Biophysical Investigations of Complement Receptor 2 (CD21 and CR2)-Ligand Interactions Reveal Amino Acid Contacts Unique to Each Receptor-Ligand Pair

Human complement receptor type 2 (CR2 and CD21) is a cell membrane receptor, with 15 or 16 extracellular short consensus repeats (SCRs), that promotes B lymphocyte responses and bridges innate and acquired immunity. The most distally located SCRs, SCR1–2, mediate the interaction of CR2 with its four known ligands (C3d, EBV gp350, IFNα, and CD23). To ascertain specific interacting residues on CR2, we utilized NMR studies wherein gp350 and IFNα were titrated into ¹⁵N-labeled SCR1–2, and chemical shift changes indicative of specific inter-molecular interactions were identified. With backbone assignments made, the chemical shift changes were mapped onto the crystal structure of SCR1–2. With regard to gp350, the binding region of CR2 is primarily focused on SCR1 and the inter-SCR linker, specifically residues Asn¹¹, Arg¹³, Ala²², Arg²⁸, Ser³², Arg³⁶, Lys⁴¹, Lys⁵⁷, Tyr⁶⁴, Lys⁶⁷, Tyr⁶⁸, Arg⁸³, Gly⁸⁴, and Arg⁸⁹. With regard to IFNα, the binding is similar to the CR2-C3d interaction with specific residues being Arg¹³, Tyr¹⁶, Arg²⁸, Ser⁴², Lys⁴⁸, Lys⁵⁰, Tyr⁶⁸, Arg⁸³, Gly⁸⁴, and Arg⁸⁹. We also report thermodynamic properties of each ligand-receptor pair determined using isothermal titration calorimetry. The CR2-C3d interaction was characterized as a two-mode binding interaction with K_d values of 0.13 and 160 μm, whereas the CR2-gp350 and CR2-IFNα interactions were characterized as single site binding events with affinities of 0.014 and 0.035 μm, respectively. The compilation of chemical binding maps suggests specific residues on CR2 that are uniquely important in each of these three binding interactions.     

Osteopontin Is Cleaved at Multiple Sites Close to Its Integrin-binding Motifs in Milk and Is a Novel Substrate for Plasmin and Cathepsin D

Osteopontin (OPN) is a highly modified integrin-binding protein present in most tissues and body fluids where it has been implicated in numerous biological processes. A significant regulation of OPN function is mediated through phosphorylation and proteolytic processing. Proteolytic cleavage by thrombin and matrix metalloproteinases close to the integrin-binding Arg-Gly-Asp sequence modulates the function of OPN and its integrin binding properties. In this study, seven N-terminal OPN fragments originating from proteolytic cleavage have been characterized from human milk. Identification of the cleavage sites revealed that all fragments contained the Arg–Gly–Asp¹⁴⁵ sequence and were generated by cleavage of the Leu¹⁵¹–Arg¹⁵², Arg¹⁵²–Ser¹⁵³, Ser¹⁵³–Lys¹⁵⁴, Lys¹⁵⁴–Ser¹⁵⁵, Ser¹⁵⁵–Lys¹⁵⁶, Lys¹⁵⁶–Lys¹⁵⁷, or Phe¹⁵⁸–Arg¹⁵⁹ peptide bonds. Six cleavages cannot be ascribed to thrombin or matrix metalloproteinase activity, whereas the cleavage at Arg¹⁵²–Ser¹⁵³ matches thrombin specificity for OPN. The principal protease in milk, plasmin, hydrolyzed the same peptide bond as thrombin, but its main cleavage site was identified to be Lys¹⁵⁴–Ser¹⁵⁵. Another endogenous milk protease, cathepsin D, cleaved the Leu¹⁵¹–Arg¹⁵² bond. OPN fragments corresponding to plasmin activity were also identified in urine showing that plasmin cleavage of OPN is not restricted to milk. Plasmin, but not cathepsin D, cleavage of OPN increased cell adhesion mediated by the α_Vβ₃- or α₅β₁-integrins. Similar cellular adhesion was mediated by plasmin and thrombin-cleaved OPN showing that plasmin can be a potent regulator of OPN activity. These data show that OPN is highly susceptible to cleavage near its integrin-binding motifs, and the protein is a novel substrate for plasmin and cathepsin D.     

Paired basic/Furin-like proprotein convertase cleavage of Pro-BMP-1 in the trans-Golgi network

Bone morphogenetic protein (BMP)-1 is a zinc-dependent metalloproteinase that cleaves a variety of extracellular matrix substrates, including type I procollagen. Little is known about the site of action of BMP-1, although the extracellular matrix seems likely to be it. BMP-1 is synthesized with an N-terminal prodomain. The removal of the prodomain presumably activates the proteinase. In this study we show that the prodomain is cleaved in the trans-Golgi network (TGN) and by furin-like/paired basic proprotein convertases. Inhibitors of furin resulted in the secretion of pro-BMP-1, which could not cleave procollagen. Recombinant furin cleaved the prodomain from pro-BMP-1. Site-directed mutagenesis of the prodomain cleavage site (RSRR) to RSAA resulted in efficient secretion of pro-BMP-1. Therefore, prodomain cleavage was not required for secretion. Using peptide N-glycosidase and neuraminidase digestion to determine the post-translational status of pro-BMP-1 during its conversion to BMP-1, we showed that BMP-1 first appears in the TGN during sialylation of the molecule. Furthermore, immunofluorescence studies using an antibody to the nascent N terminus of BMP-1 showed localization to the TGN and plasma membrane. The observation that BMP-1 occurs inside the cell raises the possibility that BMP-1 might begin to cleave its substrates prior to secretion to the extracellular matrix.     

Neutrophil Elastase Activates Protease-activated Receptor-2 (PAR2) and Transient Receptor Potential Vanilloid 4 (TRPV4) to Cause Inflammation and Pain

Proteases that cleave protease-activated receptor-2 (PAR₂) at Arg³⁶↓Ser³⁷ reveal a tethered ligand that binds to the cleaved receptor. PAR₂ activates transient receptor potential (TRP) channels of nociceptive neurons to induce neurogenic inflammation and pain. Although proteases that cleave PAR₂ at non-canonical sites can trigger distinct signaling cascades, the functional importance of the PAR₂-biased agonism is uncertain. We investigated whether neutrophil elastase, a biased agonist of PAR₂, causes inflammation and pain by activating PAR₂ and TRP vanilloid 4 (TRPV4). Elastase cleaved human PAR₂ at Ala⁶⁶↓Ser⁶⁷ and Ser⁶⁷↓Val⁶⁸_. Elastase stimulated PAR₂-dependent cAMP accumulation and ERK1/2 activation, but not Ca²⁺ mobilization, in KNRK cells. Elastase induced PAR₂ coupling to Gα_s but not Gα_q in HEK293 cells. Although elastase did not promote recruitment of G protein-coupled receptor kinase-2 (GRK2) or β-arrestin to PAR₂, consistent with its inability to promote receptor endocytosis, elastase did stimulate GRK6 recruitment. Elastase caused PAR₂-dependent sensitization of TRPV4 currents in Xenopus laevis oocytes by adenylyl cyclase- and protein kinase A (PKA)-dependent mechanisms. Elastase stimulated PAR₂-dependent cAMP formation and ERK1/2 phosphorylation, and a PAR₂- and TRPV4-mediated influx of extracellular Ca²⁺ in mouse nociceptors. Adenylyl cyclase and PKA-mediated elastase-induced activation of TRPV4 and hyperexcitability of nociceptors. Intraplantar injection of elastase to mice caused edema and mechanical hyperalgesia by PAR₂- and TRPV4-mediated mechanisms. Thus, the elastase-biased agonism of PAR₂ causes Gα_s-dependent activation of adenylyl cyclase and PKA, which activates TRPV4 and sensitizes nociceptors to cause inflammation and pain. Our results identify a novel mechanism of elastase-induced activation of TRPV4 and expand the role of PAR₂ as a mediator of protease-driven inflammation and pain.     

Identification of the site of binding of sulfated, low molecular weight lignins on thrombin

Sulfated, low molecular weight lignins (LMWLs), designed recently as macromolecular mimetics of the low molecular weight heparins (LMWHs), were found to exhibit a novel allosteric mechanism of inhibition of human thrombin, factor Xa and plasmin, which translates into potent human blood anticoagulation potential. To identify the site of binding of sulfated LMWLs, a panel of site-directed thrombin mutants was studied. Substitution of alanine for Arg⁹³ or Arg¹⁷⁵ induced a 7–8-fold decrease in inhibition potency, while Arg¹⁶⁵Ala, Lys¹⁶⁹Ala, Arg¹⁷³Ala and Arg²³³Ala thrombin mutants displayed a 2–4-fold decrease. Other exosite 2 residues including those that play an important role in heparin binding, such as Arg¹⁰¹, Lys²³⁵, Lys²³⁶ and Lys²⁴⁰, did not induce any deficiency in sulfated LMWL activity. Thrombin mutants with multiple alanine substitution of basic residues showed a progressively greater defect in inhibition potency. Comparison of thrombin, factor Xa, factor IXa and factor VIIa primary sequences reiterated Arg⁹³ and Arg¹⁷⁵ as residues likely to be targeted by sulfated LMWLs. The identification of a novel site on thrombin with capability of allosteric modulation is expected to greatly assist the design of new regulators based on the sulfated LMWL scaffold.     

Biophysical comparison of BMP-2, ProBMP-2, and the free pro-peptide reveals stabilization of the pro-peptide by the mature growth factor

Pro-forms of growth factors have received intensive scientific attention recently because in some cases different biological activities have been ascribed compared with the mature growth factors. Examples are the pro-apoptotic role of the nerve growth factor (NGF) proform (proNGF) or the latency of the transforming growth factor (TGF)-beta pro-form (proTGF-beta). To investigate a possible biological function of the pro-form of bone morphogenetic protein (BMP)-2, a member of the TGF-beta family, mature BMP-2, proBMP-2, and the isolated pro-peptide were recombinantly produced in Escherichia coli cells, and a biophysical comparison was performed. Protocols were developed that allowed efficient refolding and subsequent purification of the proteins. ProBMP-2 could be processed to an N-terminally truncated form of BMP-2, digit removed BMP-2 (drBMP-2), that possessed biological activity, i.e. it induced ectopic bone formation. Bone inducing activity was also displayed by proBMP-2. The three proteins were characterized both by fluorescence and CD spectroscopy. From these analyses, predominant beta-sheet secondary structural elements in the pro-peptide were deduced. The thermodynamic stability of the pro-peptide was determined by chemical unfolding assays. As in the case of NGF/proNGF, the mature part of BMP-2 stabilized the structure of the pro-peptide moiety. However, in contrast to NGF/proNGF, the pro-peptide did not stimulate oxidative folding of the mature part in vitro.     

Expression of sarco/endoplasmic reticulum Ca ATPase (SERCA) 3 proteins in two major conformational states in native human cell membranes

The SERCA family includes 3 genes (SERCA1-3), each of which giving rise to various isoforms. To date, detailed structural data is only available for the SERCA1a isoform. Here, limited trypsinolysis of either human platelet membranes or recombinant SERCA3a in HEK-293 cells followed by Western blotting using antibodies covering different regions of the SERCA3(a) protein revealed two, kinetically distinct, Early (ETF) and Late (LTF) Tryptic Fragmentations. The ETF uses many tryptic sites while the LTF uses a unique tryptic site. Using site-directed mutagenesis: i) Arg³³⁴, Arg³⁹⁶ and Arg⁶³⁸ were directly assigned to the ETF and ii) Arg¹⁹⁸ was assigned as the only tryptic site to the LTF. Arg⁶⁷¹, Lys⁷¹²/Lys⁷¹³ and Lys⁷²⁸ were also found to modulate the ETF. SERCA inhibitors Tg and tBHQ induced modest inhibition of the ETF. In contrast, the addition of CaCl₂, EGTA or AlF₄⁻ strikingly modified the ETF without any effect on the LTF. Trypsinolysis of the other recombinant SERCA3b-3f isoforms revealed: i) same ETF and LTF as SERCA3a, with variations of the length of the C-terminal fragments; ii) Arg¹⁰⁰² as an additional tryptic site in SERCA3b-3e isoforms. Taken together, the two distinct SERCA3 fragmentation profiles sign the co-expression of SERCA3 proteins in two conformational states in cell membranes.     

Limited tryptic digestion of recombinant human interleukin-2: Structure-binding relationships with the α chain of the interleukin-2 receptor

The surface topography and structural features of interleukin-2 (IL-2) in relation to its interaction with the α subunit of its receptor (IL-2Rα) have been probed by limited tryptic digestion followed by detailed structural analyses. Four sensitive cleavage sites in IL-2 (Lys⁸, Lys⁹, Lys³⁵, and Arg³⁸) were identified as surface amino acids, suggesting that they are potential binding sites for IL-2Rα. To examine the involvement of these residues in IL-2Rα binding, a truncated IL-2 molecule lacking the amino-terminal residues through Arg³⁸ was generated and it was found to be incapable of binding IL-2Rα in a solid-phase receptor binding sequencing assay. These studies have led to the conclusion that the IL-2Rα contact region of IL-2 includes residues Lys³⁵ and Arg³⁸. This finding is supported by the refined three-dimensional structure of IL-2 in which these residues are located outside of the compact bundle of four helices and thus are readily available for interaction with IL-2Rα.     

Dual vulnerability of tau to calpains and caspase-3 proteolysis under neurotoxic and neurodegenerative conditions

Axonally specific microtubule-associated protein tau is an important component of neurofibrillary tangles found in AD (Alzheimer''s disease) and other tauopathy diseases such as CTE (chronic traumatic encephalopathy). Such tau aggregate is found to be hyperphosphorylated and often proteolytically fragmented. Similarly, tau is degraded following TBI (traumatic brain injury). In the present study, we examined the dual vulnerability of tau to calpain and caspase-3 under neurotoxic and neurodegenerative conditions. We first identified three novel calpain cleavage sites in rat tau (four-repeat isoform) as Ser¹³⁰↓Lys¹³¹, Gly¹⁵⁷↓Ala¹⁵⁸ and Arg³⁸⁰↓Glu³⁸¹. Fragment-specific antibodies to target the major calpain-mediated TauBDP-35K (35 kDa tau-breakdown product) and the caspase-mediated TauBDP-45K respectively were developed. In rat cerebrocortical cultures treated with excitotoxin [NMDA (N-methyl-d-aspartate)], tau is significantly degraded into multiple fragments, including a dominant signal of calpain-mediated TauBDP-35K with minimal caspase-mediated TauBDP-45K. Following apoptosis-inducing EDTA treatment, tau was truncated only to TauBDP-48K/45K-exclusively by caspase. Cultures treated with another apoptosis inducer STS (staurosporine), dual fragmentation by calpain (TauBDP-35K) and caspase-3 (TauBDP-45K) was observed. Tau was also fragmented in injured rat cortex following TBI in vivo to BDPs of 45–42 kDa (minor), 35 kDa and 15 kDa, followed by TauBDP-25K. Calpain-mediated TauBDP-35K-specific antibody confirmed robust signals in the injured cortex, while caspase-mediated TauBDP-45K-specific antibody only detected faint signals. Furthermore, intravenous administration of a calpain-specific inhibitor SNJ-1945 strongly suppressed the TauBDP-35K formation. Taken together, these results suggest that tau protein is dually vulnerable to calpain and caspase-3 proteolysis under different neurotoxic and injury conditions.     

Spare interactions of highly potent [Arg14,Lys15]nociceptin for cooperative induction of ORL1 receptor activation

[Arg¹⁴,Lys¹⁵]Nociceptin is a very potent for ORL1 receptor, showing a few times stronger binding activity and much more enhanced biological activity than endogenous nociceptin. This synergistic outcome has been suggested to be due to the interaction with the receptor aromatic and/or acidic amino acid residues crucial to receptor activation. In order to identify such receptor residues in the second ORL1 extracellular loop, we prepared a series of recombinant mutant receptors. The mutant receptor Gln205Ala was found to be as active as wild-type ORL1 for both nociceptin and [Arg¹⁴,Lys¹⁵]nociceptin. In contrast, Asp206Ala and Tyr207Ala exhibited considerably reduced activity for [Arg¹⁴,Lys¹⁵]nociceptin, exhibiting no synergistic activity enhancement. These results suggest that Asp206 and Tyr207 are directly involved in the interaction with nociceptin-[Arg¹⁴,Lys¹⁵]. Trp208Ala was found to bind strongly both nociceptin and [Arg¹⁴,Lys¹⁵]nociceptin, although it elicited no biological activity. All these results indicate that the consecutive amino acid residues Asp206, Tyr207, and Trp208 are critical to the activation of the ORL1 receptor, but not to nociceptin-binding.     

Endopeptidase Cleavage Generates a Functionally Distinct Isoform of C1q/Tumor Necrosis Factor-related Protein-12 (CTRP12) with an Altered Oligomeric State and Signaling Specificity

Adipose tissue-derived adipokines are an important class of secreted metabolic regulators that mediate tissue cross-talk to control systemic energy balance. We recently described C1q/TNF-related protein-12 (CTRP12), a novel insulin-sensitizing adipokine that regulates glucose metabolism in liver and adipose tissue. However, the biochemical properties of CTRP12 and its naturally occurring cleaved isoform have not been characterized. Here, we show that CTRP12 is a secreted hormone subjected to multiple functionally relevant posttranslational modifications at highly conserved residues. For example, Asn³⁹ is glycosylated, whereas Cys⁸⁵ mediates the assembly of higher order oligomeric structure. Endopeptidase cleavage at Lys⁹¹ generates a cleaved globular gCTRP12 isoform, the expression of which is increased by insulin. PCSK3/furin was identified as the major proprotein convertase expressed by adipocytes that mediates the endogenous cleavage of CTRP12. Cleavage at Lys⁹¹ is context-dependent: mutation of the charged Arg⁹³ to Ala on the P2′ position enhanced cleavage, and triple mutations (K90A/K91A/R93A) abolished cleavage. Importantly, the two isoforms of CTRP12 differ in oligomeric structures and are functionally distinct. The full-length protein forms trimers and larger complexes, and the cleaved isoform consisted of predominantly dimers. Whereas full-length fCTRP12 strongly activated Akt signaling in H4IIE hepatocytes and 3T3-L1 adipocytes, gCTRP12 preferentially activated MAP kinase (ERK1/2 and p38 MAPK) signaling. Further, only fCTRP12 improved insulin-stimulated glucose uptake in adipocytes. These results reveal a novel mechanism controlling signaling specificity and function of a hormone via cleavage-dependent alteration in oligomeric state.     

A New Paradigm for Enzymatic Control of α-Cleavage and β-Cleavage of the Prion Protein

The cellular form of the prion protein (PrP^C) is found in both full-length and several different cleaved forms in vivo. Although the precise functions of the PrP^C proteolytic products are not known, cleavage between the unstructured N-terminal domain and the structured C-terminal domain at Lys-109↓His-110 (mouse sequence), termed α-cleavage, has been shown to produce the anti-apoptotic N1 and the scrapie-resistant C1 peptide fragments. β-Cleavage, residing adjacent to the octarepeat domain and N-terminal to the α-cleavage site, is thought to arise from the action of reactive oxygen species produced from redox cycling of coordinated copper. We sought to elucidate the role of key members of the ADAM (a disintegrin and metalloproteinase) enzyme family, as well as Cu²⁺ redox cycling, in recombinant mouse PrP (MoPrP) cleavage through LC/MS analysis. Our findings show that although Cu²⁺ redox-generated reactive oxygen species do produce fragmentation corresponding to β-cleavage, ADAM8 also cleaves MoPrP in the octarepeat domain in a Cu²⁺- and Zn²⁺-dependent manner. Additional cleavage by ADAM8 was observed at the previously proposed location of α-cleavage, Lys-109↓His-110 (MoPrP sequencing); however, upon addition of Cu²⁺, the location of α-cleavage shifted by several amino acids toward the C terminus. ADAM10 and ADAM17 have also been implicated in α-cleavage at Lys-109↓His-110; however, we observed that they instead cleaved MoPrP at a novel location, Ala-119↓Val-120, with additional cleavage by ADAM10 at Gly-227↓Arg-228 near the C terminus. Together, our results show that MoPrP cleavage is far more complex than previously thought and suggest a mechanism by which PrP^C fragmentation responds to Cu²⁺ and Zn²⁺.     

Furin-cleaved Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Is Active and Modulates Low Density Lipoprotein Receptor and Serum Cholesterol Levels

Proprotein convertase subtilisin/kexin 9 (PCSK9) regulates plasma LDL cholesterol levels by regulating the degradation of LDL receptors. Another proprotein convertase, furin, cleaves PCSK9 at Arg²¹⁸-Gln²¹⁹ in the surface-exposed “218 loop.” This cleaved form circulates in blood along with the intact form, albeit at lower concentrations. To gain a better understanding of how cleavage affects PCSK9 function, we produced recombinant furin-cleaved PCSK9 using antibody Ab-3D5, which binds the intact but not the cleaved 218 loop. Using Ab-3D5, we also produced highly purified hepsin-cleaved PCSK9. Hepsin cleaves PCSK9 at Arg²¹⁸-Gln²¹⁹ more efficiently than furin but also cleaves at Arg²¹⁵-Phe²¹⁶. Further analysis by size exclusion chromatography and mass spectrometry indicated that furin and hepsin produced an internal cleavage in the 218 loop without the loss of the N-terminal segment (Ser¹⁵³–Arg²¹⁸), which remained attached to the catalytic domain. Both furin- and hepsin-cleaved PCSK9 bound to LDL receptor with only 2-fold reduced affinity compared with intact PCSK9. Moreover, they reduced LDL receptor levels in HepG2 cells and in mouse liver with only moderately lower activity than intact PCSK9, consistent with the binding data. Single injection into mice of furin-cleaved PCSK9 resulted in significantly increased serum cholesterol levels, approaching the increase by intact PCSK9. These findings indicate that circulating furin-cleaved PCSK9 is able to regulate LDL receptor and serum cholesterol levels, although somewhat less efficiently than intact PCSK9. Therapeutic anti-PCSK9 approaches that neutralize both forms should be the most effective in preserving LDL receptors and in lowering plasma LDL cholesterol.     

The binding of Arg- and Lys-peptides to single standed polyribonucleotides and its effect on the polymer conformation

The interactions between basic oligopeptides (Lys₂, Lys₃, Arg₂, and Arg₃) and single stranded polynucleotides (poly(A), poly(C), poly(I) and poly(U) were investigated at low ion concentration by UV spectroscopy, circular dichroism and field jump relaxation. Various domains of binding were detected: 1) High concentrations (up to 1 mM) of some peptides induce opalescencs followed by coacervation- Arg₃ causes coacervation in all polynucleotides used, yet Lys₃ only in poly(I). In the case of poly(I) the threshold concentration for coacervation is much lower for Arg₃ (150 μM) than for Lys₃ (500 μM). 2) Medium concentrations (?10 μM) of Arg₃ and Lys₃ induce helix formation in poly(U). In the case of poly(I) cooperative helix formation is only induced by Lys₃, but not by Arg₃. 3) The onset of peptide association is observed at very low peptide concentrations (?1 μM) already by using the field jump method. The association is reflected by a relaxation process, that can be described by a single exponential within experimental accuracy. Measurements of relaxation time constants as a function of the peptide concentration provide information on the association constants K, the number of nucleotide residues per binding place n and the rate constants k_R and k_D. Using a simple model with independent and “separate” binding sites, K for Arg₃ and Lys₃ is found to be in the range of 10⁶ to 10⁷ M^?1. In the case of Arg₂ and K is lower by a factor of about 10. For various polynucleotides K_Arg3 is slightly higher than K_Lys3. except in the case of poly(I), where K_Arg3/K_Lys3 ≈ 5. Similar data are obtained by application of a “sphere model” (see below). These results provide quantitative evidence for specific hydrogen bonding between the guanidino group of Arg and inosine. They also explain the absence of helix formation for poly(I) + Arg₃: Arg blocks the hydrogen bonding sites of inosine. Thus cooperative coupling leads in this case to a considerable amplification of specificity in the peptide-polynucleotide interaction Both field jump and stopped flow data demonstrate a high mobility of the peptide lisands along the polymer, resulting in a redistribution being fast compared with the overall binding step. Based on this result the relaxation data are analysed by a “sphere” model, which considers a) excluded binding under the condition of fast Ugand distribution along the lattice and b) the connection of sites into a polymer sphere. The rate constants obtained by this model are in the range of 4 × 10¹¹ M^?1 s^?1. These high values reflect the large reaction distance for polymers of chain lengths around 1000. A comparison with rate constants obtained previously for oligomer complexes indicates that the recombination rate is approximately a function of the square root of the nucleotide chain length, which is directly related to the mean radius of coiled polymers.     

Identification of candidate angiogenic inhibitors processed by matrix metalloproteinase 2 (MMP-2) in cell-based proteomic screens: disruption of vascular endothelial growth factor (VEGF)/heparin affin regulatory peptide (pleiotrophin) and VEGF/Connective tissue growth factor angiogenic inhibitory complexes by MMP-2 proteolysis

Matrix metalloproteinases (MMPs) exert both pro- and antiangiogenic functions by the release of cytokines or proteolytically generated angiogenic inhibitors from extracellular matrix and basement membrane remodeling. In the Mmp2^−/− mouse neovascularization is greatly reduced, but the mechanistic aspects of this remain unclear. Using isotope-coded affinity tag labeling of proteins analyzed by multidimensional liquid chromatography and tandem mass spectrometry we explored proteome differences between Mmp2^−/− cells and those rescued by MMP-2 transfection. Proteome signatures that are hallmarks of proteolysis revealed cleavage of many known MMP-2 substrates in the cellular context. Proteomic evidence of MMP-2 processing of novel substrates was found. Insulin-like growth factor binding protein 6, follistatin-like 1, and cystatin C protein cleavage by MMP-2 was biochemically confirmed, and the cleavage sites in heparin affin regulatory peptide (HARP; pleiotrophin) and connective tissue growth factor (CTGF) were sequenced by matrix-assisted laser desorption ionization-time of flight mass spectrometry. MMP-2 processing of HARP and CTGF released vascular endothelial growth factor (VEGF) from angiogenic inhibitory complexes. The cleaved HARP N-terminal domain increased HARP-induced cell proliferation, whereas the HARP C-terminal domain was antagonistic and decreased cell proliferation and migration. Hence the unmasking of cytokines, such as VEGF, by metalloproteinase processing of their binding proteins is a new mechanism in the control of cytokine activation and angiogenesis.     

Mapping of Post-translational Modifications of Transition Proteins,TP1 and TP2, and Identification of Protein Arginine Methyltransferase 4 and Lysine Methyltransferase 7 as Methyltransferase for TP2

In a unique global chromatin remodeling process during mammalian spermiogenesis, 90% of the nucleosomal histones are replaced by testis-specific transition proteins, TP1, TP2, and TP4. These proteins are further substituted by sperm-specific protamines, P1 and P2, to form a highly condensed sperm chromatin. In spermatozoa, a small proportion of chromatin, which ranges from 1 to 10% in mammals, retains the nucleosomal architecture and is implicated to play a role in transgenerational inheritance. However, there is still no mechanistic understanding of the interaction of chromatin machinery with histones and transition proteins, which facilitate this selective histone replacement from chromatin. Here, we report the identification of 16 and 19 novel post-translational modifications on rat endogenous transition proteins, TP1 and TP2, respectively, by mass spectrometry. By in vitro assays and mutational analysis, we demonstrate that protein arginine methyltransferase PRMT4 (CARM1) methylates TP2 at Arg⁷¹, Arg⁷⁵, and Arg⁹² residues, and lysine methyltransferase KMT7 (Set9) methylates TP2 at Lys⁸⁸ and Lys⁹¹ residues. Further studies with modification-specific antibodies that recognize TP2K88me1 and TP2R92me1 modifications showed that they appear in elongating to condensing spermatids and predominantly associated with the chromatin-bound TP2. This work establishes the repertoire of post-translational modifications that occur on TP1 and TP2, which may play a significant role in various chromatin-templated events during spermiogenesis and in the establishment of the sperm epigenome.     

Substrate conformation directs selective enzymic cleavage of β-lipotropin into β-endorphin

It is shown that whilst the Arg⁵¹-Trp⁵² and Arg⁶⁰-Tyr⁶¹ peptide bonds of β-lipotropin are preferentially split by trypsin-Sepharose in aqueous solution, in a secondary structure promoting environment the Arg⁶⁰-Tyr⁶¹ peptide bond is almost exclusively cleaved. It is suggested that the specificity of intracellular precursor processing may be directed by the conformation of the substrate.     

Neuroinflammation-Induced Interactions between Protease-Activated Receptor 1 and Proprotein Convertases in HIV-Associated Neurocognitive Disorder

The proprotein convertases (PCs) furin, PC5, PACE4, and PC7 cleave secretory proteins after basic residues, including the HIV envelope glycoprotein (gp160) and Vpr. We evaluated the abundance of PC mRNAs in postmortem brains of individuals exhibiting HIV-associated neurocognitive disorder (HAND), likely driven by neuroinflammation and neurotoxic HIV proteins (e.g., envelope and Vpr). Concomitant with increased inflammation-related gene expression (interleukin-1β [IL-1β]), the mRNA levels of the above PCs are significantly increased, together with those of the proteinase-activated receptor 1 (PAR1), an inflammation-associated receptor that is cleaved by thrombin at ProArg₄₁↓ (where the down arrow indicates the cleavage location), and potentially by PCs at Arg₄₁XXXXArg₄₆↓. The latter motif in PAR1, but not its R46A mutant, drives its interactions with PCs. Indeed, PAR1 upregulation leads to the inhibition of membrane-bound furin, PC5B, and PC7 and inhibits gp160 processing and HIV infectivity. Additionally, a proximity ligation assay revealed that furin and PC7 interact with PAR1. Reciprocally, increased furin expression reduces the plasma membrane abundance of PAR1 by trapping it in the trans-Golgi network. Furthermore, soluble PC5A/PACE4 can target/disarm cell surface PAR1 through cleavage at Arg₄₆↓. PACE4/PC5A decreased calcium mobilization induced by thrombin stimulation. Our data reveal a new PC-PAR1-interaction pathway, which offsets the effects of HIV-induced neuroinflammation, viral infection, and potentially the development of HAND.     

Two amino acid changes in the R3 repeat cause functional divergence of two clustered <Emphasis Type="Italic">MYB10</Emphasis> genes in peach

R2R3-MYB genes play a pivotal role in regulating anthocyanin accumulation. Here, we report two tandemly duplicated R2R3-MYB genes in peach, PpMYB10.1 and PpMYB10.2, with the latter showing lower ability to induce anthocyanin accumulation than the former. Site-directed mutation assay revealed two amino acid changes in the R3 repeat, Arg/Lys⁶⁶ and Gly/Arg⁹³, responsible for functional divergence between these two PpMYB10 genes. Anthocyanin-promoting activity of PpMYB10.2 was significantly increased by a single amino acid replacement of Arg⁹³ with Gly⁹³. However, either the Gly⁹³ → Arg⁹³ or Arg⁶⁶ → Lys⁶⁶ substitutions alone showed little impact on anthocyanin-promoting activity of PpMYB10.1, but simultaneous substitutions caused a significant decrease. Reciprocal substitution of Arg/Gly⁹³ could significantly alter binding affinity to PpbHLH3, while the Arg⁶⁶ → Lys⁶⁶ substitution is predicted to affect the folding of the MYB DNA-binding domain, instead of PpbHLH3-binding affinity. Overall, the change of anthocyanin-promoting activity was accompanied with that of bHLH-binding affinity, suggesting that DNA-binding affinity of R2R3-MYBs depends on their bHLH partners. Our study is helpful for understanding of functional evolution of R2R3-MYBs and their interaction with DNA targets.     

Nucleic acids potentiate Factor VII-activating protease (FSAP)-mediated cleavage of platelet-derived growth factor-BB and inhibition of vascular smooth muscle cell proliferation

FSAP (Factor VII-activating protease) can cleave and inactivate PDGF-BB (platelet-derived growth factor-BB) and thereby inhibits VSMC (vascular smooth-muscle cell) proliferation. The auto-activation of FSAP is facilitated by negatively charged polyanions such as heparin, dextransulfate or extracellular ribonucleic acids. Since auto-activation is essential for the anti-proliferative function of FSAP, the influence of nucleic acids as cofactors for the FSAP-mediated inhibition of PDGF-BB was investigated. Natural or artificial RNA was an effective cofactor for FSAP mediated PDGF-BB degradation, whereas the effect of DNA was weak. RNA-induced cleavage of PDGF-BB was inhibited by serine protease inhibitors. The pattern of PDGF-BB cleavage was identical with either heparin or RNA as a cofactor. One of the cleavage sites in PDGF-BB was at the positions 160-162 (R160KK162), which is an important region for receptor binding and activation. In VSMCs, PDGF-BB-stimulated DNA synthesis was inhibited by FSAP in the presence of RNA. RNA was more effective than DNA and the cofactor activity of RNA was neutralized after pretreatment with RNase. FSAP binding to RNA protected the nucleic acid from degradation by RNase. These data are relevant to situations where extracellular nucleic acids released from necrotic or apoptotic cells could activate local FSAP, leading to inhibition of PDGF-BB.