPCRs) is controlled by dynamic molecular interactions that often involve recognition of the distinct sequence signals on the cargo receptors. We reported previously that the RXR-type dibasic motif in the distal C-terminal tail of an HIV coreceptor GPR15 negatively regulates the cell surface expression by mediating the coatomer protein I complex-dependent retrograde transport to the endoplasmic reticulum (ER). Here we demonstrate that another pair of basic residues (Arg(310)-Arg(311)) in the membrane-proximal region of the C-terminal tail plays a pivotal role in mediating the anterograde trafficking of GPR15. The Ala mutation of the C-terminal membrane-proximal basic residues (MPBRs) (R310/311A) abolished the O-glycosylation and cell surface expression of GPR15. The subcellular fractionation and immunocytochemistry assays indicated that the R310/311A mutant was more localized in the ER but much less in the trans-Golgi when compared with the wild-type GPR15, suggesting the positive role of Arg(310)-Arg(311) in the ER-to-Golgi transport of GPR15. Sequence analysis on human GPCRs showed that the basic residues are frequent in the membrane-proximal region of the C-terminal tail. Similar to GPR15, mutation of the C-terminal MPBRs resulted in a marked reduction of the cell surface expression in multiple different GPCRs. Our results suggest that the C-terminal MPBRs are critically involved in mediating the anterograde trafficking of a broad range of membrane proteins, including GPCRs.

Cathepsin H functions as an aminopeptidase in secretory vesicles for production of enkephalin and galanin peptide neurotransmitters.

Peptide neurotransmitters function as key intercellular signaling molecules in the nervous system. These peptides are generated in secretory vesicles from proneuropeptides by proteolytic processing at dibasic residues, followed by removal of N- and/or C-terminal basic residues to form active peptides. Enkephalin biosynthesis from proenkephalin utilizes the cysteine protease cathepsin L and the subtilisin-like prohormone convertase 2 (PC2). Cathepsin L generates peptide intermediates with N-terminal basic residue extensions, which must be removed by an aminopeptidase. In this study, we identified cathepsin H as an aminopeptidase in secretory vesicles that produces (Met)enkephalin (ME) by sequential removal of basic residues from KR-ME and KK-ME, supported by in vivo knockout of the cathepsin H gene. Localization of cathepsin H in secretory vesicles was demonstrated by immunoelectron microscopy and immunofluorescence deconvolution microscopy. Purified human cathepsin H sequentially removes N-terminal basic residues to generate ME, with peptide products characterized by nano-LC-MS/MS tandem mass spectrometry. Cathepsin H shows highest activities for cleaving N-terminal basic residues (Arg and Lys) among amino acid fluorogenic substrates. Notably, knockout of the cathepsin H gene results in reduction of ME in mouse brain. Cathepsin H deficient mice also show a substantial decrease in galanin peptide neurotransmitter levels in brain. These results illustrate a role for cathepsin H as an aminopeptidase for enkephalin and galanin peptide neurotransmitter production.

Solution structure of the E3 ligase HOIL‐1 Ubl domain

The E3 ligases HOIL-1 and parkin are each comprised of an N-terminal ubiquitin-like (Ubl) domain followed by a zinc-binding region and C-terminal RING-In-between-RING-RING domains. These two proteins, involved in the ubiquitin-mediated degradation pathway, are the only two known E3 ligases to share this type of multidomain architecture. Further, the Ubl domain of both HOIL-1 and parkin has been shown to interact with the S5a subunit of the 26S proteasome. The solution structure of the HOIL-1 Ubl domain was solved using NMR spectroscopy to compare it with that of parkin to determine the structural elements responsible for S5a intermolecular interactions. The final ensemble of 20 structures had a β-grasp Ubl-fold with an overall backbone RMSD of 0.59 ± 0.10 Å in the structured regions between I55 and L131. HOIL-1 had a unique extension of both β1 and β2 sheets compared to parkin and other Ubl domains, a result of a four-residue insertion in this region. A similar 15-residue hydrophobic core in the HOIL-1 Ubl domain resulted in a comparable stability to the parkin Ubl, but significantly lower than that observed for ubiquitin. A comparison with parkin and other Ubl domains indicates that HOIL-1 likely uses a conserved hydrophobic patch (W58, V102, Y127, Y129) found on the β1 face, the β3-β4 loop and β5, as well as a C-terminal basic residue (R134) to recruit the S5a subunit as part of the ubiquitin-mediated proteolysis pathway.