Target+Sp17+-+Protein+serine-threonine+phosphatase+(Trypanosoma+brucei)

Human African trypanosomiasis, also known as sleeping sickness, is a vector-borne parasitic disease. It is caused by infection with protozoan parasites belonging to the genus //Trypanosoma //. They are transmitted to humans by tsetse fly (Glossina genus) bites which have acquired their infection from human beings or from animals harbouring human pathogenic parasites. The clinical course of human African trypanosomiasis has two stages. In the first stage, the parasite is found in the peripheral circulation, but it has not yet invaded the central nervous system. Once the parasite crosses the blood-brain barrier and infects the central nervous system, the disease enters the second stage. The subspecies that cause African trypanosomiasis have different rates of disease progression, and the clinical features depend on which form of the parasite (T. b. rhodesiense or T. b. gambiense) is causing the infection. However, infection with either form will eventually lead to coma and death if not treated. https://www.cdc.gov/parasites/sleepingsickness/disease.html
 * *Target (protein/gene name): **Protein serine/threonine phosphatase 1
 * *NCBI Gene # or RefSeq#: **3656900
 * *Protein ID (NP or XP #) or Wolbachia#: **XP_844513.1
 * *Organism (including strain): **Trypanosoma brucei (TREU927)
 * Etiologic Risk Group (see link below): **RG2
 * */ Disease Information (sort of like the Intro to your Mini Research Write up): **


 * Link to TDR Targets page (if present): **http://www.tdrtargets.org/targets/view?gene_id=19262
 * Link to Gene Database page: **http://tritrypdb.org/tritrypdb/app/record/gene/Tb05.5K5.30

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4083114/ https://www.ncbi.nlm.nih.gov/pubmed/21363968
 * Essentiality of this protein: **The Trypanosoma brucei protein serine/threonine phosphatase PP1 is essential in conserving the intracellular organisation of the procyclic tryanosome cell. Knockdown of this protein leads to a coordinated rearrangement of cellular organelles and compartments. The inhibition of this protein leads to a significant loss of fitness in both bloodstream and procyclic forms in 3-6 days.
 * Is it a monomer or multimer as biological unit ** **:** dimer

Each PP1 enzyme contains both a catalytic subunit and a regulatory subunit. The catalytic subunit consists of a 30-kD single-domain protein that can form complexes with other regulatory subunits. The catalytic subunit is highly conserved among all eukaryotes, thus suggesting a common catalytic mechanism. The catalytic subunit can from complexes with various regulatory subunits. These regulatory subunits play an important role in substrate specificity as well as compartmentalization. The catalytic subunit of PP1 forms an α/β fold with a central β-sandwich arranged between two α-helical domains. The interaction of the three β-sheets of the β-sandwich creates a channel for catalytic activity, as it is the site of coordination of metal ions.
 * Complex of proteins: **

TDR listed this target as having a druggability index of 0.3. Rather than target active sites, these studies have demonstrated that targeting PSP/PTP protein (substrate/regulatory) interaction sites, which are distal from the active sites, are highly viable and suitable drugs targets. Additional studies show that this approach-targeting substrate and/or regulatory protein interaction sites-also holds incredible promise for protein phosphatase 1 (PP1)-related diseases. Finally, domains outside PTP catalytic domains have also recently been demonstrated to directly alter PTP activity. Collectively, these novel insights offer new, transformative perspectives for the therapeutic targeting of PSPs by interfering with the binding of PIPs or substrates and PTPs by targeting allosteric sites outside their catalytic domains.
 * Druggable Target: **


 * [] **


 * *EC#: ** 3.1.3.16
 * Link to BRENDA EC# page: **http://www.brenda-enzymes.org/enzyme.php?ecno=3.1.3.16


 * Fig 1.** BRENDA enzyme mechanism schematic for protein serine/threonine phosphatase. Enzyme reacts with water.

Colorimetric assay of protein phosphatases using p-Nitrophenylphosphate
 * Enzyme Assay information: **

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2965627/ p-nitrophenyl phosphate tablets, Thermo Fisher Scientific, #34047, $145 1x colorimetric assay buffer (20 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 0.02% β-mercaptoethanol, 0.1 mg/ml bovine serum albumin (BSA)), Thermo Fisher Scientific, #AM9850G, #R0971, #E1219, #31350010, #AM2618, $204 5N NaOH, Sigma-Aldrich, #1310-73-2, $108.20 Standard flat-bottomed 96-well microtiter plate, Thermo Fisher Scientific, #2101, $90 microplate reader capable of reading absorbance at 405 nm, Thermo Fisher Scientific, #VL0000D0
 * Reagents and Materials:**

**-- PDB # or closest PDB entry if using homology model:** 1JK7 **Query Coverage:** 53% **Max % Identities:** 32% **% Positives:** 47% **Chain used for homology:** A
 * Structure (PDB or Homology model) **
 * -- For Homology Model option: **
 * Show pairwise alignment of your BLASTP search in NCBI against the PDB **


 * Current Inhibitors: ** Microcystin

This protein has been expressed in E. coli. www.pnas.org/content/90/22/10633.full.pdf In T. brucei, this protein is expressed primarily in the cytoplasm. It is expressed most in the the procyclic form.
 * Expression Information (has it been expressed in bacterial cells): **


 * Purification Method : ** Microcystin-Sepharose bead chromatography
 * Image of protein: **
 * Fig 2.** PyMOL representation of chain A of tumor-promoter Okadaic acid bound to protein phosphatase-1 in Homo sapiens, expressed in E. coli. A sulfate ion, manganese ion, and beta-mercaptoethanol are also shown. The secondary structures are colored differently. The protein is seen in its cartoon form. From the 1JK7 PDB file.

MDPPNVPPSP QAPPDLSPPS GSLSFRADNL VTLEMVQAVT INREKLSEWH HKIRSRLFPL VTGFFVRCRV EDSTDGRALY KVGRIKTLKT DWSVELDLLT TDEILSGRSN TNYDCISNAK LTAGEFSAFI SKVPPELLSN ISNTVHKMEE RLRMMETIIL TEPAFHGRRR DDRRQQEAQP AQAVTGLPDK YVFSQNVLLC DDEFSTFPMT VTIVDLLQNA VGQKGMTEPD TPRAGISAIT GPKSRAFDEA VASHSTMLQL QDVRNYINSP TQPPVDAARL IEMIRRSSNA QNTSFSYDNL TEFCKLVISV CSQCREVVAK EPLFIRLKSP VTVFGDIHGN FADMAYFLDK VVAFDDIMLK YTTSHLLFLG DYVDRGAFSV ECVMYLFALK VISPSKVTLL RGNHESPEVN GDMDVYGYSS FKYQCLDKFG RDRGIDVWVA VNEVFQFLPV IADIDKKIFC VHGGLPQYSG GEDKRLEILS DPSFPRIPVV QCTDPTNVAQ RMMVNDLLWS DPAPPNHQLD KYGFGPNPRG PDIKTFGSRA VDMFCERYNY QYIFRAHQEK ADGVRLSDNA RVVTIFSTSD YAGHQNGAGC ILVANGKMRM AIKKPHRHEM KETKGPVSPR TLSKSVPGMI PRLKRI http://www.genedb.org/featureSeq/Tb05.5K5.30 Fig 3. TMPred output graph for protein serine/threonine phosphatase 1 found in Trypanosoma brucei.
 * *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): **
 * *length of your protein in Amino Acids: ** 636
 * Molecular Weight of your protein in kiloDaltons: ** 71.37 kDa
 * Molar Extinction coefficient of your protein at 280 nm wavelength: **49570 L/mol*cm
 * TMpred graph Image ** (@http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.

ATGGATCCAC CTAATGTACC TCCCTCTCCT CAGGCCCCGC CGGACCTTTC GCCGCCCTCC GGAAGCCTTT CGTTCCGCGC TGACAACCTC GTCACGTTAG AAATGGTTCA GGCTGTAACC ATAAACCGTG AAAAGTTGTC TGAATGGCAT CACAAAATCC GCTCGAGGTT GTTTCCACTT GTCACTGGTT TCTTTGTGCG GTGCCGCGTG GAGGATTCAA CGGATGGCAG AGCTTTATAT AAAGTGGGAC GCATTAAAAC TCTTAAAACA GATTGGTCTG TAGAGCTTGA TTTGCTAACA ACAGATGAGA TTCTTTCGGG ACGTAGCAAT ACAAACTACG ACTGCATCAG CAACGCAAAA CTGACTGCAG GAGAGTTTAG CGCTTTTATA TCGAAGGTAC CACCAGAGTT GCTGTCTAAT ATTTCTAACA CAGTGCATAA AATGGAGGAG CGGCTGCGCA TGATGGAGAC CATCATCTTA ACGGAGCCGG CTTTCCACGG CAGACGCCGT GACGACCGGA GGCAACAGGA AGCACAACCC GCTCAGGCTG TCACGGGCCT ACCTGATAAA TATGTCTTCT CACAAAATGT GCTTTTGTGT GATGATGAGT TTTCAACCTT TCCAATGACT GTTACAATTG TTGATTTGCT ACAGAATGCG GTCGGTCAAA AGGGAATGAC GGAACCTGAC ACCCCGCGGG CAGGGATTTC CGCGATAACC GGACCGAAGT CTCGCGCGTT TGATGAGGCG GTAGCGAGTC ATAGCACGAT GCTGCAGCTT CAGGATGTAC GGAATTACAT AAATTCGCCA ACACAACCAC CAGTGGATGC GGCCCGACTT ATCGAGATGA TACGGCGCTC ATCCAATGCG CAAAACACTT CATTTTCCTA CGATAATCTC ACGGAGTTCT GCAAACTTGT TATTTCCGTT TGTAGCCAAT GTCGCGAGGT GGTGGCGAAG GAACCTTTAT TCATCCGTCT CAAGTCGCCC GTAACAGTGT TCGGTGACAT CCATGGCAAC TTCGCTGATA TGGCCTATTT CTTGGATAAA GTGGTGGCTT TTGATGATAT TATGCTCAAG TATACCACTT CGCATCTTTT GTTCCTTGGC GACTATGTAG ATCGTGGTGC CTTCTCGGTT GAGTGTGTCA TGTACCTCTT TGCACTCAAA GTTATTAGCC CATCTAAGGT TACTCTACTG CGTGGTAACC ATGAATCCCC TGAGGTAAAT GGTGATATGG ATGTGTATGG TTACAGCTCA TTCAAATATC AATGCCTCGA CAAGTTTGGA AGGGATCGTG GTATTGATGT GTGGGTTGCA GTGAATGAGG TATTTCAGTT CCTCCCCGTT ATAGCTGATA TTGATAAGAA GATATTCTGC GTTCACGGCG GCTTGCCGCA GTATTCCGGG GGTGAGGACA AACGACTTGA GATTCTTTCT GATCCTTCTT TCCCTCGTAT TCCGGTGGTT CAATGTACTG ATCCCACAAA TGTGGCACAG CGTATGATGG TGAATGACTT GTTGTGGTCC GATCCCGCAC CGCCAAACCA TCAACTTGAT AAATATGGCT TCGGGCCAAA CCCACGCGGA CCTGATATCA AAACCTTTGG TTCTCGTGCA GTTGATATGT TTTGTGAGCG CTACAACTAC CAATACATAT TTCGTGCGCA TCAAGAAAAG GCGGATGGTG TTCGGCTGTC AGATAATGCT CGCGTTGTGA CGATATTTTC CACGTCTGAC TATGCCGGCC ACCAGAATGG TGCAGGTTGT ATATTAGTGG CCAATGGTAA AATGCGCATG GCGATTAAAA AGCCCCACCG GCACGAGATG AAGGAAACGA AAGGACCGGT ATCACCTCGC ACGCTGTCGA AAAGCGTTCC AGGTATGATA CCGCGTCTTA AGCGCATTTA A ATG GAC CCT CCT AAT GTA CCA CCT TCC CCG CAA GCC CCA CCC GAC CTC AGC CCT CCG TCG GGT AGC TTG TCC TTT AGG GCT GAT AAC TTG GTC ACT CTCGAG ATG GTCCAG GCT GTG ACG ATA AAC CGT GAA AAG TTG TCT GAG TGG CAT CAC AAG ATT CGA TCC CGT TTG TTC CCT CTT GTT ACT GGT TTT TTT GTCCGA TGT CGT GTC GAG GACTCT ACC GAT GGC CGC GCC CTG TAT AAG GTG GGA CGA ATT AAA ACG CTT AAA ACT GAC TGG TCC GTC GAA CTC GAT CTC CTTACG ACT GAC GAG ATT CTT AGC GGC CGTTCC AAT ACC AAT TAC GAT TGT ATT TCA AAC GCA AAA TTG ACT GCT GGC GAG TTC AGT GCG TTT ATT TCT AAGGTC CCA CCC GAG CTC CTT TCT AAT ATT TCA AAC ACTGTC CAC AAG ATG GAA GAA AGG CTC AGG ATG ATG GAA ACG ATA ATA TTG ACA GAA CCA GCG TTTCAC GGC CGC CGT CGG GAT GAT AGG CGC CAA CAG GAG GCG CAA CCAGCG CAG GCC GTT ACG GGG CTC CCT GAT AAA TAC GTG TTT TCA CAA AAC GTG CTCCTT TGT GAT GAC GAA TTT TCA ACT TTT CCG ATG ACC GTC ACT ATT GTC GAC CTC CTT CAA AAC GCG GTA GGG CAG AAG GGA ATG ACC GAG CCC GAT ACCCCA CGG GCC GGG ATA TCC GCT ATT ACC GGC CCG AAG TCA CGC GCT TTT GAT GAA GCC GTT GCTTCG CAT AGT ACG ATG CTG CAA CTC CAA GAC GTA CGCAAT TAT ATT AAC AGT CCC ACA CAA CCT CCA GTC GAT GCC GCC CGA CTT ATT GAA ATG ATA CGT AGG AGC AGTAAT GCT CAG AAC ACG AGT TTT AGT TATGAC AAC CTG ACA GAA TTT TGT AAA CTG GTA ATA TCT GTG TGC TCG CAA TGT AGG GAA GTA GTA GCA AAG GAG CCA CTT TTCATC CGA TTG AAA TCC CCCGTG ACA GTT TTT GGG GAT ATT CAC GGT AAT TTT GCG GAT ATG GCC TAC TTT CTG GAT AAG GTT GTT GCG TTT GAC GAT ATA ATG CTT AAATAC ACA ACATCC CAT TTG CTG TTC CTC GGC GAC TAC GTT GAC CGT GGC GCG TTT TCG GTT GAG TGT GTG ATG TAT CTG TTC GCA CTG AAA GTT ATC TCA CCA AGC AAAGTA ACT CTC CTG CGA GGG AAT CAT GAA TCC CCC GAA GTG AAT GGA GAT ATG GAC GTT TAC GGG TAT AGT TCA TTC AAG TAT CAA TGC CTT GAT AAG TTTGGT CGA GACCGG GGA ATC GAT GTG TGG GTA GCC GTC AAT GAA GTA TTT CAG TTC TTG CCT GTC ATT GCC GAC ATC GAC AAG AAG ATA TTC TGT GTA CATGGC GGT CTT CCT CAA TATTCA GGT GGA GAA GAT AAG CGC CTT GAA ATT CTG TCG GAT CCC TCT TTT CCA CGA ATT CCC GTT GTG CAG TGC ACT GAC CCAACG AAT GTC GCG CAA CGC ATG ATG GTAAAT GAT CTC TTG TGG TCG GAC CCA GCA CCT CCG AAT CAC CAG CTC GAT AAG TAT GGT TTT GGC CCA AAT CCACGT GGT CCA GAC ATC AAA ACT TTT GGC TCC AGG GCGGTG GAC ATG TTC TGC GAG CGC TAT AAT TAT CAG TAT ATA TTT CGC GCG CAC CAA GAA AAA GCGGAC GGT GTG AGG CTT TCT GAT AAC GCT CGA GTT GTA ACA ATT TTTTCA ACT TCG GAT TAT GCC GGA CAT CAA AAC GGG GCA GGA TGC ATA CTG GTC GCAAAC GGC AAA ATG AGG ATG GCG ATA AAA AAG CCT CAT CGT CAT GAG ATG AAG GAAACA AAA GGG CCT GTG TCT CCG CGG ACG CTG TCC AAG AGC GTG CCGGGA ATG ATC CCT AGG CTC AAA AGG ATA TAA
 * *CDS Gene Sequence (paste as text only): **
 * *GC% Content for gene: ** 48%
 * *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): **
 * *GC% Content for gene (codon optimized): ** 47.6%

Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): ( link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.

Primer design results for 'tail' primers (this is just 2 sequences): **