HSP25 Vaccination Attenuates Atherogenesis via Upregulation of LDLR Expression, Lowering of PCSK9 Levels and Curbing of Inflammation.

[Figure: see text].

Analyzing Recombinant Protein Production in Pichia pastoris with Targeted Proteomics.

New mass spectrometry approaches enable antibody-independent tracking of protein production. Herein, we outline an antibody-independent mass spectrometry method for tracking recombinant protein production in the methylotrophic yeast Pichia pastoris system.

Application of immobilized ATP to the study of NLRP inflammasomes.

The NLRP proteins are a subfamily of the NOD-like receptor (NLR) innate immune sensors that possess an ATP-binding NACHT domain. As the most well studied member, NLRP3 can initiate the assembly process of a multiprotein complex, termed the inflammasome, upon detection of a wide range of microbial products and endogenous danger signals and results in the activation of pro-caspase-1, a cysteine protease that regulates multiple host defense pathways including cytokine maturation. Dysregulated NLRP3 activation contributes to inflammation and the pathogenesis of several chronic diseases, and the ATP-binding properties of NLRPs are thought to be critical for inflammasome activation. In light of this, we examined the utility of immobilized ATP matrices in the study of NLRP inflammasomes. Using NLRP3 as the prototypical member of the family, P-linked ATP Sepharose was determined to be a highly-effective capture agent. In subsequent examinations, P-linked ATP Sepharose was used as an enrichment tool to enable the effective profiling of NLRP3-biomarker signatures with selected reaction monitoring-mass spectrometry (SRM-MS). Finally, ATP Sepharose was used in combination with a fluorescence-linked enzyme chemoproteomic strategy (FLECS) screen to identify potential competitive inhibitors of NLRP3. The identification of a novel benzo[d]imidazol-2-one inhibitor that specifically targets the ATP-binding and hydrolysis properties of the NLRP3 protein implies that ATP Sepharose and FLECS could be applied other NLRPs as well.

Characterization of heat shock protein 27 in extracellular vesicles: a potential anti-inflammatory therapy.

Previously, we reported that elevated serum levels of heat shock protein 27 (HSP27) are predictive of a lower risk of having a heart attack, stroke, or death from cardiovascular disease. Moreover, augmenting HSP27 (or the murine ortholog, HSP25) attenuated experimental atherogenesis, reduced inflammation, and lowered cholesterol levels. Recently, we noted that HSP27 activates NF-κB via TLR-4, resulting in attenuation of plaque inflammation; however, the precise anti-atherosclerosis mechanisms mediated by extracellular HSP27 are incompletely understood. Our purpose in this study was to investigate the existence of HSP27 in extracellular vesicles (EVs) and whether HSP27 elicited atheroprotective effects on target cells. Here, we provide evidence that HSP27 localizes to EVs derived from THP-1 cells using transmission electron microscopy (TEM) and immunogold labeling, Western blotting, ELISA, and fluorescence-activated cell sorting. TEM imaging indicated that HSP27 is found at the exosomal membrane. Multiple reactor monitor-mass spectrometric analysis of large vesicles, which included microparticles and exosomes, isolated from human plasma, also led to detection of HSP27 using the unique signature peptide, R.LFDQAFGLPR.L. Studies using THP-1 and human embryonic kidney cells show that HSP27-laden exosomes significantly stimulated NF-κB activation ( P < 0.001) and release of IL-10 ( P < 0.0001), suggesting that HSP27 may be important exosomal cargo with beneficial anti-inflammatory effects.-Shi, C., Ulke-Lemée, A., Deng, J., Batulan, Z., O'Brien, E. R. Characterization of heat shock protein 27 in extracellular vesicles: a potential anti-inflammatory therapy.

Quantification of Inflammasome Adaptor Protein ASC in Biological Samples by Multiple-Reaction Monitoring Mass Spectrometry.

Inflammation is an integral component of many diseases, including chronic kidney disease (CKD). ASC (apoptosis-associated speck-like protein containing CARD, also PYCARD) is the key inflammasome adaptor protein in the innate immune response. Since ASC specks, a macromolecular condensate of ASC protein, can be released by inflammasome-activated cells into the extracellular space to amplify inflammatory responses, the ASC protein could be an important biomarker in diagnostic applications. Herein, we describe the development and validation of a multiple reaction monitoring mass spectrometry (MRM-MS) assay for the accurate quantification of ASC in human biospecimens. Limits of detection and quantification for the signature DLLLQALR peptide (used as surrogate for the target ASC protein) were determined by the method of standard addition using synthetic isotope-labeled internal standard (SIS) peptide and urine matrix from a healthy donor (LOQ was 8.25 pM, with a ~ 1000-fold linear range). We further quantified ASC in the urine of CKD patients (8.4 ± 1.3 ng ASC/ml urine, n = 13). ASC was positively correlated with proteinuria and urinary IL-18 in CKD samples but not with urinary creatinine. Unfortunately, the ASC protein is susceptible to degradation, and patient urine that was thawed and refrozen lost 85% of the ASC signal. In summary, the MRM-MS assay provides a robust means to quantify ASC in biological samples, including clinical biospecimens; however, sample collection and storage conditions will have a critical impact on assay reliability.

Renal immune surveillance and dipeptidase-1 contribute to contrast-induced acute kidney injury.

Radiographic contrast agents cause acute kidney injury (AKI), yet the underlying pathogenesis is poorly understood. Nod-like receptor pyrin containing 3-deficient (Nlrp3-deficient) mice displayed reduced epithelial cell injury and inflammation in the kidney in a model of contrast-induced AKI (CI-AKI). Unexpectedly, contrast agents directly induced tubular epithelial cell death in vitro that was not dependent on Nlrp3. Rather, contrast agents activated the canonical Nlrp3 inflammasome in macrophages. Intravital microscopy revealed diatrizoate (DTA) uptake within minutes in perivascular CX3CR1+ resident phagocytes in the kidney. Following rapid filtration into the tubular luminal space, DTA was reabsorbed and concentrated in tubular epithelial cells via the brush border enzyme dipeptidase-1 in volume-depleted but not euvolemic mice. LysM-GFP+ macrophages recruited to the kidney interstitial space ingested contrast material transported from the urine via direct interactions with tubules. CI-AKI was dependent on resident renal phagocytes, IL-1, leukocyte recruitment, and dipeptidase-1. Levels of the inflammasome-related urinary biomarkers IL-18 and caspase-1 were increased immediately following contrast administration in patients undergoing coronary angiography, consistent with the acute renal effects observed in mice. Taken together, these data show that CI-AKI is a multistep process that involves immune surveillance by resident and infiltrating renal phagocytes, Nlrp3-dependent inflammation, and the tubular reabsorption of contrast via dipeptidase-1.

Quantitation of myosin regulatory light chain phosphorylation in biological samples with multiple reaction monitoring mass spectrometry.

The 20-kDa regulatory light chain of myosin II plays an important role in regulating smooth muscle contractile force. LC20 is phosphorylated canonically by myosin light chain kinase in a Ca2+/calmodulin-dependent manner at S19. The diphosphorylation of LC20 at T18 and S19 has been observed in smooth muscle tissues. Given that the phosphorylation of LC20 is positively correlated with tension development, the molar stoichiometry of LC20 phosphorylation is commonly profiled as a measure of smooth muscle contractility. Herein, we describe a novel multiple reaction monitoring (MRM)-mass spectrometry (MS) approach for the quantification of LC20 phosphorylation at T18 and S19. Unique precursor as well as y- and b-ion transitions were identified for unphosphorylated LC20-(TS), monophosphorylated LC20-(TpS) and diphosphorylated LC20-(pTpS) peptides. The MRM-MS assay could accurately define molar phosphorylation stoichiometries of S19 and T18 over a broad range (i.e., 0-2 mol P/mol LC20). Correlations of the results for two quantification techniques indicate that the MRM-MS assay performs equally to Phos-tag SDS-PAGE for the determination of LC20 phosphorylation stoichiometry in arterial tissue samples. The MRM-MS technique provides a robust alternative to antibody-based detection systems for the quantification of LC20 phosphorylation.

Tools and protocol for quantification of myosin phosphorylation with MRM-MS.

The phosphorylation of myosin regulatory light chain (LC20) at Thr18 and Ser19 is positively correlated with tension development in smooth muscle tissue, and the molar stoichiometry of LC20 phosphorylation is commonly profiled as a measure of smooth muscle contractility. We provide details for a newly applied multiple reaction monitoring (MRM)-mass spectrometry (MS) method for the quantification of LC20 phosphorylation at Thr18 and Ser19. This MRM-MS method provides a robust alternative to antibody-based detection systems (such as Phos-Tag SDS-PAGE) for the quantification of LC20 phosphorylation.

Binding of smoothelin-like 1 to tropomyosin and calmodulin is mutually exclusive and regulated by phosphorylation.

BACKGROUND: The smoothelin-like 1 protein (SMTNL1) can associate with tropomyosin (Tpm) and calmodulin (CaM), two proteins essential to the smooth muscle contractile process. SMTNL1 is phosphorylated at Ser301 by protein kinase A during calcium desensitization in smooth muscle, yet the effect of SMTNL1 phosphorylation on Tpm- and CaM-binding has yet to be investigated. RESULTS: Using pull down studies with Tpm-Sepharose and CaM-Sepharose, we examined the interplay between Tpm binding, CaM binding, phosphorylation of SMTNL1 and calcium concentration. Phosphorylation greatly enhanced the ability of SMTNL1 to associate with Tpm in vitro; surface plasmon resonance yielded a 10-fold enhancement in K D value with phosphorylation. The effect on CaM binding is more complex and varies with the availability of calcium. CONCLUSIONS: Combining both CaM and Tpm with SMTNL1 shows that the binding to both is mutually exclusive.

Assessment of endothelin and copeptin as biomarkers for vasovagal syncope.

BACKGROUND: The diagnosis of vasovagal syncope continues to be difficult despite the use of accurate histories, tilt testing and implantable loop recorders. A circulating biomarker might be useful to facilitate diagnoses. Both endothelin-1 and vasopressin are increased during positive tilt tests resulting in syncope. Copeptin is a stable cleavage product of vasopressin formation. We conducted a pilot study to assess the utility of endothelin-1 and copeptin as circulating biomarkers of vasovagal syncope. METHODS: Three populations were studied: syncope patients, epilepsy patients and controls. Vasovagal syncope diagnosis was ascertained with the Calgary Syncope Score and epilepsy diagnosis was confirmed with EEG. Plasma levels of endothelin-1 were measured using by ELISA and copeptin levels were determined using an EIA kit. RESULTS: Asymptomatic control subjects had mean age 35 ± 11 years (7/22 male); epileptic subjects had mean age 32 ± 7 years (4/15 male); and syncope subjects had mean age 33 ± 16 years (4 of 21 male). Circulating plasma levels of endothelin-1 and copeptin were no different among the three groups. Mean concentrations of endothelin-1 were as follows: syncope, 23 ± 32 pg/mL; controls, 21 ± 17 pg/mL; and epileptics, 18 ± 12 pg/mL. Mean concentrations of copeptin were as follows: syncope, 1·29 ± 0·79 ng/mL; controls, 1·25 ± 0·79 ng/mL; and seizures, 1·23 ± 0·45 ng/mL. There were no significant correlations between syncope frequency and copeptin or endothelin-1 levels. CONCLUSION: Circulating plasma endothelin-1 and copeptin levels are not significantly different among populations of controls, syncope patients and seizure patients.

In situ analysis of smoothelin-like 1 and calmodulin interactions in smooth muscle cells by proximity ligation.

The smoothelin-like 1 (SMTNL1) protein is the newest member of the smoothelin family of muscle proteins. Two calmodulin (CaM)-binding domains (CBD1 for Ca-CaM; CBD2 for apo-CaM) have been described for the SMTNL1 protein using in vitro assays. We now demonstrate in situ associations of SMTNL1 and CaM in A7r5 smooth muscle cells using the proximity ligation assay (PLA). We quantified CaM-SMTNL1 proximity events accurately after taking into account variations in protein expression levels. The refined method allows quantification of in situ proximity after transient transfection with an associated error of <10%. The proximity of SMTNL1 and CaM in A7r5 cells could be reduced by scrambling the amino acid sequence and mutation of large hydrophobic amino acids of CBD1. The truncation of CBD2 did not influence SMTNL1 proximity to CaM. Ultimately, we conclude that SMTNL1 forms complex interactions with CaM in smooth muscle cells, with a role for CBD1 and possibly the intrinsically disordered region.

Two domains of the smoothelin-like 1 protein bind apo- and calcium-calmodulin independently.

The smoothelin-like 1 protein (SMTNL1) is a modulator of smooth and skeletal muscle contractility and can bind to calmodulin and tropomyosin. Calmodulin is the major calcium sensor of eukaryotic cells and it can cycle between calcium-free (apo-CaM) and calcium-bound (Ca-CaM) forms. Bioinformatic screening of the SMTNL1 sequence predicted a second CaM-binding region (CBD1) that is located N-terminal to the previously defined apo-CaM-binding site (CBD2). Pull-down assays, surface plasmon resonance, isothermal calorimetry and NMR techniques were used to determine that CBD1 associated preferentially to Ca-CaM while CBD2 bound preferentially to apo-CaM. Mutation of hydrophobic residues abolished Ca-CaM-binding to CBD1 while acidic residues in CBD2 were necessary for apo-CaM-binding to CBD2. The dissociation constant (Kd) for Ca-CaM-binding to a CBD1 peptide was 26∗10(-6)M while the value for binding to a longer protein construct was 0.5∗10(-6)M. The binding of SMTNL1 to both apo-CaM and Ca-CaM suggests that endogenous CaM is continuously associated with SMTNL1 to allow for quick response to changes in intracellular calcium levels. We also found that the intrinsically disordered N-terminus of SMTNL1 can reduce binding to apo-CaM and increase binding to Ca-CaM. This finding suggests that an additional CaM-binding region may exist and/or that intramolecular interactions between the N-terminus and the folded C-terminus reduce apo-CaM-binding to CBD2. Intriguingly, CBD1 is located close to the SMTNL1 phosphorylation site and tropomyosin-binding region. We discuss the possibility that all three signals are integrated at the region surrounding CBD1.

Cross-talk between Rho-associated kinase and cyclic nucleotide-dependent kinase signaling pathways in the regulation of smooth muscle myosin light chain phosphatase.

Ca(2+) sensitization of smooth muscle contraction depends upon the activities of protein kinases, including Rho-associated kinase, that phosphorylate the myosin phosphatase targeting subunit (MYPT1) at Thr(697) and/or Thr(855) (rat sequence numbering) to inhibit phosphatase activity and increase contractile force. Both Thr residues are preceded by the sequence RRS, and it has been suggested that phosphorylation at Ser(696) prevents phosphorylation at Thr(697). However, the effects of Ser(854) and dual Ser(696)-Thr(697) and Ser(854)-Thr(855) phosphorylations on myosin phosphatase activity and contraction are unknown. We characterized a suite of MYPT1 proteins and phosphospecific antibodies for specificity toward monophosphorylation events (Ser(696), Thr(697), Ser(854), and Thr(855)), Ser phosphorylation events (Ser(696)/Ser(854)) and dual Ser/Thr phosphorylation events (Ser(696)-Thr(697) and Ser(854)-Thr(855)). Dual phosphorylation at Ser(696)-Thr(697) and Ser(854)-Thr(855) by cyclic nucleotide-dependent protein kinases had no effect on myosin phosphatase activity, whereas phosphorylation at Thr(697) and Thr(855) by Rho-associated kinase inhibited phosphatase activity and prevented phosphorylation by cAMP-dependent protein kinase at the neighboring Ser residues. Forskolin induced phosphorylation at Ser(696), Thr(697), Ser(854), and Thr(855) in rat caudal artery, whereas U46619 induced Thr(697) and Thr(855) phosphorylation and prevented the Ser phosphorylation induced by forskolin. Furthermore, pretreatment with forskolin prevented U46619-induced Thr phosphorylations. We conclude that cross-talk between cyclic nucleotide and RhoA signaling pathways dictates the phosphorylation status of the Ser(696)-Thr(697) and Ser(854)-Thr(855) inhibitory regions of MYPT1 in situ, thereby regulating the activity of myosin phosphatase and contraction.

Intrinsically disordered N-terminus of calponin homology-associated smooth muscle protein (CHASM) interacts with the calponin homology domain to enable tropomyosin binding.

The calponin homology-associated smooth muscle (CHASM) protein plays an important adaptive role in smooth and skeletal muscle contraction. CHASM is associated with increased muscle contractility and can be localized to the contractile thin filament via its binding interaction with tropomyosin. We sought to define the structural basis for the interaction of CHASM with smooth muscle tropomyosin as a first step to understanding the contribution of CHASM to the contractile capacity of smooth muscle. Herein, we provide a structure-based model for the tropomyosin-binding domain of CHASM using a combination of hydrogen/deuterium exchange mass spectrometry (HDX-MS) and NMR analyses. Our studies provide evidence that a portion of the N-terminal intrinsically disordered region forms intramolecular contacts with the globular C-terminal calponin homology (CH) domain. Ultimately, cooperativeness between these structurally dissimilar regions is required for CHASM binding to smooth muscle tropomyosin. Furthermore, it appears that the type-2 CH domain of CHASM is required for tropomyosin binding and presents a novel function for this protein domain.

Chemical genetics of zipper-interacting protein kinase reveal myosin light chain as a bona fide substrate in permeabilized arterial smooth muscle.

Zipper-interacting protein kinase (ZIPK) has been implicated in Ca(2+)-independent smooth muscle contraction, although its specific role is unknown. The addition of ZIPK to demembranated rat caudal arterial strips induced an increase in force, which correlated with increases in LC(20) and MYPT1 phosphorylation. However, because of the number of kinases capable of phosphorylating LC(20) and MYPT1, it has proven difficult to identify the mechanism underlying ZIPK action. Therefore, we set out to identify bona fide ZIPK substrates using a chemical genetics method that takes advantage of ATP analogs with bulky substituents at the N(6) position and an engineered ZIPK capable of utilizing such substrates. (32)P-Labeled 6-phenyl-ATP and ZIPK-L93G mutant protein were added to permeabilized rat caudal arterial strips, and substrate proteins were detected by autoradiography following SDS-PAGE. Mass spectrometry identified LC(20) as a direct target of ZIPK in situ for the first time. Tissues were also exposed to 6-phenyl-ATP and ZIPK-L93G in the absence of endogenous ATP, and putative ZIPK substrates were identified by Western blotting. LC(20) was thereby confirmed as a direct target of ZIPK; however, no phosphorylation of MYPT1 was detected. We conclude that ZIPK is involved in the regulation of smooth muscle contraction through direct phosphorylation of LC(20).

Mapping and functional characterization of the murine smoothelin-like 1 promoter.

BACKGROUND: Smoothelin-like 1 (SMTNL1, also known as CHASM) plays a role in promoting relaxation as well as adaptive responses to exercise, pregnancy and sexual development in smooth and skeletal muscle. Investigations of Smtnl1 transcriptional regulation are still lacking. Thus, in this study, we identify and characterize key regulatory elements of the mouse Smtnl1 gene. RESULTS: We mapped the key regulatory elements of the Smtnl1 promoter region: the transcriptional start site (TSS) lays -44 bp from the translational start codon and a TATA-box motif at -75 bp was conserved amongst all mammalian Smtnl1 promoters investigated. The Smtnl1 proximal promoter enhances expression up to 8-fold in smooth muscle cells and a second activating region lays 500 bp further upstream. Two repressing motifs were present (-118 to -218 bp and -1637 to -1869 bp). The proximal promoter is highly conserved in mammals and contains a mirror repeat sequence. In silico analysis suggests many transcription factors (notably MyoD) could potentially bind within the Smtnl1 proximal promoter sequence. CONCLUSION: Smtnl1 transcript was identified in all smooth muscle tissues examined to date, albeit at much lower levels than found in skeletal muscle. It is unlikely that multiple SMTNL1 isoforms exist since a single Smtnl1 transcription start site was identified in both skeletal and intestinal smooth muscle. Promoter studies suggest restrictive control of Smtnl1 expression in non-muscle cells.

Tropomyosin-binding properties of the CHASM protein are dependent upon its calponin homology domain.

The calponin homology-associated smooth muscle protein (CHASM) can modulate muscle contractility, and its biological action may involve an interaction with the contractile filament. In this study, we demonstrate an interaction between CHASM and tropomyosin. Deletion constructs of CHASM were generated, and pull-down assays revealed a minimal deletion construct that could bind tropomyosin. Removal of the calponin homology (CH) domain or expression of the CH domain alone did not enable binding. The interaction was characterized by microcalorimetry with a dissociation constant of 2.0x10(-6) M. Confocal fluorescence microscopy also showed green fluorescent protein (GFP)-CHASM localization to filamentous structures within smooth muscle cells, and this targeting was dependent upon the CH domain.

Clostridium difficile toxin-induced inflammation and intestinal injury are mediated by the inflammasome.

BACKGROUND & AIMS: Clostridium difficile-associated disease (CDAD) is the leading cause of nosocomial diarrhea in the United States. C difficile toxins TcdA and TcdB breach the intestinal barrier and trigger mucosal inflammation and intestinal damage. The inflammasome is an intracellular danger sensor of the innate immune system. In the present study, we hypothesize that TcdA and TcdB trigger inflammasome-dependent interleukin (IL)-1beta production, which contributes to the pathogenesis of CDAD. METHODS: Macrophages exposed to TcdA and TcdB were assessed for IL-1beta production, an indication of inflammasome activation. Macrophages deficient in components of the inflammasome were also assessed. Truncated/mutated forms of TcdB were assessed for their ability to activate the inflammasome. The role of inflammasome signaling in vivo was assessed in ASC-deficient and IL-1 receptor antagonist-treated mice. RESULTS: TcdA and TcdB triggered inflammasome activation and IL-1beta secretion in macrophages and human mucosal biopsy specimens. Deletion of Nlrp3 decreased, whereas deletion of ASC completely abolished, toxin-induced IL-1beta release. TcdB-induced IL-1beta release required recognition of the full-length toxin but not its enzymatic function. In vivo, deletion of ASC significantly reduced toxin-induced inflammation and damage, an effect that was mimicked by pretreatment with the IL-1 receptor antagonist anakinra. CONCLUSIONS: TcdA and TcdB trigger IL-1beta release by activating an ASC-containing inflammasome, a response that contributes to toxin-induced inflammation and damage in vivo. Pretreating mice with the IL-1 receptor antagonist anakinra afforded the same level of protection that was observed in ASC-/- mice. These data suggest that targeting inflammasome or IL-1beta signaling may represent new therapeutic targets in the treatment of CDAD.

Opportunities to Target Specific Contractile Abnormalities with Smooth Muscle Protein Kinase Inhibitors.

Smooth muscle is a major component of most hollow organ systems (e.g., airways, vasculature, bladder and gut/gastrointestine); therefore, the coordinated regulation of contraction is a key property of smooth muscle. When smooth muscle functions normally, it contributes to general health and wellness, but its dysfunction is associated with morbidity and mortality. Rho-associated protein kinase (ROCK) is central to calcium-independent, actomyosin-mediated contractile force generation in the vasculature, thereby playing a role in smooth muscle contraction, cell motility and adhesion. Recent evidence supports an important role for ROCK in the increased vasoconstriction and remodeling observed in various models of hypertension. This review will provide a commentary on the development of specific ROCK inhibitors and their clinical application. Fasudil will be discussed as an example of bench-to-bedside development of a clinical therapeutic that is used to treat conditions of vascular hypercontractility. Due to the wide spectrum of biological processes regulated by ROCK, many additional clinical indications might also benefit from ROCK inhibition. Apart from the importance of ROCK in smooth muscle contraction, a variety of other protein kinases are known to play similar roles in regulating contractile force. The zipper-interacting protein kinase (ZIPK) and integrin-linked kinase (ILK) are two well-described regulators of contraction. The relative contribution of each kinase to contraction depends on the muscle bed as well as hormonal and neuronal stimulation. Unfortunately, specific inhibitors for ZIPK and ILK are still in the development phase, but the success of fasudil suggests that inhibitors for these other kinases may also have valuable clinical applications. Notably, the directed inhibition of ZIPK with a pseudosubstrate molecule shows unexpected effects on the contractility of gastrointestinal smooth muscle.

The nuclear PP1 interacting protein ZAP3 (ZAP) is a putative nucleoside kinase that complexes with SAM68, CIA, NF110/45, and HNRNP-G.

The targeting of protein kinases and phosphatases is fundamental to their roles as cellular regulators. The type one serine/threonine protein phosphatase (PP1) is enriched in the nucleus, yet few nuclear PP1 targeting subunits have been described and characterized. Here we show that the human protein, ZAP3 (also known as ZAP), is localized to the nucleus, that it is expressed in all mammalian tissues examined, and docks to PP1 through an RVRW motif located in its highly conserved carboxy-terminus. Proteomic analysis of a ZAP3 complex revealed that in addition to binding PP1, ZAP3 complexes with CIA (or nuclear receptor co-activator 5) and the RNA binding proteins hnRNP-G, SAM68 and NF110/45, but loses affinity for SAM68 and hnRNP-G upon digestion of endogenous nucleic acid. Bioinformatics has revealed that the conserved carboxy-terminus is orthologous to T4- and mammalian polynucleotide kinases with residues necessary for kinase activity maintained throughout evolution. Furthermore, the substrate binding pocket of uridine-cytidine kinase (or uridine kinase) has localized sequence similarity with ZAP3, suggesting uridine or cytidine as possible ZAP3 substrates. Most polynucleotide kinases have a phosphohydrolase domain in conjunction with their kinase domain. In ZAP3, although this domain is present, it now appears degenerate and functions to bind PP1 through an RVRW docking site located within the domain.