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WO2008066612A2 - Procédés et cibles pour identifier des matières pour réguler des troubles de la peau à caractère hyper prolifératif - Google Patents

Procédés et cibles pour identifier des matières pour réguler des troubles de la peau à caractère hyper prolifératif Download PDF

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Publication number
WO2008066612A2
WO2008066612A2 PCT/US2007/022097 US2007022097W WO2008066612A2 WO 2008066612 A2 WO2008066612 A2 WO 2008066612A2 US 2007022097 W US2007022097 W US 2007022097W WO 2008066612 A2 WO2008066612 A2 WO 2008066612A2
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Prior art keywords
protein
gene
identifying
identified
expression
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WO2008066612A8 (fr
Inventor
Thomas Larry Dawson, Jr.
Charles Winston Saunders
Jun Xu
Raymond Alan Grant
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Procter and Gamble Co
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Procter and Gamble Co
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/5743—Specifically defined cancers of skin, e.g. melanoma
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • This Application contains three data tables (designated as Table 1 , Table 2, and Table 3 in the specification) as appendices on one separate compact disc, which are herein incorporated by reference in their entirety. Two additional discs containing the three data tables are also provided as required. The compact disc and its' respective duplicates are identical in their contents. The compact disc contains three ASCI (.txt) files.
  • the present invention relates to methods of identifying target genes and materials for regulating proliferative skin conditions that are caused by or associated with, at least in part, Malassezia, or more particularly Malassezia globosa, including multiple hyper-proliferative skin conditions ("HPSCs"), particularly dandruff and/or seborrheic dermatitis ("D/SD").
  • HPSCs hyper-proliferative skin conditions
  • D/SD seborrheic dermatitis
  • the invention also relates to methods for the treatment of proliferative skin-related conditions, including HPSCs, using the genes or proteins of the invention as targets for intervention.
  • D/SD and other HPSCs are the result of a convergence of three factors - Malassezia fungi, sebaceous secretions, and a susceptible host.
  • the Malassezia feed on oils secreted by the sebaceous glands consume the fatty acids necessary for their proliferation, and release other irritating free fatty acids.
  • the released free fatty acids penetrate into the scalp skin outer layer, the stratum corneum, and break up the skin's natural barrier function.
  • the skin senses this barrier breach and attempts repair, resulting in the natural barrier function.
  • the skin senses this barrier breach and attempts repair, resulting in the excess proliferation and enhanced oil secretion which characterizes the hyper-proliferative skin condition.
  • the Malassezia also secrete a variety of other enzymes to feed on the scalp.
  • Seborrheic dermatitis is a more severe form of dandruff seen in 3-5% of individuals, resulting from the same etiologic factors, but expressed in individuals with a higher sensitivity. Seborrheic dermatitis is often seen on the sides of the nose (nasolabial folds), the eyebrows, and in men in the beard and mustache. Since dandruff and seborrheic dermatitis respond to the same treatments, these conditions are believed to be caused by the same etiologic factors.
  • the applicants have applied new biotechnology to find new, proprietary ways to reduce HPSCs without necessarily killing the fungus. Furthermore, the applicants have used techniques similar to police forensic identification to identify an HPSC-associated Malassezia species, Malassezia globosa, and decoded its DNA sequence for the purpose of developing new treatments for HPSCs. In addition, applicants set forth in Table 2 the list of Malassezia genes and proteins believed by applicants to be newly identified. This allows the study of every biological process used by the fungus.
  • Applicants have identified the biological pathways most used by the fungus; lipases, proteases, phospholipases, MAP kinases, ceramidase, and peroxide generators, based on multiple laboratory investigations and the DNA sequence. This allows high throughput screens against these proteins to identify materials that can interrupt their function, and thereby the generation of the toxic metabolites.
  • the present invention relates to identifying materials that modulate gene expression or the activity of a protein involved in the metabolism of Malassezia.
  • the present invention identifies such genes and proteins as targets to identify materials that modulate their expression or activity and thereby regulate M ⁇ / ⁇ ysez/ ⁇ -associated conditions.
  • the invention provides for a method of screening materials useful for regulating an HPSC comprising the steps of: (a) contacting a protein of the invention to a material; and (b) measuring binding or activity of the protein, wherein binding of the material to the protein or a modulation in the activity of the protein indicates that the material may be useful for regulating an HPSC.
  • the invention provides for a method of identifying materials useful for regulating an HPSC, comprising the steps of: (a) contacting a cell population producing a protein identified in Table 2 to a material; and (b) measuring the activity of the protein, wherein a modulation in the activity of the protein indicates that the material may be useful for regulating an HPSC.
  • the invention provides for a method of screening materials useful for modulating expression of a gene or a family of genes involved in regulating an HPSC comprising the steps of: (a) exposing a gene of the invention to a material; and (b) measuring expression of the gene, wherein a modulation in the expression of the gene indicates the material may be useful for regulating an HPSC.
  • the invention provides for a method of screening materials useful for regulating HPSCs, comprising: (a) selecting a material that binds or regulates the activity or the expression of a protein of the invention; (b) further determining whether the material regulates HPSCs in an in vitro or human-based in vivo HPSC model system; and (c) identifying a material that modulates an HPSC in the HPSC model system as a material for regulating an HPSC.
  • the invention provides a method for diagnosing a condition associated with an HPSC, comprising detecting the level of expression of, or assaying for activity of a protein encoded by, a gene of Table 2 on skin or in an in vitro model or a cell culture system, wherein the difference in expression and/or activity compared to expression and/or activity in healthy/control skin or in an in vitro model or a cell culture system, is indicative of an HPSC.
  • the present invention also provides methods of monitoring the effectiveness of treatment, or monitoring the progression/regression of a condition that is associated with an HPSC, comprising: (a) administering a therapeutic or cosmetic composition; (b) preparing a gene or gene family expression profile and/or assaying for an activity of a protein encoded by a gene or a member of a gene family identified in Table 2 on skin or in an in vitro model or a cell culture system at various time intervals during the treatment, and (c) comparing the subject expression profile and/or activity to the expression profile and/or activity to each other and to control datasets.
  • the profiles of gene expression or protein activity and their comparison to datasets from control samples would be indicative of effectiveness of the treatment of the HPSC.
  • Table 1 is the DNA sequence of the Malassezia globosa genome. TABLE 2. Table 2 sets forth the Malassezia globosa genes and proteins believed by applicants to be newly identified, including the location of genes and the translated proteins that are coded by those genes, and the assigned functions of those genes.
  • Table 3 sets forth the Malassezia globosa genes and proteins believed by applicants to be newly identified.
  • the invention comprises various molecules: genes that are DNA; transcripts that are RNA; nucleic acids that regulate their expression such as antisense molecules, siRNAs, micro RNAs; molecules that may be used to detect them, such as DNA or RNA probes; primers that may be used to identify and isolate related genes; and proteins and polypeptides, and materials that regulate or modulate them.
  • molecule is used herein to describe all or some of the entities of the invention. It is to be construed in the context in which it is used.
  • RNA processing RNA precursors
  • translational control e.g., through control of initiation, provision of RNA precursors, RNA processing
  • fundamental biological processes such as cell cycle, cell differentiation, and cell death, are often characterized by the variations in the expression levels of groups of genes and their translation products.
  • Changes in gene expression may also be associated with changes in HPSC symptoms. Thus, changes in the expression levels of particular genes or gene families may serve as signposts for the presence and progression of various conditions. Monitoring changes in gene expression may also provide certain advantages during material screening. Often materials are screened for their ability to interact with a major target without regard to other effects the materials have on cells.
  • the present inventors have examined various HPSCs to identify the role of Malassezia in such conditions. Further work in the art reveals this causal or exacerbating role in other HPSCs. Analysis of Malassezia biology and gene expression may provide novel targets for the treatment of D/SD and other HPSCs. This may also provide useful markers for diagnostic uses as well as markers that may be used to monitor Malassezia associated conditions, progression, material efficacy, and material metabolism.
  • the expression profiles may be used to identify genes that are differentially expressed under different conditions.
  • the present invention may be used to identify families of genes that are differentially expressed.
  • gene families includes, but is not limited to, the specific genes identified by accession numbers herein, as well as related sequences.
  • Related sequences may be, for example, sequences having a high degree of sequence homology with an identified sequence either at the nucleotide level or at the amino acid level.
  • a high degree of sequence identity is seen to be from 65%- 100% sequence identity at the nucleotide level to the genes, in another embodiment from 80%- 100%, in still another embodiment from 85%-100%, in yet another embodiment from 90%-100%, in an alternate embodiment from 95%- 100%, and in still another embodiment from 98%- 100% sequence identity to the genes.
  • a high degree of similarity is seen to be from 50%- 100%, in another embodiment from 70% - 100% similarity, in yet another embodiment from 75%- 100% similarity, in still another embodiment from 80% - 100% similarity, in an alternate embodiment from 85%- 100% similarity, in yet another embodiment from 90% - 100% similarity, in still another embodiment from 95%- 100% similarity, and in still another embodiment from 98%- 100% sequence similarity.
  • Methods are known in the art for determining homologies and identities between various sequences, some of which are described herein.
  • related sequences include homologs and orthologs from different organisms.
  • a homologous gene may be of different length and may comprise regions with differing amounts of sequence identity or similarity to a specifically identified sequence.
  • genes and proteins from species other than those listed in the sequence listing could be useful in the present invention.
  • One of skill in the art would further recognize that by using probes from the known species' sequences, cDNA or genomic sequences homologous to the known sequence could be obtained from the same or alternate species by known cloning methods. Such homologs and orthologs are contemplated to be useful as genes and proteins of the invention.
  • variants it is intended to mean similar sequences.
  • conservative variants may include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the polypeptides of the invention.
  • Naturally occurring allelic and splice variants may be identified with the use of known techniques, e.g., with polymerase chain reaction (PCR), single nucleotide polymorphism (SNP) analysis, and hybridization techniques. In order to isolate orthologs and homologs, generally stringent hybridization conditions are utilized dictated by specific sequence, sequence length, guanine + cytosine (GC) content and other parameters.
  • Variant nucleotide sequences also include synthetically derived nucleotide sequences, e.g., derived by using site-directed mutagenesis. Variants may contain additional sequences from the genomic locus alone or in combination with other sequences.
  • the molecules of the invention can also include truncated and/or mutated proteins wherein regions of the protein not required for ligand binding or signaling have been deleted or modified. Similarly, they may be mutated to modify their ligand binding or signaling activities. Such mutations may involve non-conservative mutations, deletions, or additions of amino acids or protein domains. Variant proteins may or may not retain biological activity. Such variants may result from, e.g., genetic polymorphism or from human manipulation.
  • Fragments and variants of genes and proteins of the invention are also encompassed by the present invention.
  • fragment it is intended to mean a portion of the nucleotide or protein sequence. Fragments may retain the biological activity of the native protein. Fragments of a nucleotide sequence are also useful as hybridization probes and primers or to regulate expression of a gene, e.g., antisense, siRNA, or micro RNA.
  • a biologically active portion may be prepared by isolating a portion of a nucleotide sequence, expressing the isolated portion (e.g., by recombinant expression), and assessing the activity of the encoded protein.
  • Fusions of a protein or a protein fragment to a different polypeptide are also contemplated.
  • the fusion partner may be a signal (or leader) polypeptide sequence that co-translationally or post-translationally directs transfer of the protein from its site of synthesis to another site (e.g., the yeast ⁇ -factor leader).
  • a fusion partner may be added to facilitate purification or identification of the protein of the invention (e.g., poly-His, Flag peptide, or fluorescent proteins).
  • the molecules of the invention may be prepared by various methods including, but not limited to, cloning, PCR-based cloning, site-directed mutagenesis, mutagenesis, DNA shuffling, DNA and/or RNA isolation and/or purification, oligonucleotide probe design, and nucleotide sequence alterations known in the art.
  • Libraries of recombinant polynucleotides may also be generated from a population of related sequences comprising regions that have substantial sequence identity and may be recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding a domain of interest may be shuffled between a gene of the invention and other known genes to obtain a new gene coding for a protein with an altered property of interest, e.g. a dominant negative mutation (Ohba et al. (1998) MoI. Cell. Biol. 18:51199-51207, Matsumoto et al. (2001) J. Biol. Chem. 276: 14400-14406).
  • sequence identity also referred to herein as “sequence identity” or “percent identity,” may be determined by aligning two sequences or subsequences over a comparison window, wherein the portion of the sequence in the comparison window may optionally comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which may comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which an identical residue (e.g., nucleic acid base or amino acid) occurs in both sequences, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
  • Percent sequence identity may be calculated by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482-485 (1981); or by the homology alignment algorithm of Needleman & Wunsch, J. MoI. Biol. 48:443-445 (1970); either manually or by computerized implementations of these algorithms (GAP & BESTFIT in the GCG Wisconsin Software Package, Genetics Computer Group; various BLASTs from National Center for Biotechnology Information (NCBI), NIH).
  • a preferred method for determining homology or sequence identity is by BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx (Karlin et al. (1990) Proc. Natl. Acad. Sci. USA 87, 2264-2268 and Altschul, (1993) J. MoI. Evol. 36, 290-300), which are tailored for sequence similarity searching.
  • BLAST Basic Local Alignment Search Tool
  • these various genes and proteins may exhibit sequence variations.
  • the length of the sequence to be compared may be less than the full-length sequence.
  • Molecules of the invention may be prepared for various uses including, but not limited to: purifying the protein or nucleic acid product, generating antibodies, for use as reagents in screening assays, and for use as pharmaceutical compositions. Some embodiments may be carried out using an isolated gene or a protein, while other embodiments may require use of cells that express the gene and/or protein.
  • the cells may endogenously express it; may have been stimulated to increase endogenous expression; or have been genetically engineered to express the molecule.
  • Expression of a protein of interest may be determined by, for example, detection of the polypeptide with an appropriate antibody ⁇ e.g., Western blot), use of a DNA probe to detect mRNA encoding the protein ⁇ e.g., northern blot or various PCR-based techniques), or measuring binding of an agent selective for the polypeptide of interest ⁇ e.g., a suitably-labeled selective ligand).
  • the present invention further provides recombinant molecules that contain a coding sequence of, or a variant form of, a molecule of invention.
  • a coding DNA sequence is operably linked to other DNA sequences of interest including, but not limited to, various control sequences for integration, replication, transcription, expression, and/or modification.
  • a vector of the present invention may be capable of directing the replication or insertion into the host chromosome, and preferably directing the expression of the gene.
  • Control elements that are used for regulating the expression of a gene include, but are not limited to, inducible or constitutive promoters, secretion signals, enhancers, termination signals, ribosome-binding sites, and other regulatory elements.
  • the inducible promoter is readily controlled, such as being responsive to a nutrient or an antibiotic.
  • the vector harboring a nucleic acid molecule may include a prokaryotic or eukaryotic replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extra-chromosomally in a prokaryotic or eukaryotic host cell, such as a bacterial or fungal host cell.
  • a prokaryotic or eukaryotic replicon may also include a gene whose expression confers a detectable characteristic (e.g., resistance to ampicillin).
  • Vectors may further include a prokaryotic or eukaryotic or bacteriophage promoter capable of directing the expression (transcription and translation) of the coding gene sequences in a bacterial or fungal host cell, such as E. coli, S ⁇ cch ⁇ romyces, or Cryptococcus.
  • Promoter sequences compatible with bacterial or fungal hosts may be provided in plasmid vectors containing convenient restriction sites for insertion of a DNA sequence of the present invention, e.g., pCDNAl, pCDNA3.
  • Expression vectors compatible with eukaryotic cells may also be used to form a recombinant molecule that contains a sequence of interest.
  • Commercially available vectors often contain both prokaryotic and eukaryotic replicons and control sequences, for an easy switch from prokaryotic to eukaryotic cell to ES cells for generating transgenic cells (e.g., pCDNA series from InvitrogenTM).
  • Eukaryotic cell expression vectors used to construct the recombinant molecules of the present invention may further include a selectable marker that is effective in a eukaryotic cell (e.g., neomycin resistance).
  • the selectable marker may be present on a separate plasmid, the two vectors introduced by co-transfection of the host cell, and transfectants selected by culturing in the appropriate material for the selectable marker.
  • Vectors may also contain fusion proteins or tag sequences that facilitate purification or detection of the expressed protein.
  • the present invention further provides host cells transformed with a recombinant molecule of the invention.
  • the host cell may be a prokaryote, e.g. , a bacterium, or a eukaryote.
  • eukaryotic host cell lines include, but are not limited to, Sf-9, Sf-21, CHO cells, ATCC CCL61, NIH-3T3, S. cerevisi ⁇ e, and BHK cells. In many instances, primary cell cultures may be preferred.
  • Transformation of appropriate host cells with a molecule of the present invention may be accomplished by known methods that depend on the host system employed.
  • electroporation and salt treatment methods may be employed, while for transformation of eukaryotic cells, electroporation, cationic lipids, or salt treatment methods may be employed (See Sambrook et ⁇ l. (1989) supra).
  • Viral vectors including, but not limited to, retroviral and adenoviral vectors have also been developed that facilitate transfection of primary or terminally differentiated cells.
  • Other techniques may also be used that introduce DNA into cells, e.g., liposome, gold particles, or direct injection of the DNA expression vector (as a projectile), containing the gene of interest, into human tissue.
  • Successfully transformed cells may be cloned to produce stable clones.
  • Cells from these clones may be harvested, lysed and their content examined for the presence of the recombinant molecules using known methods.
  • nucleic acid samples which may be DNA and/or RNA, used in the methods and assays of the invention may be prepared by available methods. Methods of isolating total mRNA are known. For example, methods of isolation and purification of nucleic acids are described in detail in Chapter 3 of Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization with Nucleic Acid Probes, Elsevier Press (1993). Such samples include RNA samples, but may also include cDNA synthesized from an mRNA sample isolated from a cell or tissue of interest. Such samples also include DNA amplified from the cDNA, and RNA transcribed from the amplified DNA.
  • Biological samples containing nucleic acids or proteins may be of any biological tissue or fluid or cells, or may be cells grown in vitro, such as cell lines and tissue culture cells.
  • the sample may be any suitable "clinical sample.”
  • suitable clinical samples can include, but are not limited to, sputum, blood, blood-cells (e.g., white cells), various tissues or organs or parts thereof, or fine needle biopsy samples, surface swabs, cup scrubs, hair plucks, razor biopsies, urine, peritoneal fluid, and pleural fluid, or cells therefrom from humans.
  • Biological samples may also include sections of tissues, such as frozen sections or formaldehyde- fixed sections taken for histological purposes from humans.
  • the present invention provides a method of identifying a target protein that causes or is associated with an HPSC.
  • the method comprises: (a) isolating a protein from a biological sample, (b) generating a sequence of the isolated protein, and (c) comparing the sequence of the isolated protein to the protein sequence identified in Table 2 to identify and assign a function to the isolated protein.
  • the present invention provides a method of identifying a protein identified in Table 2 as a target protein.
  • the method comprises: identifying a protein as a target protein if the protein possesses one or more criteria selected from the group consisting of: relative abundance; shared function with other proteins having a role in other microbe-associated diseases; and proteins whose function is deleterious to skin.
  • relative abundance means that the protein is a member of the 50 most abundant proteins as measured by either silver or coomassie staining of a one or two dimensional gel; if the protein is a member of the 50 most abundant proteins as measured by at least one of the aforementioned methods, then the protein meets the relative abundance criterion.
  • a protein having a "shared function” with other proteins is defined as a protein which has homology to other proteins with identity greater than 30%, and/or a BLAST bit score greater than 100, and/or contains similar functional domains such as those defined in the PFAM database.
  • a "role in other microbe-associated diseases” is defined as known or postulated to have a role.
  • a "protein whose function is deleterious to skin” means those proteins known or postulated to be deleterious to skin.
  • the present invention provides a method of identifying a gene or protein identified in Table 2 as a target gene or protein that causes and/or is associated with an HPSC.
  • the method comprises: (a) assigning a function to a gene or protein identified in Table 1, (b) grouping genes or proteins of similar function into families, and (c) identifying a gene or protein that is a member of a family having 3 or more members as a target gene or protein. Isolation of Other Related Nucleic Acid Molecules
  • the present invention provides a method of quantifying Malassezia that comprises: (a) designing an oligonucleotide, wherein said oligonucleotide comprises a sequence identified in Table 3, and (b) probing a biological sample with said oligonucleotide.
  • a single oligonucleotide comprising a sequence identified in Table 3 is used to probe nucleic acid isolated from a biological sample.
  • a pair of oligonucleotides comprising sequences identified in Table 1 are used to probe nucleic acid isolated from a biological sample using quantitative polymerase chain reaction.
  • three or more oligonucleotides comprising sequences identified in Table 3 are used to probe nucleic acid isolated from a biological sample using quantitative technology such as, TaqManTM (TaqManTM available from Applied Biosystems, Division Headquarters, 850 Lincoln Centre Drive, Foster City, CA 94404).
  • the present invention provides a method of preparing a gene expression profile for one or more genes identified in Table 3 that comprises: (a) isolating RNA from a biological sample, (b) constructing a labeled nucleic acid probe from the isolated RNA, and (c) hybridizing the labeled nucleic acid probe to a target nucleic acid sequence identified in Table 3.
  • target oligonucleotides comprising sequences identified in Table 3 are attached to a microchip, such as AffymetrixTM (AffymetrixTM microchip is available from Affymetrix, 3420 Central Expressway, Santa Clara, CA 95051). The microchips are then probed with the labeled nucleic acid probes described herein.
  • target oligonucleotides comprising sequences identified in Table 3 are attached to a microarray that can include, but is not limited to, glass slides, nylon and/or nitrocellulose membranes.
  • the identification of the fungal nucleic acid molecules of Table 3 allows a skilled artisan to isolate nucleic acid molecules that encode other members of the gene family in addition to the sequences herein described. Further, the presently disclosed nucleic acid molecules allow a skilled artisan to isolate nucleic acid molecules that encode other members of the gene families.
  • a skilled artisan may use the proteins of Table 3 or fragments thereof to generate antibody probes to screen expression libraries prepared from appropriate cells.
  • the fragments may contain amino acid insertion and substitution.
  • Polyclonal or monoclonal antibodies generated against proteins, fragments of proteins, or modified proteins or fragments of proteins from Table 3 may be used to probe a cDNA or genomic expression library, such as lambda gtl 1 library, to obtain the appropriate coding sequence for other members of the protein family.
  • the cloned cDNA sequence may be expressed as a fusion protein, expressed using its own control sequences, or expressed by constructs using control sequences appropriate to the particular host used for expression of a protein.
  • coding sequences herein described may be synthesized and used as a probe to retrieve DNA encoding a member of the protein family from any organism.
  • Oligomers e.g., containing 18-20 nucleotides, may be prepared and used to screen genomic DNA or cDNA libraries to obtain hybridization under stringent conditions or conditions of sufficient stringency to eliminate an undue level of false positives.
  • pairs of oligonucleotide primers may be prepared for use in a polymerase chain reaction (PCR) to clone a nucleic acid molecule.
  • PCR polymerase chain reaction
  • Various PCR formats are known in the art and may be adapted for use in isolating other nucleic acid molecules.
  • Materials that may be screened in accordance with the assays of the invention include, but are not limited to, libraries of known materials, including natural products, such as plant extracts. Also included are synthetic chemicals, biologically active materials, e.g., proteins, nucleic acids, and peptides, including, but not limited to, members of random peptide libraries and combinatorial chemistry derived molecular libraries made of D- or L- configuration amino acids, and phosphopeptides, antibodies (including, but not limited to, polyclonal, monoclonal, chimeric, human, anti-idiotypic or single chain antibodies, and Fab, F(ab') 2 and Fab expression library fragments, and epitope-binding fragments thereof); and other organic and inorganic molecules.
  • synthetic chemicals e.g., proteins, nucleic acids, and peptides, including, but not limited to, members of random peptide libraries and combinatorial chemistry derived molecular libraries made of D- or L- configuration amino acids, and phosphopeptides
  • test materials In addition to the more traditional sources of test materials, computer modeling and searching technologies permit the rational selection of test materials by utilizing structural information from the ligand binding sites of proteins of the present invention. Such rational selection of test materials may decrease the number of test materials that must be screened in order to identify a therapeutic material.
  • Knowledge of the protein sequences of the present invention may allow for generation of models of their binding sites that may be used to screen for potential ligands. This process may be accomplished in manners known in the art.
  • a preferred approach involves generating a sequence alignment of the protein sequence to a template (derived from the crystal structures or NMR-based model of a similar protein(s)), conversion of the amino acid structures, and refining the model by molecular mechanics and visual examination.
  • a model may also be generated by building models of the hydrophobic helices. Mutational data that point towards contact residues may also be used to position the helices relative to each other so that these contacts are achieved. During this process, docking of the known ligands into the binding site cavity within the helices may also be used to help position the helices by developing interactions that would stabilize the binding of the ligand.
  • the model may be completed by refinement using molecular mechanics and loop building using standard homology modeling techniques. General information regarding modeling may be found in Schoneberg, T. et.
  • the model may be used in conjunction with one of several computer programs to narrow the number of materials to be screened, e.g., the DOCK program (UCSF Molecular Design Institute, 533 Parnassus Ave, U-64, Box 0446, San Francisco, California 94143-0446) or FLEXX (Tripos Inc., 1699 South Hanley Rd., St. Louis, Missouri).
  • DOCK program U-64, Box 0446, San Francisco, California 94143-0446
  • FLEXX Tripos Inc., 1699 South Hanley Rd., St. Louis, Missouri.
  • One may also screen databases of commercial and/or proprietary materials for steric fit and rough electrostatic complementarity to the binding site.
  • genes of the present invention may play a role in regulating HPSCs enables various methods of screening one or more materials to identify materials that may be used for prophylactic or therapeutic treatment of HPSCs, including D/SD.
  • the materials be selective for proteins or nucleic acids of the invention.
  • the in vitro screen be carried out using a protein or nucleic acid of the invention with an amino acid sequence that is, e.g., in one embodiment from 50% to 100% similar, in an alternate embodiment from 70% to 100% similar, in another embodiment from 75% to 100% similar, in yet another embodiment from 80% to 100% similar, in still another embodiment from 85% to 100% similarity, in yet another embodiment from 90% to 100% similar, in still another embodiment from 95%- 100% similar, and in an alternate embodiment from 98%- 100% similar to a protein or nucleotide sequence described in Table 3.
  • the methods of the present invention may be amenable to high throughput applications; however, use of as few as one material in the method is encompassed by the term "screening".
  • Screening This in vitro screen provides a means by which to select a range of materials, i.e., the materials that merit further investigation. For example, materials that activate a protein of the invention at concentrations of less than 200 ⁇ M might be further tested in a model, whereas those above that threshold may not be further tested.
  • kits comprising a protein of the invention or cells expressing a protein of the invention, which may be packaged in a variety of containers, e.g., vials, tubes microtitre plates, bottles and the like.
  • Other reagents may be included with the kit, e.g., positive and negative control samples, and buffers.
  • the invention provides a method to identify materials that bind to a protein or nucleic acid of the invention.
  • Methods to determine binding of a material to a protein or nucleic acid are known in the art.
  • the assays include incubating a protein or nucleic acid of the invention with a labeled material, known to bind to the protein or nucleic acid, in the presence or absence of a test material and determining the amount of bound labeled material.
  • the source of a protein or nucleic acid of the invention may either be cells expressing the protein or nucleic acid, cell lysates, or partially purified proteins, or protein extracts.
  • the labeled material may be a known ligand or a ligand analog labeled such that it may be measured, preferably quantitatively (e.g., labeled with 125 1, 35 S-methionine, or a fluorescent tag, or peptide or a fluorescent protein fusion). Such methods of labeling are known in the art. Test materials that bind to a protein of the invention may reduce ligand bound to the protein, thereby reducing the signal level compared to control samples. Variations of this technique have been described by Keen, M., Radioligand Binding Methods for Membrane Preparations and Intact cells in Receptor Signal Transduction Protocols, R.A.J. Challis, (Ed.), Humana Press Inc., Totoway NJ. (1997).
  • the invention provides methods for screening test materials to identify materials that modulate or regulate the activity of a protein or nucleic acid of the invention.
  • the assays may be cell-based, or may comprise proteins, isolated proteins, cell lysates, or extracts.
  • the cell population comprises a native Malassezia culture.
  • the cell population comprises a modified prokaryotic or eukaryotic cell that produces a protein identified in Table 3.
  • Assays can include contacting cells, proteins, isolated proteins, cell lysates, or extracts that contain a protein or nucleic acid of the invention with a test material or a control substance and measuring activation of the protein or nucleic acid by measuring the expression or activity of components of the intended activity.
  • lysates of the cells may be prepared and assayed for transcription, translation, or modification of a protein, e.g., phosphorylation, or glycosylation, or induction of second messengers like cAMP.
  • a protein e.g., phosphorylation, or glycosylation, or induction of second messengers like cAMP.
  • Many high- throughput assays are available that measure the response without the need of lysing the cells, e.g. calcium imaging.
  • cAMP induction may be measured with the use of recombinant constructs containing the cAMP responsive element linked to any of a variety of reporter genes.
  • reporter genes include, but are not limited to, chloramphenicol acetyltransferase (CAT), luciferase, glucuronide synthetase, growth hormone, fluorescent proteins, or alkaline phosphatase.
  • CAT chloramphenicol acetyltransferase
  • luciferase luciferase
  • glucuronide synthetase growth hormone
  • fluorescent proteins or alkaline phosphatase
  • specific phospho-tyrosine or phospho-serine antibodies may be utilized to measure the level of phosphorylation of a signaling protein after the exposure to a test material, whereby a significant deviation in phosphorylation levels compared to control samples would indicate inhibition or activation of a protein of the invention.
  • a protein's (e.g., receptor) responses subside, or become desensitized, after prolonged exposure to an agonist.
  • the protein of interest may be an enzyme and thus the effect of the binding of the test materials could be measured in terms of changes in the enzymatic activity.
  • changes in intracellular calcium concentration [Ca 2+ ] are generally indicative of activation of many signaling cascades.
  • a method for identifying a material for regulating an HPSC comprises: (a) contacting a material with a cell population that expresses a gene identified in Table 3; (b) determining and comparing the level of expression of the gene in the cell population that is contacted with the material to the level of expression of the gene in the cell population that is not contacted with the material; and (c) identifying those materials that modulate the expression of the gene in the cell population that is contacted with the material compared to the expression of the gene in the cell population that is not contacted with the material as a material for regulating HPSCs.
  • the cell population comprises a native Malassezia culture.
  • the cell population comprises a modified prokaryotic or eukaryotic cell that expresses a gene identified in Table 3.
  • the method for identifying a material for regulating an HPSC comprises; (a) contacting a material with a cell population expressing a protein identified in Table 3; (b) determining and comparing the level of expression of the protein in the cell population that is contacted with the material to the level of expression of the protein in the cell population that is not contacted with the material; and (c) identifying those materials that modulate the expression of the protein in the cell population that is contacted with the material compared to the expression of the protein in the cell population that is not contacted with the material as a material for regulating HPSCs.
  • the cell population comprises a native Malassezia culture.
  • the cell population comprises a modified prokaryotic or eukaryotic cell that produces a protein identified in Table 3.
  • a method for identifying a material for regulating an HPSC comprises: (a) contacting a material with a cell lysate or extract comprising a protein or partially purified protein identified in Table 3; (b) determining and comparing the level of activity of the protein or partially purified protein in the cell lysate or extract that is contacted with the material to the level of activity of the protein or partially purified protein in the cell lysate or extract that is not contacted with the material; and (c) identifying those materials that modulate the activity of the protein or partially purified protein in the cell lysate or extract that is contacted with the material compared to the activity in the cell lysate or extract comprising a protein or partially purified protein that is not contacted with the material as a material for regulating an HPSC, and (d) optionally administering any material identified as a material for regulating an HPSC to a human, and determining whether the material regulates an HPSCs in the human, wherein a material that regulates a Mal
  • the cell lysate or extract is derived from a native Malassezia culture.
  • the cell lysate or extract is derived from a modified prokaryotic or eukaryotic cell that expresses a protein identified in Table 3. Screening for Materials Using Cell Culture, Tissue, ex-vivo human skin, and human-based HPSC models
  • Materials selected from one or more test materials by an in vitro assay, as described above, may be further tested for their ability to regulate Malassezia associated conditions in various models.
  • models include in vitro cell culture models.
  • the model comprises a native Malassezia culture, or a lysate or extract of a native Malassezia culture.
  • the model comprises a modified prokaryotic or eukaryotic cell that expresses a protein identified in Table 3.
  • the model comprises a lysate or extract that is derived from a modified prokaryotic or eukaryotic cell that express a protein identified in Table 3.
  • Such additional levels of screening are useful to further narrow the range of candidate materials that merit additional investigation, e.g., clinical trials.
  • the overall activity of a protein of the invention may be increased by overexpressing the gene for that protein. Overexpression will increase the total cellular protein activity, and thereby the function.
  • the gene or genes of interest are inserted into a vector suitable for expression in the model cell. These vectors include, but are not limited to, bacteriophages, adenoviruses, adenovirus associated viruses, retroviruses, and herpes virus vectors. Other techniques may also be used that introduce DNA into cells (e.g., liposome, gold particles, or direct injection of the DNA expression vector (as a projectile) containing the gene of interest into the target cell).
  • the materials that modulate or regulate the genes and/or proteins identified in Table 3 may be used in a method for the treatment of a Malassezia associated condition.
  • the treatment can comprise contact of a human having a microbial cell population on the skin.
  • the cell population comprises native Malassezia.
  • the cell population comprises a modified prokaryotic or eukaryotic cell that produces a protein identified in Table 3.
  • the term "regulate” is used as would be by one skilled in the art.
  • the meaning of the term “regulate” includes, but is not limited to, up-regulate or down-regulate, to fix, to bring order or uniformity, to govern, or to direct by various means.
  • a material may be used in a method for the treatment of an HPSC.
  • treatment is used herein to mean that administration of a material of the present invention mitigates a condition in a host.
  • treatment includes, but is not limited to: preventing a condition from occurring in a host, particularly when the host is predisposed to acquiring the condition, but has not yet been diagnosed with the condition; inhibiting the condition; and/or alleviating or reversing the condition.
  • the term "prevent” does not require that the condition be completely thwarted.
  • prevent(ing) refers to the ability of the skilled artisan to identify a population that is susceptible to conditions, such that administration of the materials of the present invention may occur prior to onset of a condition. The term does not imply that the condition state be completely avoided.
  • the materials identified by the screening methods of the present invention may be administered in conjunction with other materials.
  • Safety and therapeutic efficacy of materials identified may be determined by standard procedures using in vitro or human-based in vivo technologies. Materials that exhibit large therapeutic indices may be preferred, although materials with lower therapeutic indices may be useful if the level of side effects is acceptable.
  • the data obtained from the in vitro and human- based in vivo toxicological and pharmacological techniques may be used to formulate the range of doses.
  • Effectiveness of a material may further be assessed in clinical trials of humans with HPSCs.
  • cosmetically acceptable carrier is intended to include all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with cosmetic administration.
  • the use of such media and agents for cosmetically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active material, such media may be used in the compositions of the invention. Supplementary active materials may also be incorporated into the compositions.
  • a personal care (e.g., cosmetic) composition of the present invention is formulated to be compatible with its intended route of administration.
  • Personal care compositions of the present invention can be in any suitable form. All forms of topical personal care compositions are contemplated and can include, for instance, creams, gels, lotions, emulsions, colloids, solutions, suspensions, ointments, milks, sprays, liquids, sticks, solids, powders, compacts, pencils, spray- on formulations, brush-on formulations, cloths, wipes, and the like.
  • Non-limiting examples of topical personal care compositions can include, without limitation, lipstick, mascara, rouge, foundation, blush, eyeliner, lipliner, lip gloss, facial or body powder, sunscreens and blocks, nail polish, mousse, sprays, styling gels, tonics, nail conditioner, bath and shower gels, shampoos, conditioners, cream rinses, hair dyes and coloring products, leave-on conditioners, sunscreens and sunblocks, lip balms, skin conditioners, cold creams, moisturizers, hair sprays, soaps, body scrubs, exfoliants, astringents, depilatories and permanent waving solutions, antidandruff formulations, antisweat and antiperspirant compositions, shaving, preshaving and after shaving products, moisturizers, deodorants, cold creams, cleansers, skin gels, and rinses.
  • the composition can be applied topically through the use of a patch or other delivery device. Delivery devices can include, but are not limited to, those that can be heated or cooled, as well as those that utilize
  • the genes and gene expression information provided in Table 3 may be used as diagnostic markers for the prediction or identification of the condition of a sample tissue.
  • a tissue sample may be assayed by any of the methods described above, and the expression levels for a gene or member of a gene family from Table 3 may be compared to the expression levels found in a control subject.
  • the expression level may also be compared to the expression levels observed in sample tissues exhibiting a similar condition, which may aid in its diagnosis.
  • the comparison of expression data, as well as available sequences or other information may be done by researcher or diagnostician or may be done with the aid of a computer and databases as described above. Such methods may be used to diagnose or identify conditions characterized by abnormal expression of the genes that are described in Table 3.
  • the methods of the present invention may be particularly useful in diagnosing or monitoring effectiveness of treatment regimen.
  • Materials that modulate the expression of one or more genes or gene families or proteins identified in Table 3 and/or modulate the activity of one or more of the proteins encoded by one or more of the genes or members of a gene family identified in Table 3 will be useful in diagnosis, monitoring, and evaluation of mammal responses to treatment regimen.
  • M. globosa, strain # 7966 is obtained from: The Centraalbureau voor Schimmelcultures (CBS), Uppsalalaan 8, 3584 CT Utrecht, The Netherlands. Obtaining the Raw Sequence Data
  • Genomic DNA is prepared from lab cultured M. globosa cells using standard methods that are known by individuals skilled in the art.
  • the genomic DNA is sheared and cloned into bacterial plasmid vectors.
  • the resulting plasmids are isolated and portion sequenced by standard methods that are known by individuals skilled in the art. Genome assembly
  • the genome is assembled using Phrap (Gordon et al., 1998).
  • the assembly is edited in Consed (Gordon et al., 1998).
  • the linkage information between the contigs, using the pair-reads information displayed in Consed, is recorded and is the basis upon which to generate super contigs.
  • Gene Finding A set of 2000 EST (Expression Sequence Tag) sequences is generated and assembled using Phrap.
  • the EST sequences are aligned back to the genome using EST_GENOME (Mott, 1997).
  • a set of training set genes is manually curated based on the alignments.
  • the training set genes provide information of start, stop, splicing sites and exon coding sequences.
  • NCBI nr database (ftp://ftp.ncbi.nih.gov/blast/db/FASTA/nr.gz) is searched against the assembled contigs using tblastn (Altschul et al., 1997). Proteins sequences with bit score (>200) and with protein identity > 60% are used to align to the genome using GeneWise (Birney et al., 2004). The alignment is then manually curated to create additional training sequences. Based on the curated training sequences, gene finding models is created for Geneld (Guigo, 1998) and TwinScan (Korf et al., 2001).
  • the identified genes are translated into protein sequences and searched against the nr database using blastp (Altschul et al., 1997), and Pfam (Bateman et al., 2002) and TigrFam databases (http://www.tigr.org/TIGRFAMs/) using HMMER(Eddy, 1998).
  • the function assignment is based on the following successive steps. Once the function of the gene has been assigned, the gene is removed from the query gene set and later steps will not be applied to it.
  • genes match equivalogs (explained at http://www.tigr.org/TIGRFAMs/Explanations.shtml) from TigrFam with a score above the trusted cut-off, the function annotation of the equivalog is transferred to the matching gene. If a gene matches proteins in the nr database, with a bit score > 200 and an identity greater than 30%, then the function of the matching gene is transferred to the gene (when multiple genes satisfy the condition, genes that are annotated by Swiss-Prot (Bairoch and Apweiler, 1997) are given higher priority, otherwise the function of the best match protein that is not annotated with hypothetical or unknown are assigned to the gene ).
  • ESTs The locations of EST to the genome are determined by using the program EST GENOME. Mapping of ESTs to the called genes is determined by blastn (Altschul et al., 1997). Comparative Genomics
  • Genome comparison are done using blastn, blastp and MUMMER (Kurtz et al., 2004).
  • Blastn is used to compare similarity at nucleotide level between the two genomes of interest.
  • Blastp is used to compare the proteome similarity.
  • MUMMER is used to align the two genomes.
  • PCR primer design is performed using Primer3 (Rozen and Skaletsky, 2000).
  • Affymetrix microarray gene sequence preparation is based on the instructions from Affymetrix customer array expression program. Identification of Gene Clusters in the Same Functional Group
  • Another aspect of the present invention relates to an expression system comprising the lipase polynucleotide.
  • Such expression systems include recombinant expression vectors comprising the lipase polynucleotide, as well as hosts which have been genetically engineered with such recombinant expression vectors ("recombinant host").
  • recombinant expression vector refers to a DNA construct used to express a polynucleotide which encodes a desired polypeptide (for example, the lipase polypeptide) and which includes a transcriptional subunit comprising an assembly of 1) genetic elements having a regulatory role in gene expression, for example, promoters and enhancers, 2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and 3) appropriate transcription and translation initiation and termination sequences.
  • recombinant expression vectors of the present invention can be constructed.
  • the nature of the vector is not critical to the invention, and any vector may be used, including plasmid, virus, bacteriophage, and transposon.
  • Possible vectors for use in the present invention include, but are not limited to, chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
  • Additional useful vectors include, but are not limited to: for mammalian cells, pcDNA-1 (Invitrogen, San Diego, Calif.) and pSV-SPORT 1 (Gibco-BRL, Gaithersburg, Md.); for insect cells, pBlueBac 111 or pBlueBacHis baculovirus vectors (Invitrogen, San Diego, Calif.); and for bacterial cells, pET-3 (Novagen, Madison, Wis.). Any other vector may be used as well, as long as it is replicable and viable in the host.
  • the lipase polynucleotide can be present in the vector operably linked to regulatory elements.
  • the lipase polynucleotide may be inserted into the vector by a variety of procedures.
  • the polynucleotide is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed within the scope of those skilled in the art.
  • the vector may preferably comprise an expression element or elements operably linked to the lipase polynucleotide to provide for expression thereof at suitable levels.
  • Any of a wide variety of expression elements may be used.
  • the expression element or elements may, for example, be selected from promoters, enhancers, ribosome binding sites, operators and activating sequences.
  • Such expression elements may be regulatable, for example, inducible (via the addition of an inducer).
  • Representative examples of useful promoters include, but are not limited to: LTR (long terminal repeat from a retrovirus) or SV40 promoter, the E.
  • the expression vector preferably also contains a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector may also include appropriate sequences for amplifying expression.
  • the expression vector further contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell cultures, or such as tetracycline or ampicillin resistance for prokaryotic cell cultures.
  • selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell cultures, or such as tetracycline or ampicillin resistance for prokaryotic cell cultures.
  • Useful expression vectors for bacterial use are constructed by inserting the lipase polynucleotide with suitable translation initiation and termination signals in operable reading frame with a functional promoter.
  • the vector will preferably contain one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host.
  • useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017).
  • cloning vector pBR322 ATCC 37017
  • Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEMl (Promega Biotec, Madison, Wis., U.S.A.). These pBR322 "backbone" sections are combined with an appropriate promoter and the LIPASE polypeptide to be expressed.
  • Suitable bacterial vectors include: pQE70, pQE60, and pQE-9 (Qiagen); pbs, pDIO, phagescript, psiX174, pBluescript SK, pbsks, pNH8A, pNHl ⁇ a, pNH18A, and pNH47A (Stratagene); and ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia).
  • Useful expression vectors for use with yeast can comprise a yeast replication origin or fragments of DNA which are required for integration into the host's chromosomal DNA, a selectable marker, a suitable promoter and enhancer, and also any necessary ribosome binding sites, a polyadenilation site, transcriptional termination sequences and 5' flanking nontranscribed sequences.
  • Suitable yeast expression vectors include, but are not limited to, pPIC3, pPIC3K, pPIC3.5K, pPIC9, pPIC9K, pAO815, pHIL-D2, pHIL-Sl, pPICZaA, pPICZaB, and pPICZaC (Invitrogen) preferably for Pichia pastoris; pYES2 (Invitrogen), and the pRS series vectors (STRATAGENE) preferably for Saccharomyces cerevisiae.
  • Mammalian expression vectors will preferably comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences.
  • DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the necessary nontranscribed genetic elements.
  • Suitable mammalian vectors include: pWLNEO, pOG44, pXTl, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia).
  • the recombinant expression vector containing the lipase polynucleotide as well as an appropriate promoter or control sequence may be employed to transform an appropriate host to permit the recombinant host to express the lipase polypeptide.
  • Recombinant hosts may include bacterial, fungal, insect, plant or mammalian cells which have been transformed with a recombinant expression vector of the present invention. Recombinant hosts may also include entire plants, or insects which have been transformed with the recombinant expression vector. Representative examples of appropriate hosts for in vitro production include: bacterial cells such as E.
  • yeast or fungal cells such as Pichia pastoris, Candida boidinii, and Saccharamyces cervisiae
  • insect cells such as Drosophila and Sf9
  • animal cells such as the COS-7 lines (described by Gluzman, Cell, 23:175 (1981)), C127, 3T3, CHO and BHK
  • human cells such as HeLa.
  • recombinant hosts for in vivo production include, but are not limited to, insects including silk worm larvae; and plants. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
  • transformation means introducing DNA into a cell or an organism so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integrant.
  • transformation is done using standard techniques appropriate to such cells.
  • the calcium treatment employing calcium chloride, as described by Cohen, S. N., Proc. Natl. Acad. Sci. (USA), 69:2110 (1972); Mandel et al., J. MoI. Biol., 53:154 (1970); and Lilgestrom et al., Gene, 40:241-246 (1985), is generally used for prokaryotes or other cells that contain substantial cell-wall barriers.
  • introduction of the expression vector into the host can be effected by calcium phosphate transfection, DEAE- Dextran mediated transfection, or electroporation as set forth in "Basic Methods in Molecular Biology,” (D. L. Davis and I. M. Battey, (1986)).
  • other methods for introducing DNA into cells such as by nuclear injection or by protoplast fusion may also be used.
  • One aspect of the invention relates to a process for producing an isolated lipase polypeptide via recombinant technology.
  • the process comprises inserting the lipase polynucleotide into a suitable recombinant expression vector (as described above), and then transforming a suitable host with this recombinant expression vector (as described above).
  • the transformed host is subsequently used to express the lipase polypeptide, followed by purification of the resulting lipase polypeptide from the host.
  • the recombinant lipase polypeptide is produced so that the lipase polypeptide is secreted from the host cell (Mileto, D., S. Brocaa, M. Lotti, M. Takagi, C. Alquati, and L. Alberghina, Characterization of the Candida rugosa Lipase System and Overexpression of the Lipl Isoenzyme in a Non-conventional Yeast, Chemistry of Physics of Lipids (1998) 93: 47- 55).
  • This may be done by fusing a signal sequence from a different secreted protein with the lipase polypeptide (Qasim, M. A., P. J Ganz, C. W. Saunders, K. S. Bateman, M. N. G. James, and M. Laskowski, Jr., Interscaffolding Additivity. Association of P ⁇ Variants of Eglin c and of
  • the lipase polypeptide may be produced within the host cell.
  • the lipase polypeptide is produced as part of a fusion protein (Tang, S., K. Sun, G. Sun, T. Chang, and G. Lee, Recombinant Expression of the Candida rugosa Iip4 Lipase in Escherichia coli, Protein Expression and Purification (2000) 20: 308-313).
  • the method comprises culturing a yeast host cell which has been transformed with a recombinant expression vector comprising the lipase polynucleotide.
  • the cultured yeast host cell is subsequently used to produce the lipase polypeptide, followed by purification of the resulting lipase polypeptide from the cultured medium.
  • An embodiment of this procedure includes fusing the lipase polynucleotide with the gene for the Saccharomyces cerevisae ⁇ -factor such that the ⁇ -factor signal sequence (Kurjan, J., and 1.
  • the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
  • the hosts could be modified to produce lipase polypeptide which accumulates within the cells.
  • Such cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • Microbial cells employed in expression of the lipase polypeptide can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents; such methods are well known to those skilled in the art.
  • the lipase polypeptide can be recovered and purified from the recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
  • HPLC high performance liquid chromatography
  • the lipase polypeptide may be generated through chemical synthesis, using methods well known to those skilled in the art for chemically synthesizing proteins (see, e.g., Wilce, J. A., S. G. Love, S. J. Richardson, P. F. Alewood, and D. J. Craik, Synthesis of an analog of the thyroid hormone-binding protein transthyretin via regioselective chemical ligation).
  • the lipase polynucleotide may be generated through chemical synthesis using methods well known to those skilled in the art (see, e.g., Traub, P. C, C. Schmidt-Dannert, J. Schmitt, and R. D. Schmid, Gene Synthesis, Expression in E. coli and in vitro refolding of Pseudomonas sp. KWI 56 and Chromobacterium viscosum lipases and their chaperones, Applied Microbiology and Biotechnology (2001) 55: 198-204.) Such an approach allows the optimization of the synthetic gene for host-specific conditions, such as codon usage.
  • RNA-STAT60 Tel-Test, Friendswood, TX
  • Retsch Wigger, Bavaria
  • MM300 Bead-Beater Mill using 5mm stainless steel beads. Chloroform is added to the lysate and the mixture is shaken for 1-2 minutes. The aqueous phase, containing crude nucleic acids, is removed and precipitated in 100% isopropanol. Nucleic acids are pelleted by centrifugation and the pellets are washed with 70% ethanol and then resuspended in 200 ul of DEPC-water (diethyl pyrocarbonate treated water).
  • DEPC-water diethyl pyrocarbonate treated water
  • RNA is then purified using Qiagen (Hilden, Germany), RNEasy Cleanup minicolumns and the manufacturer's recommended protocol. Quantity of RNA is determined by UV spectroscopy and quality is determined using an Agilent (Palo Alto, CA) Bioanalyzer 2100.
  • RNA GeneChip target 5-10 ug of purified total RNA is converted to cRNA GeneChip target using the protocol provided by Affymetrix. 20 ug of cRNA target are fragmented and hybridized, washed, and scanned according to the Affymetrix Expression Analysis protocol. Complete protocols for target synthesis and GeneChip processing can be found at: www.affymetrix.com/support/download/manuals/expression s2 manual.pdf GeneChip Analysis:
  • GeneChip scans are converted to tabular data using the Affymetrix MAS5.0 algorithm, which is described in: www.afrymetrix.corn/Auth/support/downloads/manuals/mas manual.zip.
  • Data quality is determined using a variety of statistical measures, including t-tests, scatter biplots, and principal components analysis, depending upon the source and character of the data. Once the data quality are confirmed, the data are analyzed and visualized using any suitable commercially-available tools, such as Affymetrix Data Mining ToolTM (DMT)(Affymetrix, Santa Clara, California), Spotf ⁇ reTM (Sommerville, MA), and OmnivizTM (Maynard, MA).
  • DMT Affymetrix Data Mining ToolTM
  • Spotf ⁇ reTM Spotf ⁇ reTM
  • OmnivizTM Maynard, MA
  • Cell wall-associated proteins Cells grown in either rich or minimal media are pelleted by centrifugation and washed once with 50 mM Tris-HCl (pH 8.0). The cell pellet is resuspended in Tris buffer, and for most experiments, cells are washed further by pelleting through a cushion of 20% sucrose. The washed pellets are resuspended in 2 volumes of 2X SDS-containing Laemmli sample buffer or a buffer containing high amounts of EDTA and beta-mercaptoethanol. Cell suspensions are vortexed for 1 hr at room temperature. SDS-extracted samples are run directly on ID gels.
  • EDTA-extracted samples are either (1) TCA-precipitated, solubilized in SDS sample buffer (ID gels) or IEF sample buffer (2D gels), or (2) dialyzed, lyophilized, and solubilized in digestion buffer in preparation for multidimensional liquid chromatography (MDLC).
  • ID gels SDS sample buffer
  • IEF sample buffer 2D gels
  • MDLC multidimensional liquid chromatography
  • Proteins are identified by one of several techniques: (a) Edman protein sequencing of intact, blotted proteins, (b) ES-MS or MALDI-MS of tryptic peptides derived from the gel bands or spots, or (c) MDLC of tryptic peptides followed by ES-MS. Edman sequencing. Proteins are electrophoresed under denaturing conditions on 10- 20% mini-gels (Invitrogen, Carlsbad, CA). Gels are electroblotted to PVDF membranes (Fluorotrans, Pall Life Sciences, East Hills, NY) and stained with Coomassie blue. Bands of interest are excised from the blot and subjected to NH2-terminal sequencing on a Procise 494 HT protein sequencer (Applied Biosystems, Foster City, CA) according to standard procedures.
  • Mass spectrometry Secreted or cell wall-associated proteins from M. globosa are identified using CapLC-MS/MS on an electrospray hybrid quadruple time-of-flight mass spectrometer (Q-TOF, Waters, Milford, MA). Tryptic digests of ID- or 2D-gel isolated proteins are first loaded onto the CapLC system equipped with a reversed-phase capillary column (75 urn x 10 cm, C 18, New Objective, Wobura, MA); peptides are separated with a gradient of 5-80% acetonitrile containing 0.02% trifluoroacetic acid at 0.5 ul/min over 60 minutes. Peptides eluted from the column are detected automatically in data-dependent MS/MS scan mode.
  • Q-TOF electrospray hybrid quadruple time-of-flight mass spectrometer
  • MS data is then used to search an in-house M. globosa protein database using MascotTM software (Matrix Science, London, UK).
  • the protein digests are also analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF, Voyager-DE STR, Applied Biosystems, Foster City, CA). Proteins are identified by peptide mass fingerprinting using the MascotTM software and the in-house M. globosa protein database.
  • This example shows how to screen for inhibitors of the lipase polypeptide.
  • the assay is performed in a deep well 96-well plate (costar® 4412). To each well, 270 ⁇ l of 25 mM MES, pH 5.5, 90 mM NaCI, 8 mM KCI, 1 mM CaC12 is added. 10 ⁇ l of olive oil (lipid source), diluted three-fold into dimethyl sulfoxide (DMSO), is then added. A candidate inhibitor (15 ⁇ l) is then added. Then 5 ⁇ l of lipase polypeptide is added. Caps are then used to cover the microtiter plate.
  • DMSO dimethyl sulfoxide
  • the solution is left for one hour at room temperature, with vigorous shaking on a VX-2500 Multi-tube Vortexer (Scientific Products). Following the one-hour period, the microtiter plate is spun briefly in a centrifuge to force the liquid into the bottom of the well.
  • Tris Tris (hydroxymethyl) amino methane
  • a portion (90 ⁇ l) is transferred to a standard microtiter plate (costar® 3596), and 90 ⁇ l of a glycerol detection solution is added.
  • the glycerol detection solution contains: 100 mM Tris HCl pH 7.6, 10 mM MgC12, 2 mM 4-aminoantipyrine, 3 mM N- ethyl-N-(3-sulfopropyl)-m anisidine, 1 mM adenosine 5'-triphosphate, 20 units/ml peroxidase, 8 units/ml glycerol-3-phosphate oxidase, 0.5 units/ml glycerol kinase.
  • the mixture is rotated on a Lab-Line Lab Rotator for 15 minutes at room temperature.
  • the optical density at 540 nm is measured as an indication of the amount of glycerol produced in the assay.
  • glycerol generated in the assay To know the absolute amount of glycerol generated in the assay, one can generate a standard curve with known glycerol concentrations. A comparison is made between the amount of glycerol generated by the lipase polypeptide in the absence of the candidate inhibitor and the amount of glycerol generated by the lipase polypeptide in the presence of the candidate inhibitor. An inhibitor-dependent reduction in glycerol levels of at least two-fold indicates inhibition of the ability of the lipase polypeptide to break down lipids.

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Abstract

La présente invention propose des procédés pour identifier des gènes et des protéines qui peuvent aider à réguler des troubles de la peau à caractère prolifératif provoqués, au moins en partie, par la Malassezia globosa, y compris des troubles de la peau à caractère hyper prolifératif. Les gènes identifiés peuvent être utilisés en tant que marqueurs pour le début et la progression des troubles, et pour mesurer l'efficacité d'un traitement. La présente invention propose également des procédés pour cribler des agents qui sont capables de réguler des troubles de la peau à caractère prolifératif, y compris des troubles de la peau à caractère hyper prolifératif. La présente invention propose également des procédés pour identifier des matières cosmétiques et/ou thérapeutiques qui peuvent avoir une incidence ou traiter de manière cosmétique divers troubles en régulant l'expression et l'activité de gènes et de protéines identifiés.
PCT/US2007/022097 2006-10-16 2007-10-16 Procédés et cibles pour identifier des matières pour réguler des troubles de la peau à caractère hyper prolifératif Ceased WO2008066612A2 (fr)

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Cited By (2)

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US9671410B2 (en) 2011-01-16 2017-06-06 The Procter & Gamble Company Biomarker-based methods for identifying and formulating compositions that improve skin quality and reduce the visible signs of aging in skin
US9808408B2 (en) 2010-01-17 2017-11-07 The Procter & Gamble Company Biomarker-based methods for formulating compositions that improve skin quality and reduce the visible signs of aging in skin for individuals in a selected population

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CN111812265B (zh) * 2020-07-10 2021-05-18 甘肃省药品检验研究院 一种同时测定染发剂中32种染料的检测方法

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9808408B2 (en) 2010-01-17 2017-11-07 The Procter & Gamble Company Biomarker-based methods for formulating compositions that improve skin quality and reduce the visible signs of aging in skin for individuals in a selected population
US10172771B2 (en) 2010-01-17 2019-01-08 The Procter & Gamble Company Biomarker-based methods for formulating compositions that improve skin quality and reduce the visible signs of aging in skin for individuals in a selected population
US9671410B2 (en) 2011-01-16 2017-06-06 The Procter & Gamble Company Biomarker-based methods for identifying and formulating compositions that improve skin quality and reduce the visible signs of aging in skin

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