Polypyrimidine tract binding protein 1 (PTBP1) contains a novel regulatory sequence, the rBH3, that binds the prosurvival protein MCL1.

The maturation of RNA from its nascent transcription to ultimate utilization (e.g., translation, miR-mediated RNA silencing, etc.) involves an intricately coordinated series of biochemical reactions regulated by RNA-binding proteins (RBPs). Over the past several decades, there has been extensive effort to elucidate the biological factors that control specificity and selectivity of RNA target binding and downstream function. Polypyrimidine tract binding protein 1 (PTBP1) is an RBP that is involved in all steps of RNA maturation and serves as a key regulator of alternative splicing, and therefore, understanding its regulation is of critical biologic importance. While several mechanisms of RBP specificity have been proposed (e.g., cell-specific expression of RBPs and secondary structure of target RNA), recently, protein-protein interactions with individual domains of RBPs have been suggested to be important determinants of downstream function. Here, we demonstrate a novel binding interaction between the first RNA recognition motif 1 (RRM1) of PTBP1 and the prosurvival protein myeloid cell leukemia-1 (MCL1). Using both in silico and in vitro analyses, we demonstrate that MCL1 binds a novel regulatory sequence on RRM1. NMR spectroscopy reveals that this interaction allosterically perturbs key residues in the RNA-binding interface of RRM1 and negatively impacts RRM1 association with target RNA. Furthermore, pulldown of MCL1 by endogenous PTBP1 verifies that these proteins interact in an endogenous cellular environment, establishing the biological relevance of this binding event. Overall, our findings suggest a novel mechanism of regulation of PTBP1 in which a protein-protein interaction with a single RRM can impact RNA association.

Reviewing PTBP1 Domain Modularity in the Pre-Genomic Era: A Foundation to Guide the Next Generation of Exploring PTBP1 Structure-Function Relationships.

Polypyrimidine tract binding protein 1 (PTBP1) is one of the most well-described RNA binding proteins, known initially for its role as a splicing repressor before later studies revealed its numerous roles in RNA maturation, stability, and translation. While PTBP1's various biological roles have been well-described, it remains unclear how its four RNA recognition motif (RRM) domains coordinate these functions. The early PTBP1 literature saw extensive effort placed in detailing structures of each of PTBP1's RRMs, as well as their individual RNA sequence and structure preferences. However, limitations in high-throughput and high-resolution genomic approaches (i.e., next-generation sequencing had not yet been developed) precluded the functional translation of these findings into a mechanistic understanding of each RRM's contribution to overall PTBP1 function. With the emergence of new technologies, it is now feasible to begin elucidating the individual contributions of each RRM to PTBP1 biological functions. Here, we review all the known literature describing the apo and RNA bound structures of each of PTBP1's RRMs, as well as the emerging literature describing the dependence of specific RNA processing events on individual RRM domains. Our goal is to provide a framework of the structure-function context upon which to facilitate the interpretation of future studies interrogating the dynamics of PTBP1 function.

The GTPase Rab27b regulates the release, autophagic clearance, and toxicity of α-synuclein.

α-Synuclein (αsyn) is the primary component of proteinaceous aggregates termed Lewy bodies that pathologically define synucleinopathies including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). αsyn is hypothesized to spread through the brain in a prion-like fashion by misfolded protein forming a template for aggregation of endogenous αsyn. The cell-to-cell release and uptake of αsyn are considered important processes for its prion-like spread. Rab27b is one of several GTPases essential to the endosomal-lysosomal pathway and is implicated in protein secretion and clearance, but its role in αsyn spread has yet to be characterized. In this study, we used a paracrine αsyn in vitro neuronal model to test the impact of Rab27b on αsyn release, clearance, and toxicity. shRNA-mediated knockdown (KD) of Rab27b increased αsyn-mediated paracrine toxicity. Rab27b reduced αsyn release primarily through nonexosomal pathways, but the αsyn released after Rab27b KD was of higher-molecular-weight species, as determined by size-exclusion chromatography. Rab27b KD increased intracellular levels of insoluble αsyn and led to an accumulation of endogenous light chain 3 (LC3)-positive puncta. Rab27b KD also decreased LC3 turnover after treatment with an autophagosome-lysosome fusion inhibitor, chloroquine, indicating that Rab27b KD induces a defect in autophagic flux. Rab27b protein levels were increased in brain lysates obtained from postmortem tissues of individuals with PD and DLB compared with healthy controls. These data indicate a role for Rab27b in the release, clearance, and toxicity of αsyn and, ultimately, in the pathogenesis of synucleinopathies.

Specific inhibition of DPY30 activity by ASH2L-derived peptides suppresses blood cancer cell growth.

DPY30 facilitates H3K4 methylation by directly binding to ASH2L in the SET1/MLL complexes and plays an important role in hematologic malignancies. However, the domain on DPY30 that regulates cancer growth is not evident, and the potential of pharmacologically targeting this chromatin modulator to inhibit cancer has not been explored. Here we have developed a peptide-based strategy to specifically target DPY30 activity. We have designed cell-penetrating peptides derived from ASH2L that can either bind to DPY30 or show defective or enhanced binding to DPY30. The DPY30-binding peptides specifically inhibit DPY30's activity in interacting with ASH2L and enhancing H3K4 methylation. Treatment with the DPY30-binding peptides significantly inhibited the growth of MLL-rearranged leukemia and other MYC-dependent hematologic cancer cells. We also revealed subsets of genes that may mediate the effect of the peptides on cancer cell growth, and showed that the DPY30-binding peptide sensitized leukemia to other types of epigenetic inhibitors. These results strongly support a critical role of the ASH2L-binding groove of DPY30 in promoting blood cancers, and demonstrate a proof-of-principle for the feasibility of pharmacologically targeting the ASH2L-binding groove of DPY30 for potential cancer inhibition.

14-3-3 Proteins Reduce Cell-to-Cell Transfer and Propagation of Pathogenic α-Synuclein.

α-Synuclein (αsyn) is the key protein that forms neuronal aggregates in the neurodegenerative disorders Parkinson's disease (PD) and dementia with Lewy bodies. Recent evidence points to the prion-like spread of αsyn from one brain region to another. Propagation of αsyn is likely dependent on release, uptake, and misfolding. Under normal circumstances, this highly expressed brain protein functions normally without promoting pathology, yet the underlying endogenous mechanisms that prevent αsyn spread are not understood. 14-3-3 proteins are highly expressed brain proteins that have chaperone function and regulate protein trafficking. In this study, we investigated the potential role of the 14-3-3 proteins in the regulation of αsyn spread using two models of αsyn spread. In a paracrine αsyn model, 14-3-3θ promoted release of αsyn complexed with 14-3-3θ. Despite higher amounts of released αsyn, extracellular αsyn showed reduced oligomerization and seeding capability, reduced internalization, and reduced toxicity in primary mixed-gender mouse neurons. 14-3-3 inhibition reduced the amount of αsyn released, yet released αsyn was more toxic and demonstrated increased oligomerization, seeding capability, and internalization. In the preformed fibril model, 14-3-3 θ reduced αsyn aggregation and neuronal death, whereas 14-3-3 inhibition enhanced αsyn aggregation and neuronal death in primary mouse neurons. 14-3-3s blocked αsyn spread to distal chamber neurons not exposed directly to fibrils in multichamber, microfluidic devices. These findings point to 14-3-3s as a direct regulator of αsyn propagation, and suggest that dysfunction of 14-3-3 function may promote αsyn pathology in PD and related synucleinopathies.SIGNIFICANCE STATEMENT Transfer of misfolded aggregates of α-synuclein from one brain region to another is implicated in the pathogenesis of Parkinson's disease and other synucleinopathies. This process is dependent on active release, internalization, and misfolding of α-synuclein. 14-3-3 proteins are highly expressed chaperone proteins that interact with α-synuclein and regulate protein trafficking. We used two different models in which toxicity is associated with cell-to-cell transfer of α-synuclein to test whether 14-3-3s impact α-synuclein toxicity. We demonstrate that 14-3-3θ reduces α-synuclein transfer and toxicity by inhibiting oligomerization, seeding capability, and internalization of α-synuclein, whereas 14-3-3 inhibition accelerates the transfer and toxicity of α-synuclein in these models. Dysfunction of 14-3-3 function may be a critical mechanism by which α-synuclein propagation occurs in disease.

UBC9 Mutant Reveals the Impact of Protein Dynamics on Substrate Selectivity and SUMO Chain Linkages.

SUMO, a conserved ubiquitin-like protein, is conjugated to a multitude of cellular proteins to maintain genomic integrity and resist genotoxic stress. Studies of the SUMO E2 conjugating enzyme mutant, UBC9P123L, suggested that altered substrate specificity enhances cell sensitivity to DNA damaging agents. Using nuclear magnetic resonance chemical shift studies, we confirm that the mutation does not alter the core globular fold of UBC9, while 15N relaxation measurements demonstrate mutant-induced stabilization of distinct chemical states in residues near the active site cysteine and substrate recognition motifs. We further demonstrate that the P123L substitution induces a switch from the preferential addition of SUMO to lysine residues in unstructured sites to acceptor lysines embedded in secondary structures, thereby also inducing alterations in SUMO chain linkages. Our results provide new insights regarding the impact that structural dynamics of UBC9 have on substrate selection and specifically SUMO chain formation. These findings highlight the potential contribution of nonconsensus SUMO targets and/or alternative SUMO chain linkages on DNA damage response and chemotherapeutic sensitivity.

IgA1 hinge-region clustered glycan fidelity is established early during semi-ordered glycosylation by GalNAc-T2.

GalNAc-type O-glycans are often added to proteins post-translationally in a clustered manner in repeat regions of proteins, such as mucins and IgA1. Observed IgA1 glycosylation patterns show that glycans occur at similar sites with similar structures. It is not clear how the sites and number of glycans added to IgA1, or other proteins, can follow a conservative process. GalNAc-transferases initiate GalNAc-type glycosylation. In IgA nephropathy, an autoimmune disease, the sites and O-glycan structures of IgA1 hinge-region are altered, giving rise to a glycan autoantigen. To better understand how GalNAc-transferases determine sites and densities of clustered O-glycans, we used IgA1 hinge-region (HR) segment as a probe. Using LC-MS, we demonstrated a semi-ordered process of glycosylation by GalNAc-T2 towards the IgA1 HR. The catalytic domain was responsible for selection of four initial sites based on amino-acid sequence recognition. Both catalytic and lectin domains were involved in multiple second site-selections, each dependent on initial site-selection. Our data demonstrated that multiple start-sites and follow-up pathways were key to increasing the number of glycans added. The lectin domain predominately enhanced IgA1 HR glycan density by increasing synthesis pathway exploration by GalNAc-T2. Our data indicated a link between site-specific glycan addition and clustered glycan density that defines a mechanism of how conserved clustered O-glycosylation patterns and glycoform populations of IgA1 can be controlled by GalNAc-T2. Together, these findings characterized a correlation between glycosylation pathway diversity and glycosylation density, revealing mechanisms by which a single GalNAc-T isozyme can limit and define glycan heterogeneity in a disease-relevant context.

Post-Transcriptional Regulation of Anti-Apoptotic BCL2 Family Members.

Anti-apoptotic B cell lymphoma 2 (BCL2) family members (BCL2, MCL1, BCLxL, BCLW, and BFL1) are key players in the regulation of intrinsic apoptosis. Dysregulation of these proteins not only impairs normal development, but also contributes to tumor progression and resistance to various anti-cancer therapies. Therefore, cells maintain strict control over the expression of anti-apoptotic BCL2 family members using multiple mechanisms. Over the past two decades, the importance of post-transcriptional regulation of mRNA in controlling gene expression and its impact on normal homeostasis and disease have begun to be appreciated. In this review, we discuss the RNA binding proteins (RBPs) and microRNAs (miRNAs) that mediate post-transcriptional regulation of the anti-apoptotic BCL2 family members. We describe their roles and impact on alternative splicing, mRNA turnover, and mRNA subcellular localization. We also point out the importance of future studies in characterizing the crosstalk between RBPs and miRNAs in regulating anti-apoptotic BCL2 family member expression and ultimately apoptosis.

l-2-Hydroxyglutarate remodeling of the epigenome and epitranscriptome creates a metabolic vulnerability in kidney cancer models.

Tumor cells are known to undergo considerable metabolic reprogramming to meet their unique demands and drive tumor growth. At the same time, this reprogramming may come at a cost with resultant metabolic vulnerabilities. The small molecule l-2-hydroxyglutarate (l-2HG) is elevated in the most common histology of renal cancer. Similarly to other oncometabolites, l-2HG has the potential to profoundly impact gene expression. Here, we demonstrate that l-2HG remodels amino acid metabolism in renal cancer cells through combined effects on histone methylation and RNA N6-methyladenosine. The combined effects of l-2HG result in a metabolic liability that renders tumors cells reliant on exogenous serine to support proliferation, redox homeostasis, and tumor growth. In concert with these data, high-l-2HG kidney cancers demonstrate reduced expression of multiple serine biosynthetic enzymes. Collectively, our data indicate that high-l-2HG renal tumors could be specifically targeted by strategies that limit serine availability to tumors.

Phylogenetic analysis of the MCL1 BH3 binding groove and rBH3 sequence motifs in the p53 and INK4 protein families.

B-cell lymphoma 2 (Bcl-2) proteins are central, conserved regulators of apoptosis. Bcl-2 family function is regulated by binding interactions between the Bcl-2 homology 3 (BH3) motif in pro-apoptotic family members and the BH3 binding groove found in both the pro-apoptotic effector and anti-apoptotic Bcl-2 family members. A novel motif, the reverse BH3 (rBH3), has been shown to interact with the anti-apoptotic Bcl-2 homolog MCL1 (Myeloid cell leukemia 1) and have been identified in the p53 homolog p73, and the CDK4/6 (cyclin dependent kinase 4/6) inhibitor p18INK4c, (p18, cyclin-dependent kinase 4 inhibitor c). To determine the conservation of rBH3 motif, we first assessed conservation of MCL1's BH3 binding groove, where the motif binds. We then constructed neighbor-joining phylogenetic trees of the INK4 and p53 protein families and analyzed sequence conservation using sequence logos of the rBH3 locus. This showed the rBH3 motif is conserved throughout jawed vertebrates p63 and p73 sequences and in chondrichthyans, amphibians, mammals, and some reptiles in p18. Finally, a potential rBH3 motif was identified in mammalian and osteichthyan p19INK4d (p19, cyclin dependent kinase 4 inhibitor d). These findings demonstrate that the interaction between MCL1 and other cellular proteins mediated by the rBH3 motif may be conserved throughout jawed vertebrates.

Myeloid Cell Leukemia 1 Small Molecule Inhibitor S63845 Synergizes with Cisplatin in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive cancer that lacks specific molecular targets that are often used for therapy. The refractory rate of TNBC to broad-spectrum chemotherapy remains high; however, the combination of newly developed treatments with the current standard of care has delivered promising anti-tumor effects. One mechanism employed by TNBC to avoid cell death is the increased expression of the anti-apoptotic protein, myeloid cell leukemia 1 (MCL1). Multiple studies have demonstrated that increased MCL1 expression enables resistance to platinum-based chemotherapy. In addition to suppressing apoptosis, we recently demonstrated that MCL1 also binds and negatively regulates the transcriptional activity of TP73. TP73 upregulation is a critical driver of cisplatin-induced DNA damage response, and ultimately, cell death. We therefore sought to determine if the coadministration of an MCL1-targeted inhibitor with cisplatin could produce a synergistic response in TNBC. This study demonstrates that the MCL1 inhibitor, S63845, combined with cisplatin synergizes by inducing apoptosis while also decreasing proliferation in a subset of TNBC cell lines. The use of combined MCL1 inhibitors with cisplatin in TNBC effectively initiates TAp73 anti-tumor effects on cell cycle arrest and apoptosis. This observation provides a molecular profile that can be exploited to identify sensitive TNBCs.

Identification of a novel Mcl-1 protein binding motif.

Recent characterization of Mcl-1 as the primary anti-apoptotic Bcl-2 family member expressed in solid tumors, coupled with its ability to enable therapeutic resistance, has provided the impetus for further study into how Mcl-1 is involved in apoptosis signaling. Here, we employ Sabutoclax, a potent and effective Mcl-1 antagonist, as a competing agent to screen a randomized 12-residue phage display library for peptides that bind strongly to the Bcl-2 homology 3 (BH3) binding groove of Mcl-1. Although the screen identified a number of α-helical peptides with canonical BH3 domain sequences, it also isolated a pair of unique peptide sequences. These sequences exhibit a reverse organization of conserved hydrophobic and acidic residues when compared with canonical BH3 sequences, and we therefore refer to them as reverse BH3 (rBH3) peptides. Furthermore, studies of the rBH3 peptides using NMR spectroscopy, fluorescence polarization displacement assays, and alanine scanning data all suggest that they bind to the BH3 binding groove of Mcl-1 selectively over Bcl-x(L). A search for proteins containing the rBH3 motif has identified a number of interesting Mcl-1 protein partners, some of which have previously been associated with apoptosis regulation involving Mcl-1. These findings provide insights into the development of more specific Mcl-1 antagonists and open the way to the identification of a previously unknown family of apoptosis-regulating and Mcl-1 interacting proteins.

The multiple mechanisms of MCL1 in the regulation of cell fate.

MCL1 (myeloid cell leukemia-1) is a widely recognized pro-survival member of the Bcl-2 (B-cell lymphoma protein 2) family and a promising target for cancer therapy. While the role MCL1 plays in apoptosis is well defined, its participation in emerging non-apoptotic signaling pathways is only beginning to be appreciated. Here, we synthesize studies characterizing MCL1s influence on cell proliferation, DNA damage response, autophagy, calcium handling, and mitochondrial quality control to highlight the broader scope that MCL1 plays in cellular homeostasis regulation. Throughout this review, we discuss which pathways are likely to be impacted by emerging MCL1 inhibitors, as well as highlight non-cancerous disease states that could deploy Bcl-2 homology 3 (BH3)-mimetics in the future.

MCL1 binding to the reverse BH3 motif of P18INK4C couples cell survival to cell proliferation.

Commitment to cell cycle entry and cellular duplication is a tightly coordinated and regulated process. Once initiated, a series of multiple checkpoints ensure both accurate genomic replication and chromosomal separation. In the event of unsuccessful cell division, parallel pathways exist that induce the cell to undergo programmed cell death, or apoptosis. At the center of such stress-induced, intrinsic apoptotic regulation lies the BCL2 family of pro- and anti-apoptotic regulatory proteins. In a proliferative state the balance of pro- and anti-apoptotic signaling proteins would be expected to favor an excess population of anti-apoptotic members. While the anti-apoptotic BCL2 family member, MCL1, has been identified to oversee mitotic progression, direct communication between the BCL2 family and cell proliferation has not been observed. In this study, we demonstrate a direct protein-protein interaction between MCL1 and the G1/S checkpoint protein, P18INK4C. This interaction is mediated by a reverse BH3 (rBH3) motif located in P18INK4C's C-terminal ankyrin repeat. MCL1 is further shown to decrease P18INK4C expression and thereby regulate cell cycle entry in a retinoblastoma (RB1)-dependent manner. Our findings establish a mechanism for translation independent and direct communication between the BCL2 family regulation of apoptosis and CDK4/6-RB regulation of early G1/S transition during cellular division/growth.

MCL1 binds and negatively regulates the transcriptional function of tumor suppressor p73.

MCL1, an anti-apoptotic protein that controls chemosensitivity and cell fate through its regulation of intrinsic apoptosis, has been identified as a high-impact target in anti-cancer therapeutic development. With MCL1-specific inhibitors currently in clinical trials, it is imperative that we understand the roles that MCL1 plays in cells, especially when targeting the Bcl-2 homology 3 (BH3) pocket, the central region of MCL1 that mediates apoptotic regulation. Here, we establish that MCL1 has a direct role in controlling p73 transcriptional activity, which modulates target genes associated with DNA damage response, apoptosis, and cell cycle progression. This interaction is mediated through the reverse BH3 (rBH3) motif in the p73 tetramerization domain, which restricts p73 assembly on DNA. Here, we provide a novel mechanism for protein-level regulation of p73 transcriptional activity by MCL1, while also framing a foundation for studying MCL1 inhibitors in combination with platinum-based chemotherapeutics. More broadly, this work expands the role of Bcl-2 family signaling beyond cell fate regulation.

Novel EGFR ectodomain mutations associated with ligand-independent activation and cetuximab resistance in head and neck cancer.

Epidermal growth factor receptor (EGFR) is a pro-tumorigenic receptor tyrosine kinase that facilitates growth for cancer cells that overexpress the receptor. Monoclonal anti-EGFR antibody Cetuximab (CTX) provides significant clinical benefit in patients with head and neck squamous cell carcinoma (HNSCC). Missense mutations in the ectodomain (ECD) of EGFR can be acquired under CTX treatment and mimic the effect of large deletions on spontaneous untethering and activation of the receptor. Little is known about the contribution of EGFR ECD mutations to EGFR activation and CTX resistance in HNSCC. We identified two concurrent non-synonymous missense mutations (G33S and N56K) mapping to domain I in or near the EGF binding pocket of the EGFR ECD in patient-derived HNSCC cells that were selected for CTX resistance through repeated exposure to the agent in an effort to mimic what may occur clinically. Structural modeling predicted that the G33S and N56K mutants would restrict adoption of a fully closed (tethered) and inactive EGFR conformation while not permitting association of EGFR with the EGF ligand or CTX. Binding studies confirmed that the mutant, untethered receptor displayed reduced affinity for both EGF and CTX but demonstrated sustained activation and presence at the cell surface with diminished internalization and sorting for endosomal degradation, leading to persistent downstream AKT signaling. Our results demonstrate that HNSCC cells can select for EGFR ECD mutations under CTX exposure that converge to trap the receptor in an open, ligand-independent, constitutively activated state. These mutants impede the receptor's competence to bind CTX possibly explaining certain cases of CTX treatment-induced or de novo resistance to CTX.

A cell-penetrating MARCKS mimetic selectively triggers cytolytic death in glioblastoma.

Glioblastoma (GBM) is an aggressive malignancy with limited effectiveness of standard of care therapies including surgery, radiation, and temozolomide chemotherapy necessitating novel therapeutics. Unfortunately, GBMs also harbor several signaling alterations that protect them from traditional therapies that rely on apoptotic programmed cell death. Because almost all GBM tumors have dysregulated phosphoinositide signaling as part of that process, we hypothesized that peptide mimetics derived from the phospholipid binding domain of Myristoylated alanine-rich C-kinase substrate (MARCKS) could serve as a novel GBM therapeutic. Using molecularly classified patient-derived xenograft (PDX) lines, cultured in stem-cell conditions, we demonstrate that cell permeable MARCKS effector domain (ED) peptides potently target all GBM molecular classes while sparing normal human astrocytes. Cell death mechanistic testing revealed that these peptides produce rapid cytotoxicity in GBM that overcomes caspase inhibition. Moreover, we identify a GBM-selective cytolytic death mechanism involving plasma membrane targeting and intracellular calcium accumulation. Despite limited relative partitioning to the brain, tail-vein peptide injection revealed tumor targeting in intracranially implanted GBM PDX. These results indicate that MARCKS ED peptide therapeutics may overcome traditional GBM resistance mechanisms, supporting further development of similar agents.

Regulating the BCL2 Family to Improve Sensitivity to Microtubule Targeting Agents.

Chemotherapeutic targeting of microtubules has been the standard of care in treating a variety of malignancies for decades. During mitosis, increased microtubule dynamics are necessary for mitotic spindle formation and successful chromosomal segregation. Microtubule targeting agents (MTAs) disrupt the dynamics necessary for successful spindle assembly and trigger programmed cell death (apoptosis). As the critical regulators of apoptosis, anti-apoptotic BCL2 family members are often amplified during carcinogenesis that can result in MTA resistance. This review outlines how BCL2 family regulation is positioned within the context of MTA treatment and explores the potential of combination therapy of MTAs with emerging BCL2 family inhibitors.

NMR structure and functional characterization of a human cancer-related nucleoside triphosphatase.

A screen of the human cancer genome anatomy project (CGAP) database was performed to search for new proteins involved in tumorigenesis. The resulting hits were further screened for recombinant expression, solubility and protein aggregation, which led to the identification of the previously unknown human cancer-related (HCR) protein encoded by the mRNA NM_032324 as a target for structure determination by NMR. The three-dimensional structure of the protein in its complex with ATPgammaS forms a three-layered alpha/beta sandwich, with a central nine-stranded beta-sheet surrounded by five alpha-helices. Sequence and three-dimensional structure comparisons with AAA+ ATPases revealed the presence of Walker A (GPPGVGKT) and Walker B (VCVIDEIG) motifs. Using 1D (31)P-NMR spectroscopy and a coupled enzymatic assay for the determination of inorganic phosphate, we showed that the purified recombinant protein is active as a non-specific nucleoside triphosphatase, with k(cat)=7.6x10(-3) s(-1). The structural basis for the enzymatic activity of HCR-NTPase was further characterized by site-directed mutagenesis of the Walker B motif, which further contributes to making the HCR-NTPase an attractive new target for further biochemical characterization in the context of its presumed role in human tumorigenesis.

Cold-adaptation in sea-water-borne signal proteins: sequence and NMR structure of the pheromone En-6 from the Antarctic ciliate Euplotes nobilii.

Ciliates of Euplotes species constitutively secrete pleiotropic protein pheromones, which are capable to function as prototypic autocrine growth factors as well as paracrine inducers of mating processes. This paper reports the amino acid sequence and the NMR structure of the pheromone En-6 isolated from the antarctic species Euplotes nobilii. The 63-residue En-6 polypeptide chain forms three alpha-helices in positions 18-25, 36-40 and 46-56, which are arranged in an up-down-up three-helix bundle forming the edges of a distorted trigonal pyramid. The base of the pyramid is covered by the N-terminal heptadecapeptide segment, which includes a 3(10)-turn of residues 3-6. This topology is covalently anchored by four long-range disulfide bonds. Comparison with the smaller pheromones of E. raikovi, a closely related species living in temperate waters, shows that the two-pheromone families have the same three-helix bundle architecture. It then appears that cold-adaptation of the En proteins is primarily related to increased lengths of the chain-terminal peptide segments and the surface-exposed loops connecting the regular secondary structures, and to the presence of solvent-exposed clusters of negatively charged side-chains.