FIELD OF THE INVENTION

This invention relates to the field of enzymology. In particular, the present invention is based on the discovery of mechanisms mediating the formation of amyloid-type aggregates of lipid- activated enzymes. The invention discloses a method for preparing inhibitors of said enzymes and provides peptide inhibitors having potential for therapeutical use.

BACKGROUND OF THE INVENTION

Shimizu, 2009, discloses that prostaglandins, leukotrienes, platelet-activating factor, lysophosphatidic acid, sphingosine 1-phophate, and endocannabinoids, collectively referred to as lipid mediators, play pivotal roles in human immune regulation and self-defense. These lipid mediators are produced by multistep enzymatic pathways involving lipid-activated enzymes such as phospholipases. The author summarizes that researchers need to develop specific inhibitors and receptor agonists and antagonists of the lipid-activated enzymes since these would have great potential as a therapeutic approach to disease.

In Code et al., 2008, it is disclosed that activity of phospholipase A2 (PLA2) is dependent on the process of amyloid formation of the enzyme explaining its lag-burst behaviour in enzymatic catalysis. The following route for the activation of PLA2 was discussed: 1) the soluble monomeric enzyme rapidly binds to the substrate; 2) after binding a slow dimerization of the enzyme takes place, at this stage the enzyme shows low catalytic activity; 3) formation of "molten dimers" before the burst of activity; 4) formation of protofibrillar oligomers of PLA2 with high catalytic activity; and finally 5) emergence of amyloid-like fibrils devoid of enzymatic activity. Several amyloidogenic peptides are known to enhance the activity of PLA2, such as temporin B (temB) and temporin L. It has been hypothesized that the formation of heterooligomers by PLA2 and temB would be responsible for the activation by temB of PLA2, promoting enzyme aggregation into an active conformation (Code et al., 2009). The present invention is directed to a method for producing peptide inhibitors of PLA2 and other lipid-activated enzymes by identifying aggregation-prone regions of these enzymes responsible for the formation of inactive amyloids and designing a peptide inhibitor accordingly. The present invention is able to show that this approach provides effective inhibitors of enzyme activity.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Activity of phospholipase A ₂ of bee venom in the presence of peptide inhibitor having sequence KMYFNLI (SEQ ID NO: 1).

Figure 2. Activity of phospholipase A ₂ (of human tears) in the presence of peptide inhibitor having sequence AALSYGFYG (SEQ ID NO:68).

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses a method for preparing peptides capable of efficiently inhibiting the catalytic activity of lipid-activated enzymes and provides peptides made by said method. The expression "lipid-activated enzyme" refers herein to enzymes that specifically recognize a structure or bond of a lipid and require this interaction for maximal activity. Examples of such lipids are fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids and polyketides. Examples of lipid-activated enzymes are carboxylic ester hydrolases (EC 3.1.1), such as phospholipases Al, A2, B and PAF acetylhydrolase, as well as heat shock protein 70 and sphingomyelins. Particularly, some of the lipid-activated enzymes as defined in the present invention are enzymes which generate lipid mediators, such as fatty acids, phospholipids and lysophospholipids (see Shimizu, 2009)

The mechanism of activation of phospholipase A2 as described by Code et al. (2008) requires direct protein-protein interactions. These interactions also need to involve protein sequences capable of causing aggregation and amyloid type oligomerization of the enzyme. When this type of sequences were sought by the use of a computer algorithm in the structure of bee venom phospholipase A2, a stretch of residues 78-91 fulfilling these criteria was found. A suitable computer algorithm for use in the present method is preferably selected from the group consisting of: AGGRESCAN (Conchillo-Sole et al., 2007), PASTA (Trovato et al., 2007) and TANGO (Rousseau et al, 2006; Fernandez-Escamilla et al., 2004; Linding et al., 2004). These computer algorithms are designed and generally utilized as web-based software for the prediction of aggregation-prone segments in protein sequences.

The peptide inhibitor of the invention is prepared based on the found aggregation-prone segment so that the peptide sequence is identical to the amino acid sequence of the segment. The length of the peptide may vary: the peptide can be as long as the aggregation-prone segment, or it can correspond only to a part of the segment. Preferably, the peptides are 5-12 amino acids long. Peptides of the invention can be synthesized by well-known methods (see, e.g., Atherton and Sheppard, 1989).

Further along the lines of the present invention, synthetic peptides of this structure are readily expected to inhibit proper lipid-activated enzyme, such as PLA2-PLA2, contacts. The latter was verified using a short synthetic peptide corresponding to residues 85-91 (KMYFNLI, SEQ ID NO: l) of the bee venom PLA2 enzyme, which upon preincubation with the enzyme protein was capable of causing complete inhibition (see Fig.l). Notably, the inhibition was observed at equimolar concentrations with the enzyme (2 nanomolar), making this the most potent inhibitor described so far. The same experiment was subsequently performed for the human secretory PLA2 present in tear fluid. For this enzyme, an amyloid aggregation causing region of residues 17-25 was found (AALSYGFYG, SEQ ID NO:68) and the synthetic corresponding peptide also inhibited the tear fluid PLA2 activity (Fig. 2). This particular mechanism of enzyme activity control can be expected to be very widely found in nature. Accordingly, identification of this type of sequences in lipid-activated enzymes can be used to obtain very specific and powerful synthetic peptide inhibitors.

Furthermore, the peptides can be made with additional cell membrane permeating sequences, so that the inhibitors can enter cells, i.e. with transport peptides, see, e.g., US 7,265,092 . An example of a transporter peptide is a peptide which facilitates cellular uptake of an inhibitor peptide which is chemically associated or bonded to the transporter peptide. Along these lines, we identified the following amyloid aggregation sequences in the following human enzymes: myeloperoxidase, acid sphingomyelinase, and heat shock protein 70 (see Table 1 below). Heat shock protein 70 is of particular importance as its inhibition makes cell extremely sensitive to an increase in temperature. This feature could be exploited in for instance MRI- guided HIFU therapy to more efficiently eradicate cancer.

A number of peptide inhibitor candidates for human phospholipases A were also identified with sequences fulfilling the above criteria (see Table 1). Also peptide inhibitor candidates for human PAF acetyl hydrolase were found (see Table 1).

Accordingly, the present invention is directed to a method for preparing peptide inhibitors of a lipid-activated enzyme, the method comprising the steps of: a) identifying aggregation-prone regions in amino acid sequence of said enzyme by the use of a suitable computer algorithm; b) designing a peptide based on the aggregation-prone region found in step a), wherein said peptide comprises the sequence of said region or a part thereof, c) synthesizing the peptide designed in step b); d) contacting the peptide obtained in step c) with said lipid-activated enzyme and measuring the activity of said enzyme, wherein said peptide is an inhibitor of said enzyme, if the activity of the enzyme is decreased in the presence of said peptide.

Preferably, said lipid-activated enzyme is selected from the group consisting of

phospholipases, myeloperoxidase, acid sphingomyelinase, heat shock protein 70 and PAF acetylhydrolase. Peptide inhibitor candidates already designed based on the aggregation- prone regions found from these lipid-activated enzymes are listed in Table 1 below.

The present invention also provides peptides comprising amino acid sequence set forth in any one of SEQ ID NOS:l-68. Preferably, said peptide comprises or consists of amino acid sequence KMYFNLI (SEQ ID NO: l). In another preferred embodiment said peptide comprises or consists of amino acid sequence AALSYGFYG (SEQ ID NO:68).

The present invention further includes pharmaceutical compositions comprising a pharmaceutically effective amount of one or more of the above-described peptides as active ingredient. Pharmaceutical compositions according to the invention are suitable for topical, enteral, such as oral or rectal, and parenteral administration to mammals, including man, for the treatment of bee sting or other phospolipase related condition, including cancer, rheumatoid arthritis, multiple sclerosis, bronchial asthma, intestinal polyposis or pulmonary fibrosis or in combination with one or more pharmaceutically acceptable carriers.

The inventive compounds are useful for the manufacture of pharmaceutical compositions having an effective amount the compound in conjunction or admixture with excipients or carriers suitable for topical, enteral or parenteral application. Examples include tablets and gelatin capsules comprising the active ingredient together with (a) diluents; (b) lubricants, (c) binders (tablets); if desired, (d) disintegrants; and/or (e) absorbents, colorants, flavors and sweeteners. Injectable compositions are preferably aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fatty emulsions or suspensions. The compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, the compositions may also contain other therapeutically valuable substances. The compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain preferably about 1 to 50% of the active ingredient. More generally, the present invention also relates to the use of the compounds of the invention for the manufacture of a medicament, in particular for the manufacture of a medicament for the treatment of the above-mentioned conditions and diseases.

The pharmaceutical composition contains a pharmaceutically effective amount of the present active agent along with other pharmaceutically acceptable exicipients, carriers, fillers, diluents and the like. The term therapeutically effective amount as used herein indicates an amount necessary to administer to a host to achieve a therapeutic result, especially an antidote effect.

As discussed above, the compounds of the present invention are useful for treating the above- mentioned conditions and diseases. Thus, the present invention further relates to a method of treating said conditions and diseases which comprises administering a therapeutically effective amount of a compound of the invention to a mammal, preferably a human, in need of such treatment.

The present invention also provides kits for use in treating bee stings or other phospholipase or lipid- activated enzyme related condition as mentioned above comprising an administration means and a container means containing a pharmaceutical composition of the present invention. Preferably, the container in which the composition is packaged prior to use will comprise a hermetically sealed container enclosing an amount of the lyophilized formulation or a solution containing the formulation suitable for a pharmaceutically effective dose thereof, or multiples of an effective dose. The composition is packaged in a sterile container, and the hermetically sealed container is designed to preserve sterility of the pharmaceutical formulation until use. Optionally, the container can be associated with administration means and/or instruction for use.

The publications and other materials used herein to illuminate the background of the invention, and in particular, to provide additional details with respect to its practice, are incorporated herein by reference. The present invention is further described in the following examples, which are not intended to limit the scope of the invention.

EXAMPLES

EXAMPLE 1 The sequence of mature secretory phospholipase A ₂ (Apis mellifica) of class III

(NP_001011614; XP .391951 ; GL58585172) consisting of amino acids 34- 167 (SEQ ID NO:69) of the precursor was screened for aggregation-prone segments using CSSP,

AGGRESCAN (Conchillo-Sole et al, 2007), PASTA (Trovato et al, 2007) and TANGO (Rousseau et al., 2006; Fernandez-Escamilla et al., 2004; Linding et al., 2004). The stretch of amino acids 78-91 was found corresponding to sequence SYFVGKMYFNLl (SEQ ID NO:2) comprising sequence KMYFNLI (SEQ ID NO: l) as shown below. Region identified by

PLA ₂

CSSP ; TANGO PASTA AGGRESCAN a b c d

>gi|58585172:34-167 2-4, 6-10, 77-92 78-91 p (-6.46) 5-9, 78-91 p osp olipase A2 [Apis 14-34, 37-52,

mellifera] 55-63, 78-92

IIYPGTLWCGHGNKSSGPNELG

RFKHTDACCRTHDMCPDVMSAG

ESKHGLTNTASHTRLSCDCDDK

FYDCLKNSADTISSYPVG MYF

NLIDTKCYKLEHPVTGCGE

RTEGRCLHYTVDKSKPKVYQWF

DLRKY

Mature Sequence PLA2 (bv)

'HYPGTLWCGHGNKSSGPNELGRFKHTDACCRTHDMCPDVMSAGESKHGLT TAS

HTRLSCDCDDKFYDCLKNSADTISSYFVGKMYFNIJDTKCYKLEHPVTGCGERTEG

RCLHYfVDKSKPKVYQWFDLRKY ¹³⁴

Inhibitors

1) ⁸⁵ KMYF LI ⁹¹

2) ⁷⁸ SYFVGKMYFNLI ⁹¹

EXAMPLE 2

Activity of secretory phospholipase A ₂ (Apis mellifica) of class III towards C ₂ 8-0-PHPM was measured m the presence of peptide KMYFNLI (SEQ ID NO : l). Reaction mixture contained 2nM of phospholipase A _2> 2nM or 4nM of the peptide and 1.25 μΜ C ₂₈ -0-PHPM (1 - octacosanyl-2-(6-pyren- l -yl)hexanoyl-i«-glycero-3-phosphatidylmethanol) in 2.0 ml of 5mM HEPES, 0.1 mM EDTA, 1 mM CaCl ₂ , pH 7,4 at 37 °C with stirring. The assay was performed with or without preincubation step. The enzymatic reaction was followed by measuring the pyrene monomer fluorescence intensity at 400 nm using a spectrofluorometer. Excitation wavelength was 343 nm and the excitation and emission slits were 10 nm. The results are shown in Figure 1.

EXAMPLE 3

The sequence of human phospholipase A ₂ , content of tears (P 14555), consisting of amino acids 1-144 (SEQ ID NO :70) was screened for aggregation-prone segments as described in Example 1. The stretches of amino acids 17-25 and 61-67 were found corresponding to sequences AALSYGFYG (SEQ ID NO:68) and TKFLSYK (SEQ ID NO:3), respectively, as shown below.

Inhibitor 1

( 17-2 ) AALSYGFYG-NH2

Inhibitor 2

(61-67 ) TKFLSYK- H2 EXAMPLE 4

Activity of human phospholipase A2 , content of tears (P14555), was measured in the presence of peptide AALSYGFYG (SEQ ID NO:68). Reaction mixture contained 2nM of phospholipase of human tears, 40nM or 80nM of the peptide and 1.25 μΜ C ₂ 8-0-PHPM (1- octacosanyl-2-(6-pyren-l-yl)hexanoyl-sra-glycero-3-phosphati dylmethanol) in 2.0 ml of 5mM HEPES, 0.1 mM EDTA, 1 mM CaCl ₂ , pH 7,4 at 37 °C with stirring. The assay was performed with or without preincubation step. The enzymatic reaction was followed as described in Example 2. The results are shown in Figure 2.

EXAMPLE 5

The sequences of human phospolipase A2, PAF acetylhydrolase myeloperoxidase, acid sphingomyelinase, and heat shock protein 70 were screened for aggregation-prone segments using AGGRESCAN (Conchillo-Sole et al, 2007), PASTA (Trovato et al., 2007) and TANGO (Rousseau et al., 2006; Fernandez-Escamilla et al, 2004; Linding et al, 2004). The stretches found are listed in Table 1.

Table 1 . The sequences examined and peptide candidates found.

Phospholipa.se A2. group IB fpancreas) [Homo sapiens]

Phospholipase A2, group IE (pancreas) [Homo sapiens]

>gi|76827695|gb|AAI06727.1| P ospholipase A2, group IB (pancreas) [Homo sapiens]

KLLVLAVLLTV AA ADS GIS PR

² ¼VWQFRKMIKCV]ffGSDPFLEYNNYGCYCGLGGSGTPVDELDKCCQTHDNCY DQA LDSCKFLLDN PYTHTYSYSCSGSAITCSSKNKECEAFICNCDRNAAICFSKAPYNKAHKNLDTK YCQS

l ⁰² AAICF ¹⁰⁶ (0.221)

phospholipase A2, group IIA precursor THomo sapiens]

phospholipase .42, group IIA precursor [Homo sapiens]

>gi|239915991|ref|NP_001 155201.1| phospholipase A2, membrane associated precursor [Homo sapiens]

MKTLLLLAVIMIFGLLQAHG

²¹ NLVNFHRMIKLTTGKEAALSYGFYGCHCGVGGRGSPKDATDRCCVTHDCCYKRL EKRGCG

TKFLSYKFSNSGSRITCAKQDSCRSQLCECDKAAATCFARNKTTYNKKYQYYSN HCRGSTP

RC

³⁸ AALSYGFY ⁴⁵ (5)

group l!D secretory phospholipase A2 precursor [Homo sapiens] group I.I 1) secretory phospholipase A 2 precursor [Homo sapiens]

>gi|6912596|ref|NP_036532.11 phospholipase A2, group HDD precursor [Homo sapiens]

MELALLCGLVVMAGVJPIQG

²¹ GIL^N™VKQVTGmPILSYWYGCHCGLGGRGQPK13ATDWCCQTIJDCCYDHL KTQGC SIYpaDYYRYNFSQGNIHCSDKGSWCEQQLCACDKEVAFCLKRNLDTYQKRLRFYWRPHC RG QTPGC

¹¹⁶ VAFCLK ¹²¹ (4.5)

group HE secretory phospholipase A2 precursor [Homo sapiens]

group IIE secretory phospholipase A2 precursor [Homo sapiens]

>gi|765746l|ref|NP_055404.li phospholipase A2, group HE precursor [Homo sapiens]

KSPHVLVFLCLLVALVTGN

z ^l LYQFGVMIEKMTGKSALQYr©YGCYCGIGGSHWVDQTDWCCHAHDCCYGRLEl s 5CEPKlFKYLF SVSERGIFCAGRTTCQRLTCECDKRAALCFRRNLGTYNRKYAHYPN LCTGPTPPC

²³ VQFGVMI ²⁹ (39)

8 ³ YLFSVS ⁹⁰ (20)

group IIF secretory phospholipase A2 [Homo sapiens] group HF secretory phospholipase Λ2 [Homo sapiens]

>gi|145553989|ref|NP_073730.3| group IIF secretory phospholipase A2 [Homo sapiens]

MADGA A]^ GFKK VLDRCFSG GPr<FGASCPSRTSRSSLGMK IWVAILAGSVLSTAH

G

^M SLLNLKAMVEAVTGRSAILSFVGYGCYCGLGGRGQPKDEVDWCCHArrDCCYQE LFDQGCH PWDHYDHTffiNNTEWCSDLNKTECD QTCMCDKHMVLCLMNQTYREEYRGFLNVYCQGP TPNCSIYEPPPEEVTCSHQSPAPPAPP

¹ AILSFVGY ²⁴ (62)

^M VLCLM ¹⁰² (16)

¹¹³ FLNVY ^{1 I7} (4)

group 3 secretory phospholipase A2 precursor [Homo sapiens]

group 3 secretory phospholipase . 2 precursor [Homo sapiens]

>gi|142976884|ref|NP_056530.2| group 3 secretory phospholipase A2 precursor [Homo sapiens]

MGVQAGLFGMLGFLGVALGGSPALRWYRTSCHLTKAVPGNPLGYLSFLAKDAQGLAL IHA

WDAHPJILQSCSWEDEPELTAAYGALCAHETAWGSHHTPGPELQRALATLQSQWEAC RALEE

SPAGARKKRAAGQSGVPGGGHQREKRGWTMPGTLWCGVGDSAGNSSELGWQGPDLCC RE

HDRCPQMSPLQYNYGIRNYTU^TISHCDCDTPJQQCLQNQHDSISDIVGVAFFNVLE IPCFVLE

EQEACVAWYWWGGCRMYGTVPLARLQPRTFYNASWSSRATSPTPSSRSPAPP PRQKQHLR

KGPPHQKGS PJ ^J SKANTTALQDPMVSPRLDVAPTGLQGPQGGLKPQGAR CRSFRRHLDQ

CBHQIGPREffiFQLLNSAQEPLPHCNCTRRLAOTLRLHSPPEVTNMLWELLGTTCF KLAPPLDC

VEGKNCSRDPRAIRVSARHLRRLQQRRHQLQDKGTDERQPWPSEPLRGPMSFYNQCL QLTQA

ARRPDRQQKSWSQ

⁷ LFGMLGFLGVAL ^1S (70)

4 ² LGYLSFLA ⁴⁹ (38)

2 ³¹ IVGVAEF VL ²⁴⁰ (81)

2 ⁵² ACVAWYW ²⁵⁹ (91)

Cytosolic Group IV phospholipases A ₂ (cPLA ₂ )

phospholipase A2, group IV A (cytosolic, calcium-dependent) [Homo sapiens] phospholipase A2, group IVA (cytosolic, calcium-dependent) [Homo sapiens]

>gi|56203412|emb|CAI22252.1| phospholipase A2, group IVA (cytosolic, calcium-dependent) [Homo sapiens]

MSFTOPYQHIIVEHQYSHKOTVVVLRATK^

NETFEFILDPNQENVLEITLMDANY^

PDLRFSMALCDQE TFRQQRKEHIRESMKKLLGPKNSEQLHSARDVPVVAILGSGGGFRAMVGFSGVM

ALYESGILDCATYVAGLSGSTWYMSTLYSHPDFTEKGPEEI^ELMKIWSHNPLLLLT PQ VKRYVESLW

K KSSGQPVTFTDIFGMLrGETLnd RM TTLSSL E VOTAQCPLPLFTCLHVKPDVSELMFADWVEFSP

YEIGMA YGWMAPDLFGSKFMGWVKKYEENPLHFLMGVWGSAFSILFNRVLGVSGSQSRGSTMEEE

LEMTTKfflVSNDSSDSDDESHEPKGTENEDAGSDYQSDNQASMHRMIMALVSDSAL FNTREGRAGKVH

OTMLGL LNTSYPLSPLSDFATQDSFDDDELDAAVADPDEFEIRIYEPLDVKS KIHVVDSGLTFNLPYPLI

LRPQRGVDLnSFDFSARPSDSSPPFKELLLAEKWA^

DCPTnHWLANIM¾KYRAPGVPRETEEEKEIADFE>IFDDPESPFSTFNFQYPNQA

DVI EAMVESIEYRRQNPSRCSYSLS rVEARPJT KEFLSKP A

i ⁰ FTYVVL ^2S (82)

1 ⁰⁵ ATFTV«» (6 _, 5)

¹⁸⁹ WAIL ^1M (85.5)

2 ³² WYMSTLY ²³⁸ (17)

2 ⁹⁴ IFGMLI ²⁵⁹ (9)

3 ²⁸ LFTCL ³³² (5.4)

3 ⁷² FFMGTV ³⁷⁷ (4.8)

3 ⁸⁸ FLMGVWGSAFSILF ⁴⁰¹ (55)

4 ⁶⁸ IMALY ⁴⁷³ (79)

7 ⁰ LIISF ⁷⁴ (S.2)

1 ³⁶ TIIHFVLANI ¹⁴⁵ (32)

phospholipase A2. group V precursor [Homo sapiens]

phospholipase A2, group V precursor [Homo sapiens] >gi|4505853|ref|NP_000920. l | calcium-dependent phospholipase A2 precursor [Homo sapiens] M GLLPLAWFLACSVPAVQG (sig pept)

²⁰ GLLDLKSMIEKVTG ALTNYGFYGCYCGWGGRGTPKDGTDWCCWAHDHCYGRLEEKGC MRTQSYKYRFAWGVVTCEPGPFCHVNLCACDRK^

⁹ ° FAWGVVTC ⁷⁷ (86)

group 10 secretory phospholipase A2 precursor [Homo sapiens]

group 10 secretory phospholipase A2 precursor [Homo sapiens]

>gi|4505845|ref]NP_003552.1| phospholipase A2, group X precursor [Homo sapiens]

MGPLPVCLPIMLLLLLPSLLLLLLLPGPGSGEASRILRVHRRGILELAGTVGCVGPRTPI AYMKY GCFCGLGGHGQPRDAIDWCCHGHDCCYTRAEEAGCSPKTERYSWQCVNQSVLCGPAENKC Q ELLCKCDQEIANCLAQTEYNLKYLFYPQFLCEPDSPKCD ■ .

^M TVGCV ⁵ (.161)

l ^m YSWQC ^ln (0.29)

M ⁹ YLFYP ¹⁵³ (0.111)

group XIIA secretory phospholipase A2 precursor [Homo sapiens!

group XIIA secretory phospholipase A2 precursor [Homo sapiens]

>gi|21361944|refjNP_l 10448.2| group ΧΠΑ secretory phospholipase A2 precursor [Homo sapiens] MALLS RPAETLLLLLMAAVVRCQEQAQTTDWRATLKTIPJ^GVHKIDTYLNAALDLLGGEDGL CQYKCSDGSKPFPRYGYKPSPPNGCGSPLFGVHLNIGIPSLTKCCNQHDRCYETCGKSKN DCD EEFQYCLSKICRDVQKTLGLTQHVQACETTVELLFDSVIHLGCKPYLDSQRAACRCHYEE KTD

L

⁹ LTLLLLLMAAVV ²⁰ 99

FQ ( ₀ _237)

PAF acetyl hydrolases

phospholipase A2, group VII precursor [Homo sapiens]

phospholipase A2, group VII precursor [Homo sapiens]

>gi|270133071|ref|NP_001161829.1| platelet-activating factor acetylhydrolase precursor [Homo sapiens]

MVPPKLHVLFCLCGCLAVVYPFDWQYINPVAH ^

PYSVGCTDLMFDHTN GTR _^ RLYYPSQDNDRLDTLWIPNKEYFWGLSK^GTmVLMGNTLRL

LFGSMTTPANWNSPLCTGEKYPLVWSHGLGAFRTLYSAIGIDLASHGnVAAVEHRDR SASAT

YYFKDQSAAEIGDKSWLYLRTLKQEEETHH NEQWQRAKECSQAI^LILDIDHGKPVKNALD

LKTOMEQLKDSmREKIAVIGHSFGGAT\aQTLSEDQ

HHNSEYFQYPA DaiK CYSPDKER™^

AIDLSNKASLAFLQKHLGLHKDFDQWDCLffiGDDENLIPGTNmTNQHI LQNSSGIEKYN

⁸ VLFCLCGCLAVV ¹⁹ (83; ²³³ LSLIL ²³⁷ (10)

⁴⁴ VLMAAA ⁴⁹ (15) ²⁶⁷ IAVIG ²⁷¹ (20.5)

^1O3 YFWGL ¹⁰⁷ (15.8) ³¹⁴ LFFTN ³¹⁸ (17)

1 ⁴⁶ LVVFS ¹⁵⁰ (48) ³³⁷ FTFAT ³⁶¹ (1 1.4

¹⁵ *LYSAIGI ¹⁶⁵ (9.8)

I ⁷² F1VAAV ¹⁷⁷ (87)

1 ⁸⁶ ATYYF ¹⁵⁰ (18.6)

i ⁰² SWLYL ²⁰⁶ (8.7) Myeloperoxidase

>sp |P0516 I PERM_HUMAN Myeloperoxidase OS=Homo sapiens GN=MPO PE=1 SV=1 MGVPFESSLRCMVDLGPCWAGGLTAEMKIJLLAIA^

AY^RRESI QRLRSGSASPMELLSYFKQPVAATRTATRAADYLHVALDLLERKLRSLWRRPFNVTDVLT PAQLN VLSKSSGCAYQDVGVTCPEQD YRTITGMCKMRRSPTLGASNRAFVRWLPAEYEDGFSLPYGWTPGV RHGFPVA LARAVSNEIVRFPTDQLTPDQERSL FMQWGQLLDHDLDFTPEPAARASFVTGVNCETSCVQQPPCFPLKIPPND PRI NQADCIPFFRSCPACPGSNITIRNQimLTSE^/DASMWGSEEPLARNLRNMSNQLGLI^W QRFQDNGPA LLPFDNLHDDPCLLTMRSARIPCFLAGDTRSSEMPELTSMHTLLLREHNRLATELKSLNP RWDGERLYQEARKIV GAMVQII YRDYLPLVLGPTAMRKYLPTYRSYNDSVDPRIANVFTNAFRY

GHTLIQPFMFRLDNRYQPMEPNPRVPLSRVFFAS RWLEGGIDPILRGL ATPAKLNRQNQIAVDEIRERLFEQ

AmRIGLDLPALNMQRSRDHGLPGY AWRRFCGLPQPETVGQLGTVLRNLKIiARKL EQYGTPNNIDIWMGGVSEP LKRKGRVGPLLACI IGTQFRKLRDGDRFW ENEGVFSMQQRQALAQISLPRIICDNTGITTVS N1TIFMSNSYPR DFV CSTLPALNLAS REAS

LLLALAGLLAILA'

LLSYF

²⁴⁹ SLMFMQWG ²⁵⁶

²⁷⁵ FVTGV ²⁷⁹

³³³ LTSFV ³³⁷

³⁶⁰ LGLLAV ³⁶⁵

4 ⁹ 1VGAMVQIITY ^4;

⁴⁹³ VFTNAJF ⁴⁹⁸

⁵³⁰ VFFASWRWLEGGI ⁵⁴³

Acid Sphingomyelinase

>gi| 179095|gb|AAA58377.l| acid sphingomyelinase [Homo sapiens]

MPRYGASIiRQSCPRSGREQGQDGTAGAPGLL MGLVLALiALALALALSDSRVLWAPAEAHPLSPQGHPAR

LHRIVPRLRDVFGWGNLTCPIC GLFTAINLGLKKEP VARVGSVAI LCNLL IAPPAVCQSIVHLFED

DMVEV RRSVLSPSEACGLLLGSTCGHWDIFSSWNISLPTVPKPPPKPPSPPAPGAPVSRILFLTD LHWD

HDYLEGTDPDCADPLCCRRGSGLPPASRPGAGYliGEYSKCDLPLRTLESLLSGLGP AGPFDtWYWTGDIP

AHDVWHQTRQDQLRALTTVTALVRKFLGPVP-VYPAVGNHESIPVNSFPPPFIEGNH SSRWLYEAMAKAWE

PWLPAEALRTLRIGGFYALSPYPGLRLISL MNFCSRENF LLINSTDPAGQLQWIJVGELQAAEDRGD V

HIIGHIPPGHCLKSWSPOTYRIVARYENTLAAQFFGHTHVDEFEVFYDEETLSRPLA VAFLAPSATTYIG LNPGYRVYQIDGNYSRSSHWLDHETYILNLTQANIPGAIPHWQLLYRARETYGLPNTLPT AWH LVYRM RGDMQLFQTF FLYHKGHPPSEPCGTPCRLATLCAQLSARADSPALCRHLMPDGSLPEAQSL PRPLFC

³⁰ LL MGLVLALALALALAL ⁴⁸ (99)

⁹⁵ LFTAl" (13)

²⁰¹ ILFLT ²⁽⁾⁵ (86) ' ALTTVTALV (9)

'NFWLLI ³⁹⁴ (86)

' LAVAFL ⁴⁸¹ (25)

;LFQTFWFLY ⁵⁷⁴ (95)

Heat shock protein 70

>gi 1292160 | gb j AAA02807.11 heat shock protein 70 [Homo sapiens]

MSWGIDLGFQSCYVAVARAGGIETIANEYSDRCTPACISFGPKNRSIGAAA SQVISNAKNTVQGFKRF

HGRAFSDPFVEAEKSNIAYDIVQ PTGLTGIK^TYMEEERNFTTEQV AMLLSKLKETAESVLKKPVVDC SVPCFYTDAERRSXmDATQIAGLNCLRLMNETTAVALAYGIYKQDLPRLEEKPRNWFVDM GHSAYQV

SVCAFNRG LKVIATAFDTTLGGRKFDEVXV HFCEEFGKKYKLDIKS rRALLRLSQECEKL KLMSAN

ASDLPLSIECFMNDVDVSGTMNRG TLEMCNDLLARVEPPbRSVLEQTKLKKEDIYAVEIVGGATRIPAV

KEKISKFFGKELSTTLNADEAVTRGCALQCAILSPAFKTOEFSITDWPYPISLRWNS PAEEGSSDCEVF

SKNHAAPFSKVLTFYRKEPFTLEAYYSSPQDLPYPDPAIAQFSVQKA^TPQSDGSSS KV VKVRVTSrVHGI

SVSSASLVEV

HKSEENEEPMETDQNAKEEEKMQVDQEEPHVEEQQQQTPAEN AESEEMETSQAGSKDK MDQPPQCQEG SEDQYCGPANRESAIWQIDREMLNLYIENEGKMI QDKLE ER DA NAVEEYVYEMRD KLSGEYEKFVSEDDR SFTLKLEDTENWLYEDGEDQPKQVYVDKLAELKNLGQPIKIRFQESEERPNYLK

N

¹⁴ YVAVA ¹⁸ (15.5)

n 'VTAMLL ¹²² (36)

1 ⁷⁴ TTAVALAYGIY ¹⁸

1 ⁹⁷ NVVFV ²⁰¹ (66)

2 ²² VLATAF ²²⁷ (49)

93 MLNLYI (5)

REFERENCES

Atherton, E.; Sheppard, R.C., 1989, Solid Phase peptide synthesis: a practical approach. Oxford, England: IRL Press. ISBN 0199630674.

Code, Christian, Domanov,Yegor A., Jutila, Arimatti, and Kinmmen, Paavo K. J., 2008, Amyloid-Type Fiber Formation in Control of Enzyme Action: Interfacial Activation of Phospholipase A ₂ , Biophysical Journal, 95:215-224

Code, Christian, Domanov, Yegor A., Killian, J. Antoinette, and Kinnunen, Paavo K. J., 2009, Activation of phospholipase A ₂ by temporin B: Formation of antimicrobial peptide-enzyme amyloid-type cofibrils, BBA - Biomembranes, doi: 10.1016/j.bbamem.2009.03.002

Conchillo-Sole, Oscar, de Groot, Natalia S., A iles, Francesc X., Vendrell, Josep, Daura, Xavier, and Ventura, Salvador, 2007, AGGRESCAN: a server for the prediction and evaluation of "hot spots" of aggregation in polypeptides, BMC Bioinformatics, 8:65; doi 10.1186/1471-2105-8-65

Fernandez-Escamilla AM, Rousseau F, Schymkowitz J, and Serrano L, 2004, Prediction of sequence-dependent and mutational effects on the aggrgation of peptides and proteins, Nat Biotechnol, e-pub

Linding R, Schymkowitz J, Rousseau F, diella F, and Serrano L, 2004, A comparative study of the relationship between protein structure and beta-aggregation in globular and intrinsically disordered proteins, J Mol Biol 354-353

Rousseau, Frederic, Schymkowitz, Joost, and Serrano, Luis, 2006, Protein aggregation and amyloidosis: confusion of the kinds?, Current Opinion in Structural Biology, 16:1-9

Shimizu, Takao, 2009, Lipid Mediators in Health an Disease: Enzymes and Receptors as Therapeutic Targets for the Regulation on Immunity and Inflammation, Annu. Rev.

Pharmacol. Toxicol, 49: 123-150

Trovato, Antonio, Seno, Flavio, and Tosatto, Silvio C.E., 2007, The PASTA server for protein aggregation prediction, Protein Engineering Design and Selection, 20(10):521-523;

doi: 10.1093/protein/gzm042