Synthesis and characterization of the human CC chemokine HCC-2.

Human CC chemokine 2 (HCC-2) is a novel member of the chemokine peptide family that induces chemotaxis of monocytes, T lymphocytes and eosinophils via activation of the CCR-1 and CCR-3 receptors. Fmoc chemistry was optimized and used to synthesize the biologically active 66-residue peptide HCC-2-(48-113). Introduction of the three disulfide bonds was achieved by oxidative folding in the presence of the redox system cysteine/cystine. Alternatively, a semiselective approach utilizing a mixed Acm/Trt protection scheme for disulfide formation was applied. It was found that, without participation of the two HCC-2-specific cysteine residues in positions 64 and 104, the two typical chemokine disulfides are formed predominantly during oxidative folding. In addition, the mutant [Ala64,104]HCC-2-(48-113) lacking the third disulfide bond that discriminates HCC-2 from most other chemokines was synthesized. For disulfide bond formation, oxidative folding was compared with the use of Acm/Trt protection. HCC-2-(48-113) and the mutant [Ala64,104]HCC-2-(48-113) were further analyzed by CD and one-dimensional 1H NMR-spectroscopy. Both peptides adopt a similar stable secondary and tertiary structure in solution.     

Fmoc-based synthesis of the human CC chemokine CCL14/HCC-1 by SPPS and native chemical ligation

Summary The CC chemokine CCL14/HCC-1(9–74), a 66-residue polypeptide containing two disulfide bonds, was recently discovered from a human hemofiltrate peptide library as a high-affinity ligand of the chemokine receptors CCR1 and CCR5. It has been shown to inhibit HIV infection by blocking CCR5. Using Fmoc methodology, we, report the chemical synthesis of CCL14/HCC-1 by conventional stepwise solid-phase peptide synthesis (SPPS) and, alternatively, native chemical ligation. To optimize SPPS of CCL14/HCC-1, difficult sequence regions were identified by mass spectrometry, in order to obtain a crude tetrathiol precursor suitable for oxidative disulfide formation. For synthesis of CCL14/HCC-1 by native chemical ligation, the peptide was divided into two segments, CCL14/HCC-1(9–39) and CCL14/HCC-1(40–74), the latter containing a cysteine residue at the amino-terminus. The synthesis of the thioester segment was carried out comparing a thiol linker with a sulfonamide safety-catch linker. While the use of the thiol linker led to very low overall yields of the desired thioester, the sulfonamide linker was efficient in obtaining the 31-residue thioester of CCL14/HCC-1(9–39), suggesting a superior suitability of this linker in generating larger thioesters using Fmoc chemistry. The thioester of CCL14/HCC-1 was subsequently ligated with the cysteinyl segment to the full-length chemokine. Disulfides were introduced in the presence of the redox buffer cysteine/cystine. The products of both SPPS and native chemical ligation were identical. The use of a sulfonamide safety-catch linker enables the Fmoc synthesis of larger peptide thioesters, and is thus useful to generate arrays of larger polypeptides.     

Fmoc-based synthesis of the human CC chemokine CCL14/HCC-1 by SPPS and native chemical ligation

The CC chemokine CCL14/HCC-1(9-74), a 66-residue polypeptide containing two disulfide bonds, was recently discovered from a human hemofiltrate peptide library as a high-affinity ligand of the chemokine receptors CCR1 and CCR5. It has been shown to inhibit HIV infection by blocking CCR5. Using Fmoc methodology, we report the chemical synthesis of CCL14/HCC-1 by conventional stepwise solid-phase peptide synthesis (SPPS) and, alternatively, native chemical ligation. To optimize SPPS of CCL14/HCC-1, difficult sequence regions were identified by mass spectrometry, in order to obtain a crude tetrathiol precursor suitable for oxidative disulfide formation. For synthesis of CCL14/HCC-1 by native chemical ligation, the peptide was divided into two segments, CCL14/HCC-1(9-39) and CCL14/HCC-1(40-74), the latter containing a cysteine residue at the amino-terminus. The synthesis of the thioester segment was carried out comparing a thiol linker with a sulfonamide safety-catch linker. While the use of the thiol linker led to very low overall yields of the desired thioester, the sulfonamide linker was efficient in obtaining the 31-residue thioester of CCL14/HCC-1(9-39), suggesting a superior suitability of this linker in generating larger thioesters using Fmoc chemistry. The thioester of CCL14/HCC-1 was subsequently ligated with the cysteinyl segment to the full-length chemokine. Disulfides were introduced in the presence of the redox buffer cysteine/cystine. The products of both SPPS and native chemical ligation were identical. The use of a sulfonamide safety-catch linker enables the Fmoc synthesis of larger peptide thioesters, and is thus useful to generate arrays of larger polypeptides.     

Differential CCR1-mediated chemotaxis signaling induced by human CC chemokine HCC-4/CCL16 in HOS cells

Human CC chemokine-4 (HCC-4)/CCL16 is a chemoattractant for monocytes and lymphocytes. Although HCC-4 binds to multiple CC chemokine receptors, the receptor-mediated signal transduction pathway induced by HCC-4 has not been characterized. Human osteogenic sarcoma cells stably expressing CCR1 were used to investigate HCC-4-mediated chemotaxis signaling events via CCR1. The chemotactic activity of HCC-4 as well as those of other CCR1-dependent chemokines including MIP-1alpha/CCL3, RANTES/CCL5, and Lkn-1/CCL15 was inhibited by the treatment of pertussis toxin, an inhibitor of Gi/Go protein, U73122, an inhibitor of phospholipase C (PLC), and rottlerin, a specific inhibitor of protein kinase Cdelta (PKCdelta). These results indicate that HCC-4-induced chemotaxis signaling is mediated through Gi/Go protein, PLC, and PKCdelta. SB202190, an inhibitor of p38 mitogen activated protein kinase, only blocked the chemotactic activity of HCC-4, but not those of other CCR1-dependent chemokines. SB202190 inhibited HCC-4-induced chemotaxis in a dose-dependent manner (P < 0.01). HCC-4 induces p38 activation in both a time and dose-dependent manner. However, such p38 activation was not induced by other CCR1-dependent chemokines. To further investigate the differential effect of HCC-4, the Ca2+ mobilization was examined. HCC-4 induced no intracellular Ca2+ flux in contrast to other CCR1-dependent chemokines. These results indicate that HCC-4 transduces signals differently from other CCR1-dependent chemokines and may play different roles in the immune response.     

Functional analysis of chemically synthesized derivatives of the human CC chemokine CCL15/HCC-2, a high affinity CCR1 ligand.

The CCL15 is a human CC chemokine that activates the receptors, CCR1 and CCR3. Unlike other chemokines, it contains an unusually long N-terminal domain of 31 amino acids preceding the first cysteine residue and a third disulfide bond. To elucidate the functional role of distinct structural determinants, a series of sequential amino-terminal truncated and point-mutated CCL15 derivatives as well as mutants lacking the third disulfide bond and the carboxy-terminal alpha-helix were synthesized using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. We demonstrate that a truncation of 24 amino acid residues (delta24-CCL15) converts the slightly active 92-residue delta0-CCL15 into a potent agonist of CC chemokine receptor 1 (CCR1) and a weak agonist of CCR3 in cell-based assays. The biological activity decreases from delta24-CCL15 to delta29-CCL15, and re-increases from delta29-CCL15 to delta30-CCL15. Thus, an exocyclic N-terminal region of only one amino acid residue is sufficient for efficient CCR1 activation. As none of the peptides investigated except for delta24-CCL15 activates CCR3, we suggest that CCR1 is the major receptor for CCL15 in vivo. Further we demonstrate that the third disulfide bond of CCL15 and an exchange of tyrosine in position 70 by a leucine residue, which is conserved in CXC chemokines, do not alter the interaction with CCR1. In contrast, a CCL15 derivative lacking the carboxy-terminal alpha-helix exhibits a complete loss of tertiary structure and hence loss of CCR1 agonistic and binding activity. This study demonstrates that specific protein residues in chemokines, which contribute to receptor-ligand interaction, vary significantly between chemokines and cannot be extrapolated using data from functionally related chemokines.     

Mouse liver lysosomes contain enzymatically active processed forms of Hyal-1

It has long been known that liver lysosomes contain an endoglycosidase activity able to degrade the high molecular mass glycosaminoglycan hyaluronic acid (HA). The identification and cloning of a hyaluronidase with an acidic pH optimum, Hyal-1, suggested it might be responsible for this activity. However, we previously reported that this hydrolase could only be detected in pre-lysosomal compartments of the mouse liver using a zymography technique that allows the detection of Hyal-1 activity after SDS–PAGE (“renatured protein zymography”). Present work reveals that the activity highlighted by this technique belongs to a precursor form of Hyal-1 and that the lysosomal HA endoglycosidase activity of the mouse liver is accounted for by a proteolytically processed form of Hyal-1 that can only be detected using “native protein zymography”. Indeed, the distribution of this form follows the distribution of β-galactosidase, a well-established lysosomal marker, after fractionation of the mouse liver in a linear sucrose density gradient. In addition, both activities shift toward the lower density region of the gradient when a specific decrease of the lysosomal density is induced by Triton WR-1339 injection. The fact that only native protein zymography but not renatured protein zymography is able to detect Hyal-1 activity in lysosomes points to a non-covalent association of Hyal-1 proteolytic fragments or the existence of closely linked partners supporting Hyal-1 enzymatic activity. The knockdown of Hyal-1 results in an 80% decrease of total acid hyaluronidase activity in the mouse liver, confirming that Hyal-1 is a key actor of HA catabolism in this organ.     

Rescue of HIV-1 receptor function through cooperation between different forms of the CCR5 chemokine receptor

Interaction of the human immunodeficiency virus (HIV-1) envelope glycoproteins with the CCR5 chemokine receptor, a G-protein-coupled receptor, triggers a membrane fusion process and virus entry. Cooperation for HIV-1 receptor activity was observed when two forms of CCR5 were coexpressed, either the wild-type (WT) receptor and a defective mutant with deletion of the amino-terminal (NT) extracellular domain or the latter deltaNT mutant and a human-mouse CCR5 chimera bearing the NT domain from human CCR5. Cooperation was most efficient when the two forms of CCR5 were in a 1:1 ratio. It was not observed between the CCR5 deltaNT mutant and a chimeric receptor (5444) in which the NT domain of CCR5 was in the context of another G-protein-coupled receptor, the HIV-1 receptor CXCR4. These results suggested that physical association between two forms of CCR5 was required for their cooperation. Coimmunoprecipitation experiments in transfected cell lysates indeed showed that the deltaNT CCR5 mutant formed oligomeric complexes with the WT CCR5 or the HMMM chimera but not with the CXCR4-derived chimera 5444. These observations suggest that the formation of CCR5 oligomers is a constitutive process independent from activation by chemokine ligands. The interaction of HIV-1 with independent subunits of CCR5 oligomers could favor the local recruitment of fusiogenic proteins and the formation of a fusion pore.     

Interleukin-1 beta converting enzyme requires oligomerization for activity of processed forms in vivo.

Interleukin-1 beta converting enzyme (ICE) is composed of 10' (p10) and 20 kDa (p20) subunits, which are derived from a common 45 kDa precursor. Recent crystallographic studies have shown that ICE exists as a tetramer (p20/p10)2 in the crystal lattice. We provide evidence that the p10 and p20 subunits of ICE associate as oligomers in transfected COS cells. Using intragenic complementation, we show that the activity of a p10/p10 interface mutant defective in autoprocessing can be restored by co-expression with active site ICE mutants. Different active site mutants can also complement each other by oligomerization to form active ICE. These studies indicate that ICE precursor polypeptides may associate in different quaternary structures and that oligomerization is required for autoprocessing. Furthermore, integenic complementation of active site mutants of ICE and an ICE homolog restores autoprocessing activity, suggesting that hetero-oligomerization occurs between ICE homologs.     

Regulation of the human chemokine receptor CCR1. Cross-regulation by CXCR1 and CXCR2

To investigate the regulation of the CCR1 chemokine receptor, a rat basophilic leukemia (RBL-2H3) cell line was modified to stably express epitope-tagged receptor. These cells responded to RANTES (regulated upon activation normal T expressed and secreted), macrophage inflammatory protein-1alpha, and monocyte chemotactic protein-2 to mediate phospholipase C activation, intracellular Ca(2+) mobilization and exocytosis. Upon activation, CCR1 underwent phosphorylation and desensitization as measured by diminished GTPase stimulation and Ca(2+) mobilization. Alanine substitution of specific serine and threonine residues (S2 and S3) or truncation of the cytoplasmic tail (DeltaCCR1) of CCR1 abolished receptor phosphorylation and desensitization of G protein activation but did not abolish desensitization of Ca(2+) mobilization. S2, S3, and DeltaCCR1 were also resistant to internalization, mediated greater phosphatidylinositol hydrolysis and sustained Ca(2+) mobilization, and were only partially desensitized by RANTES, relative to S1 and CCR1. To study CCR1 cross-regulation, RBL cells co-expressing CCR1 and receptors for interleukin-8 (CXCR1, CXCR2, or a phosphorylation-deficient mutant of CXCR2, 331T) were produced. Interleukin-8 stimulation of CXCR1 or CXCR2 cross-phosphorylated CCR1 and cross-desensitized its ability to stimulate GTPase activity and Ca(2+) mobilization. Interestingly, CCR1 cross-phosphorylated and cross-desensitized CXCR2, but not CXCR1. Ca(2+) mobilization by S3 and DeltaCCR1 were also cross-desensitized by CXCR1 and CXCR2 despite lack of receptor phosphorylation. In contrast to wild type CCR1, S3 and DeltaCCR1, which produced sustained signals, cross-phosphorylated and cross-desensitized responses to CXCR1 as well as CXCR2. Taken together, these results indicate that CCR1-mediated responses are regulated at several steps in the signaling pathway, by receptor phosphorylation at the level of receptor/G protein coupling and by an unknown mechanism at the level of phospholipase C activation. Moreover selective cross-regulation among chemokine receptors is, in part, a consequence of the strength of signaling (i.e. greater phosphatidylinositol hydrolysis and sustained Ca(2+) mobilization) which is inversely correlated with the receptor's susceptibility to phosphorylation. Since many chemokines activate multiple chemokine receptors, selective cross-regulation among such receptors may play a role in their immunomodulation.     

Detection of a processed pseudogene of the human MBL-associated serine protease,MASP1

Southern hybridization analysis of the MASP1 gene using an intron-specific probe detected a single band. An exon-specific probe detected several bands. PCR of genomic DNA using several exon-specific primer sets of MASP1 produced short and long products. Sequence of the shorter products corresponded to the processed pseudogene of MASP1. By fluorescence in situ hybridization, this pseudogene (MASP1P1) was mapped to 1p34.     

Functional redundancy of the human CCL4 and CCL4L1 chemokine genes

CCL4 and CCL4L1 are two CC chemokine genes located at chromosome 17q21 whose mature proteins differ at only a single amino acid. Abundant functional information exists for CCL4, however, CCL4L1 has only recently been recognized as a distinct gene, thus information describing it is wanting. The CCL4L1 protein was synthesized in Escherichia coli and compared with the CCL4 protein. Competitive binding studies using HEK-293/CCR5 cells produced comparable EC50 values for the two proteins. Similarly, chemotaxis assays with cells expressing CCR1, CCR3, or CCR5 revealed no substantial differences. CCL4L1 was somewhat more effective at inhibiting HIV-1 replication in PBMCs than was CCL4, however the difference was not statistically significant. These data combined with the observation of individual variation in CCL4L1 gene copy number [Eur. J. Immunol. 32 (2002) 3016, Genomics 83 (2004) 735] support the contention that the CCL4 and CCL4L1 proteins have redundant functions.     

O-glycosylated human MUC1 repeats are processed in vitro by immunoproteasomes

The targeting of epitopes on tumor-associated glycoforms of human MUC1 represents a primary goal in immunotherapeutic anticancer strategies. Effective immune responses to cancer cells certainly require the activation of specific cytotoxic T cell repertoires by cross-priming of dendritic cells either via immunoproteasomal or by endosomal processing of ectodomain epitopes on MUC1-positive carcinomas. Because no evidence is currently available on the capacities of human immunoproteasomes to cleave mucin-type O-glycosylated peptides, we performed in vitro studies to address the questions of whether glycosylated MUC1 repeats are cleaved by immunoproteasomes and in which way O-linked glycans control the site specificity of peptide cleavage via their localization and structures. We show for the first time that mucin-type O-glycosylated peptides are effective substrates of immunoproteasomes, however, the patterns of cleavage are qualitatively and quantitatively influenced by O-glycosylation. The nonglycosylated MUC1 repeat peptide (clusters of oligorepeats AHGVTSAPDTRPAPGSTAPP or AHGVTSAPESRPAPGSTAPA) is cleaved preferentially within or adjacent to the SAP and GST motifs with formation of a complex fragment pattern that includes major nona- and decapeptides. O-GalNAc modified peptides are largely resistant to proteolysis if these preferred cleavage sites are located adjacent to O-glycosylation, whereas peptides even with elongated glycans at more distant sites can form effective substrates yielding major glycopeptide fragments in the class I size range.     

Crystal structures of oligomeric forms of the IP-10/CXCL10 chemokine

We have determined the structure of wild-type IP-10 from three crystal forms. The crystals provide eight separate models of the IP-10 chain, all differing substantially from a monomeric IP-10 variant examined previously by NMR spectroscopy. In each crystal form, IP-10 chains form conventional beta sheet dimers, which, in turn, form a distinct tetrameric assembly. The M form tetramer is reminiscent of platelet factor 4, whereas the T and H forms feature a novel twelve-stranded beta sheet. Analytical ultracentrifugation indicates that, in free solution, IP-10 exists in a monomer-dimer equilibrium with a dissociation constant of 9 microM. We propose that the tetrameric structures may represent species promoted by the binding of glycosaminoglycans. The binding sites for several IP-10-neutralizing mAbs have also been mapped.     

Two C-terminal peptides of human CKLF1 interact with the chemokine receptor CCR4

Human chemokine-like factor 1 (CKLF1) exhibits chemotactic effects on leukocytes. A previous study demonstrated that CKLF1 is a functional ligand for human CC chemokine receptor 4 (CCR4). In this study, N-terminal amino acid sequencing of secreted CKLF1 protein showed that it contains at least two peptides, C27 and C19. To examine whether C27 or C19 play a role via CCR4, C27 and C19 were chemically synthesized and analyzed by chemotaxis, calcium mobilization, and receptor internalization assays in CCR4-tranfected HEK293 cells or Hut78 cells. The chemotaxis assay showed that C27 could induce chemotaxis to CCR4-transfected HEK293 cells or Hut78 cells while C19 had weaker chemotactic activity, especially in Hut78 cells. C27- or C19-induced chemotaxis was abolished by pertussis toxin, suggesting the involvement of a Gi/o pathway. C27- or C19-induced chemotaxis was also inhibited by an antagonist of CCR4 that show good binding potency, excellent chemotaxis inhibitory activity and selectivity toward CCR4, suggesting that their chemotactic activity specifically involved CCR4. The chemotactic response of CCR4-tranfected HEK293 cells to C27 or C19 was markedly inhibited by preincubation with TARC/CCL17. TARC/CCL17 effectively desensitized the calcium mobilization induced by C27 or C19. Similarly, both of C27 or C19 also desensitized the calcium mobilization and chemotaxis of CCR4-tranfected HEK293 cells in response to TARC/CCL17, suggesting that they might interact with a common receptor. Both C27- and C19-induced clear internalization of CCR4-EGFP. These results confirm that the secreted peptides of CKLF1, C27 and C19, have functional activation via CCR4.     

Purification of bacteriorhodopsin and characterization of mature and partially processed forms

Bacteriorhodopsin (BR) essentially free of native lipids has been prepared in a highly stable state. Purple membrane was solubilized in Triton X-100 and BR was purified by size exclusion chromatography using 3-[cholamidopropyl)dimethylammonio]-2-hydroxyl-1-propanesulfonic acid (CHAPSO) detergent at pH 5. Molar ratios of phospholipid/BR ranged from 0.4 to 0.05 corresponding to 94-98% phospholipid removal. Purified BR has an absorbance ratio (A280nm/A548nm) of 1.5-1.6 in the dark-adapted state which is the highest purified BR/protein ratio reported to date. The purified BR in CHAPSO shows maximum stability in the pH range 5.0-5.5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of native purple membrane and solubilized BR from most Halobacterium halobium JW-3 cultures show 3 higher molecular weight bands in addition to BR. Immunological staining and amino acid sequencing indicates that these additional proteins are partially processed forms of the BR precursor protein. The BR preprotein contains 13 additional amino acids on the NH2 terminus which are removed by post-translational processing in at least four steps. Isoelectric focusing separated most delipidated and non-delipidated BR samples into 8 bands. Incomplete BR post-translational processing BR is thought to be largely responsible for the multiplicity of isoelectric BR species. The principal components have pI values of 5.20 and 5.24 and both have absorption maxima at 550 nm, characteristic of detergent-solubilized BR. BR in Triton X-100 or nonylglucoside, delipidated BR in CHAPSO, and BR in intact purple membrane all have a dark-adapted ratio of 13-cis to all-trans-retinal of 1.9:1.     

Determinants of the trans-dominant negative effect of truncated forms of the CCR5 chemokine receptor

The human immunodeficiency virus, type 1 (HIV-1) entry process is triggered by interaction between the viral envelope and a seven membrane-spanning domain receptor at the cell surface, usually the CCR5 chemokine receptor. Different naturally occurring mutations in the CCR5 gene abolish receptor function, the most frequent being a 32-nucleotide deletion resulting in a truncated protein (Delta32) lacking the last three transmembrane domains (TM5-7). This mutant is retained in the endoplasmic reticulum and exerts a trans-dominant negative (TDN) effect on the wild type, preventing its exit from this compartment. This TDN effect is often considered as evidence for the oligomerization of CCR5 during transport to the cell surface. Here we use a genetic approach to define the structural determinants of the TDN effect of the Delta32 mutant. It was abolished by certain deletions and by mutations of cysteine residues preventing formation of a disulfide link between the first and second extracellular loops, suggesting that conformation of Delta32 is important for its interaction with CCR5. To circumvent this problem, we used chimeric forms of the Delta32 and wild type CCR5, consisting in substitutions with homologous domains from the mouse CCR5. All chimeric full-length receptors were expressed at the cell surface and were functional for interaction with HIV-1 or with a chemokine ligand, when assayed. The TDN effect was only observed if both the TM3 domain in CCR5 and the TM4 domain in Delta32 were from human origin, whereas the rest of the proteins could be from either origin. This suggests that the TDN effect involves some form of interaction between these transmembrane domains. Alternatively, but less likely to us, substitutions in TM4 could affect the conformation of CCR5 in the endoplasmic reticulum but not at the cell surface. However that may be, it seems that the TDN effect of the Delta32 mutant has no bearing to the issue of CCR5 dimerization and to its possible role in the processing of the receptor to the cell surface.     

Differential Distribution of Posttranslationally Modified Microtubules in Osteoclasts

The differential distribution of microtubules in osteoclasts in culture was examined by using antibodies against acetylated, tyrosinated, or detyrosinated tubulins. Tyrosinated tubulin was found throughout the cytoplasmic microtubules in all cells examined. An expanding protrusion that contained tyrosinated tubulin but none of the detyrosinated or acetylated form was seen in the immature osteoclasts. Detyrosinated or acetylated tubulin was detectable in the peripheral cytoplasm of the mature osteoclasts displaying the loss of the expanding protrusion. Although most of the microtubules were derived from the centrosome, noncentrosomal microtubules were distributed in the expanding protrusion, which was predominantly positive for tyrosinated tubulin. By tracing single microtubules, the authors found that their growing ends were always rich in tyrosinated tubulin subunits. End binding protein 1 bound preferentially to the microtubule ends. Both acetylated and tyrosinated microtubules were shown to be closely associated with podosomes. Microtubules appeared to grow over or into the podosomes; in addition, the growing ends of single microtubules could be observed to target the podosomes. Moreover, a microtubule-associated histone deacetylase 6 was localized in the podosomes of the osteoclast. On the basis of these results, the authors conclude that posttranslational modifications of microtubules may correlate with characteristic changes in podosome dynamics in osteoclasts.     

Evidence for multiple forms and modifications of human POT1

Human POT1, a widely studied telomere protector protein is perceived to be expressed as a single 70 kDa form. A survey of the literature as well as different commercially available antibodies against POT1 suggests occurrence of multiple forms of POT1. Knowledge about possible various forms of an important protein like POT1 is necessary for our understanding about its function. We have discovered that POT1 exists in at least three consistently occurring forms; 90, 70 and 45 kDa. The unexpected molecular weights of POT1 seem to be associated with SUMO1 and ubiquitin conjugation; the latter occurring at a double lysine residue at 289-KK-290. We also present evidence that the relative abundance of the different POT1 forms can be altered by experimental modulation of POT1 nuclear localization. We thus present strong evidence that there are post-translational modifications of POT1 that can affect its molecular weight as well as intracellular localization and function.