Proline-rich Segment Research Articles

Unraveling the Molecular Complexity of N-Terminus Huntingtin Oligomers: Insights into Polymorphic Structures.

Huntington's disease (HD) is a fatal neurodegenerative disorder resulting from an abnormal expansion of polyglutamine (polyQ) repeats in the N-terminus of the huntingtin protein. When the polyQ tract surpasses 35 repeats, the mutated protein undergoes misfolding, culminating in the formation of intracellular aggregates. Research in mouse models suggests that HD pathogenesis involves the aggregation of N-terminal fragments of the huntingtin protein (htt). These early oligomeric assemblies of htt, exhibiting diverse characteristics during aggregation, are implicated as potential toxic entities in HD. However, a consensus on their specific structures remains elusive. Understanding the heterogeneous nature of htt oligomers provides crucial insights into disease mechanisms, emphasizing the need to identify various oligomeric conformations as potential therapeutic targets. Employing coarse-grained molecular dynamics, our study aims to elucidate the mechanisms governing the aggregation process and resultant aggregate architectures of htt. The polyQ tract within htt is flanked by two regions: an N-terminal domain (N17) and a short C-terminal proline-rich segment. We conducted self-assembly simulations involving five distinct N17 + polyQ systems with polyQ lengths ranging from 7 to 45, utilizing the ProMPT force field. Prolongation of the polyQ domain correlates with an increase in β-sheet-rich structures. Longer polyQ lengths favor intramolecular β-sheets over intermolecular interactions due to the folding of the elongated polyQ domain into hairpin-rich conformations. Importantly, variations in polyQ length significantly influence resulting oligomeric structures. Shorter polyQ domains lead to N17 domain aggregation, forming a hydrophobic core, while longer polyQ lengths introduce a competition between N17 hydrophobic interactions and polyQ polar interactions, resulting in densely packed polyQ cores with outwardly distributed N17 domains. Additionally, at extended polyQ lengths, we observe distinct oligomeric conformations with varying degrees of N17 bundling. These findings can help explain the toxic gain-of-function that htt with expanded polyQ acquires.

Insights into the Interaction Landscape of the EVH1 Domain of Mena.

The Enabled/VASP homology 1 (EVH1) domain is a small module that interacts with proline-rich stretches in its ligands and is found in various signaling and scaffolding proteins. Mena, the mammalian homologue of Ena, is involved in diverse actin-associated events, such as membrane dynamics, bacterial motility, and tumor intravasation and extravasation. Two-dimensional (2D) 1H-15N HSQC NMR was used to study Mena EVH1 binding properties, defining the amino acids involved in ligand recognition for the physiological ligands ActA and PCARE, and a synthetic polyproline-inspired small molecule (hereafter inhibitor 6c). Chemical shift perturbations indicated that proline-rich segments bind in the conserved EVH1 hydrophobic cleft. The PCARE-derived peptide elicited more perturbations compared to the ActA-derived peptide, consistent with a previous report of a structural alteration in the solvent-exposed β7-β8 loop. Unexpectedly, EVH1 and the proline-rich segment of PTP1B did not exhibit NMR chemical shift perturbations; however, the high-resolution crystal structure implicated the conserved EVH1 hydrophobic cleft in ligand recognition. Intrinsic steady-state fluorescence and fluorescence polarization assays indicate that residues outside the proline-rich segment enhance the ligand affinity for EVH1 (Kd = 3-8 μM). Inhibitor 6c displayed tighter binding (Kd ∼ 0.3 μM) and occupies the same EVH1 cleft as physiological ligands. These studies revealed that the EVH1 domain enhances ligand affinity through recognition of residues flanking the proline-rich segments. Additionally, a synthetic inhibitor binds more tightly to the EVH1 domain than natural ligands, occupying the same hydrophobic cleft.

Conformational and Solvation Dynamics of an Amyloidogenic Intrinsically Disordered Domain of a Melanosomal Protein.

The conformational plasticity of intrinsically disordered proteins (IDPs) allows them to adopt a range of conformational states that can be important for their biological functions. The driving force for the conformational preference of an IDP emanates from an intricate interplay between chain-chain and chain-solvent interactions. Using ultrafast femtosecond and picosecond time-resolved fluorescence measurements, we characterized the conformational and solvation dynamics around the N- and C-terminal segments of a disordered repeat domain of a melanosomal protein Pmel17 that forms functional amyloid responsible for melanin biosynthesis. Our time-resolved fluorescence anisotropy results revealed slight compaction and slower rotational dynamics around the amyloidogenic C-terminal segment when compared to the proline-rich N-terminal segment of the repeat domain. The compaction of the C-terminal region was also associated with the restrained mobility of hydration water as indicated by our solvation dynamics measurements. Our findings indicate that sequence-dependent chain-solvent interactions govern both the conformational and solvation dynamics that are crucial in directing the conversion of a highly dynamic IDP into an ordered amyloid assembly. Such an interplay of amino acid composition-dependent conformational and solvation dynamics might have important physicochemical consequences in specific water-protein, ion-protein, and protein-protein interactions involved in amyloid formation and phase transitions.

Intersectin-Mediated Clearance of SNARE Complexes Is Required for Fast Neurotransmission

The rapid replenishment of release-ready synaptic vesicles (SVs) at a limiting number of presynaptic release sites is required to sustain high-frequency neurotransmission in CNS neurons. Failure to clear release sites from previously exocytosed material has been shown to impair vesicle replenishment and, therefore, fast neurotransmission. The identity of this material and the machinery that removes it from release sites have remained enigmatic. Here we show that the endocytic scaffold protein intersectin 1 clears release sites by direct SH3 domain-mediated association with a non-canonical proline-rich segment of synaptobrevin assembled into the SNARE complex for neuroexocytosis. Acute structure-based or sustained genetic interference with SNARE complex recognition by intersectin 1 causes a rapid stimulation frequency-dependent depression of neurotransmission due to impaired replenishment of release-ready SVs. These findings identify a key molecular mechanism that underlies exo-endocytic coupling during fast neurotransmitter release at central synapses.

Structure of the RBM7-ZCCHC8 core of the NEXT complex reveals connections to splicing factors.

The eukaryotic RNA exosome participates extensively in RNA processing and degradation. In human cells, three accessory factors (RBM7, ZCCHC8 and hMTR4) interact to form the nuclear exosome targeting (NEXT) complex, which directs a subset of non-coding RNAs for exosomal degradation. Here we elucidate how RBM7 is incorporated in the NEXT complex. We identify a proline-rich segment of ZCCHC8 as the interaction site for the RNA-recognition motif (RRM) of RBM7 and present the crystal structure of the corresponding complex at 2.0 Å resolution. On the basis of the structure, we identify a proline-rich segment within the splicing factor SAP145 with strong similarity to ZCCHC8. We show that this segment of SAP145 not only binds the RRM region of another splicing factor SAP49 but also the RRM of RBM7. These dual interactions of RBM7 with the exosome and the spliceosome suggest a model whereby NEXT might recruit the exosome to degrade intronic RNAs.

Topogenesis and cell surface trafficking of GPR34 are facilitated by positive-inside rule that effects through a tri-basic motif in the first intracellular loop

Protein folding, topogenesis and intracellular targeting of G protein-coupled receptors (GPCRs) must be precisely coordinated to ensure correct receptor localization. To elucidate how different steps of GPCR biosynthesis work together, we investigated the process of membrane topology determination and how it relates to the acquisition of cell surface trafficking competence in human GPR34. By monitoring a fused FLAG-tag and a conformation-sensitive native epitope during the expression of GPR34 mutant panel, a tri-basic motif in the first intracellular loop was identified as the key topogenic signal that dictates the orientation of transmembrane domain-1 (TM1). Charge disruption of the motif perturbed topogenic processes and resulted in the conformational epitope loss, post-translational processing alteration, and trafficking arrest in the Golgi. The placement of a cleavable N-terminal signal sequence as a surrogate topogenic determinant overcame the effects of tri-basic motif mutations and rectified the TM1 orientation; thereby restored the conformational epitope, post-translational modifications, and cell surface trafficking altogether. Progressive N-tail truncation and site-directed mutagenesis revealed that a proline-rich segment of the N-tail and all four cysteines individually located in the four separate extracellular regions must simultaneously reside in the ER lumen to muster the conformational epitope. Oxidation of all four cysteines was necessary for the epitope formation, but the cysteine residues themselves were not required for the trafficking event. The underlying biochemical properties of the conformational epitope was therefore the key to understand mechanistic processes propelled by positive-inside rule that simultaneously regulate the topogenesis and intracellular trafficking of GPR34.

Free-Energy Landscape of the Amino-Terminal Fragment of Huntingtin in Aqueous Solution

The first exon of Huntingtin—a protein with multiple biological functions whose misfolding is related to Huntington’s disease—modulates its localization, aggregation, and function within the cell. It is composed of a 17-amino-acid amphipathic segment (Htt17), an amyloidogenic segment of consecutive glutamines (QN), and a proline-rich segment. Htt17 is of fundamental importance: it serves as a membrane anchor to control the localization of huntingtin, it modulates huntingtin’s function through posttranslational modifications, and it controls the self-assembly of the amyloidogenic QN segment into oligomers and fibrils. Experimentally, the conformational ensemble of the Htt17 monomer, as well as the impact of the polyglutamine and proline-rich segments, remains, however, mostly uncharacterized at the atomic level due to its intrinsic flexibility. Here, we unveil the free-energy landscape of Htt17, Htt17Q17, and Htt17Q17P11 using Hamiltonian replica exchange combined with well-tempered metadynamics. We characterize the free-energy landscape of these three fragments in terms of a few selected collective variables. Extensive simulations reveal that the free energy of Htt17 is dominated by a broad ensemble of configurations that agree with solution NMR chemical shifts. Addition of Q17 at its carboxy-terminus reduces the extent of the main basin to more extended configurations of Htt17 with lower helix propensity. Also, the aliphatic carbons of Q17 partially sequester the nonpolar amino acids of Htt17. For its part, addition of Q17P11 shifts the overall landscape to a more extended and helical Htt17 stabilized by interactions with Q17 and P11, which almost exclusively form a PPII-helix, as well as by intramolecular H-bonds and salt bridges. Our characterization of Huntingtin’s amino-terminus provides insights into the structural origin of its ability to oligomerize and interact with phospholipid bilayers, processes closely linked to the biological functions of this protein.

Structural Determinants of the IF-OF Transition in Human Glucose Transporter GLUT1

Glucose transporters (GLUTs) are membrane proteins that facilitate the diffusion of glucose across the plasma membrane. Recently, the crystal structures of human GLUTs (e.g., GLUT1 and GLUT3) have been resolved in the inward-facing (IF) and outward-facing (OF) states, enabling a detailed study of their transport mechanism.In this work, we aim to characterize the IF-OF transition of GLUT1, the major glucose transporter in the human body. Starting from the GLUT1 IF state, we performed an extensive search for an optimal reaction coordinate for the transition. Employing non-equilibrium simulations, we induced the reorientation of multiple combinations of transmembrane (TM) helices of GLUT1 to trigger the transition to the OF state for each TM combination. The relevance of the reaction coordinates was then assessed by the non-equilibrium work required for the transition, as well as the induced structural rearrangement of GLUT1. As a starting point for the transitions, an OF target model of GLUT1 was first generated based on available structures of its close homologue OF-GLUT3, and further refined once the optimal reaction coordinate was obtained. Based on the minimum non-equilibrium work required to drive the IF-OF transition, we identified a specific set of TM helices whose motion is required to reach the OF state. Using this reaction coordinate, we were able to obtain previously uncharacterized stable GLUT1 OF state, and characterize multiple intermediate states connecting the IF and OF states, allowing us to identify important interhelical interactions and the gating helices suggesting a local gating mechanism apart from global large scale transition involving a proline-rich TM segment that mediates the conformational transition in GLUT1. Given the structural similarity among GLUTs, we expect that the our detailed description of the GLUT1 transition will provide a basis to study other human GLUTs.

The Neuronal Migration Factor srGAP2 Achieves Specificity in Ligand Binding through a Two-Component Molecular Mechanism

srGAP proteins regulate cell migration and morphogenesis by shaping the structure and dynamics of the cytoskeleton and membranes. First discovered as intracellular effectors for the Robo1 axon-guidance receptor, srGAPs were later identified as interacting with several other nuclear and cytoplasmic proteins. In all these cases, the srGAP SH3 domain mediates protein-protein interactions by recognizing a short proline-rich segment on the cognate-binding partner. However, as interactions between the isolated SH3 domain and a selected set of ligands show weak affinity and low specificity, it is not clear how srGAPs are precisely recruited to their signaling sites. Here, we report a two-component molecular mechanism that regulates ligand binding to srGAP2 by on the one hand dramatically tightening their association and on the other, moderately autoinhibiting and restricting binding. Our results allow the design of point mutations for better probing of srGAP2 activities, and may facilitate the identification of new srGAP2 ligands.

The proline-rich region of 18.5 kDa myelin basic protein binds to the SH3-domain of Fyn tyrosine kinase with the aid of an upstream segment to form a dynamic complex invitro.

The intrinsically disordered 18.5 kDa classic isoform of MBP (myelin basic protein) interacts with Fyn kinase during oligodendrocyte development and myelination. It does so primarily via a central proline-rich SH3 (Src homology 3) ligand (T92–R104, murine 18.5 kDa MBP sequence numbering) that is part of a molecular switch due to its high degree of conservation and modification by MAP (mitogen-activated protein) and other kinases, especially at residues T92 and T95. Here, we show using co-transfection experiments of an early developmental oligodendroglial cell line (N19) that an MBP segment upstream of the primary ligand is involved in MBP–Fyn–SH3 association in cellula. Using solution NMR spectroscopy in vitro, we define this segment to comprise MBP residues (T62–L68), and demonstrate further that residues (V83–P93) are the predominant SH3-target, assessed by the degree of chemical shift change upon titration. We show by chemical shift index analysis that there is no formation of local poly-proline type II structure in the proline-rich segment upon binding, and by NOE (nuclear Overhauser effect) and relaxation measurements that MBP remains dynamic even while complexed with Fyn–SH3. The association is a new example first of a non-canonical SH3-domain interaction and second of a fuzzy MBP complex.

Molecular characterization of WDCP, a novel fusion partner for the anaplastic lymphoma tyrosine kinase ALK.

Anaplastic lymphoma kinase (ALK) is a member of the receptor tyrosine kinase superfamily. The ALK gene is a site of frequent mutation and chromosomal rearrangement in various types of human cancers. A novel chromosomal translocation was recently identified in human colorectal cancer between the ALK gene and chromosome 2, open reading frame 44 (C2orf44), a gene of unknown function. As a first step in understanding the oncogenic properties of this fusion protein, C2orf44 cDNA was cloned and the encoded protein was characterized, which was designated as WD repeat and coiled coil containing protein (WDCP). A C-terminal proline-rich segment in WDCP was shown to mediate binding to the Src homology 3 domain of the Src family kinase hematopoietic cell kinase (Hck). Co-expression with Hck lead to tyrosine phosphorylation of WDCP. Chromatographic fractionation of WDCP-containing lysates indicates that the protein exists as an oligomer in mammalian cells. These results suggest that, in the context of the ALK-C2orf44 gene fusion, WDCP imposes an oligomeric structure on ALK that results in constitutive kinase activation and signaling.

Probing the interaction between U24 and the SH3 domain of Fyn tyrosine kinase.

The putative membrane protein U24 from HHV-6A shares a seven-residue sequence identity (which includes a PxxP motif) with myelin basic protein (MBP), a protein responsible for the compaction of the myelin sheath in the central nervous system. U24 from HHV-6A also shares a PPxY motif with U24 from the related virus HHV-7, allowing them both to block early endosomal recycling. Recently, MBP has been shown to have protein-protein interactions with a range of proteins, including proteins containing SH3 domains. Given that this interaction is mediated by the proline-rich segment in MBP, and that similar proline-rich segments are found in U24, we investigate here whether U24 also interacts with SH3 domain-containing proteins and what the nature of that interaction might be. The implications of a U24-Fyn tyrosine kinase SH3 domain interaction are discussed in terms of the hypothesis that U24 may function like MBP through molecular mimicry, potentially contributing to the disease state of multiple sclerosis or other demyelinating disorders.

Mapping of Apoptin-interaction with BCR-ABL1, and development of apoptin-based targeted therapy

Majority of chronic myeloid leukemia patients experience an adequate therapeutic effect from imatinib however, 26-37% of patients discontinue imatinib therapy due to a suboptimal response or intolerance. Here we investigated derivatives of apoptin, a chicken anemia viral protein with selective toxicity towards cancer cells, which can be directed towards inhibiting multiple hyperactive kinases including BCR-ABL1. Our earlier studies revealed that a proline-rich segment of apoptin interacts with the SH3 domain of fusion protein BCR-ABL1 (p210) and acts as a negative regulator of BCR-ABL1 kinase and its downstream targets. In this study we show for the first time, the therapeutic potential of apoptin-derived decapeptide for the treatment of CML by establishing the minimal region of apoptin interaction domain with BCR-ABL1. We further show that the apoptin decapeptide is able to inhibit BCR-ABL1 down stream target c-Myc with a comparable efficacy to full-length apoptin and Imatinib. The synthetic apoptin is able to inhibit cell proliferation in murine (32Dp210), human cell line (K562), and ex vivo in both imatinib-resistant and imatinib sensitive CML patient samples. The apoptin based single or combination therapy may be an additional option in CML treatment and eventually be feasible as curative therapy.

Solution Nuclear Magnetic Resonance Structure and Molecular Dynamics Simulations of a Murine 18.5 kDa Myelin Basic Protein Segment (S72–S107) in Association with Dodecylphosphocholine Micelles

The 18.5 kDa myelin basic protein (MBP), the most abundant splice isoform in adult mammalian myelin, is a multifunctional, intrinsically disordered protein involved in the development and compaction of the myelin sheath in the central nervous system. A highly conserved central segment comprises a membrane-anchoring amphipathic α-helix followed by a proline-rich segment that represents a ligand for SH3 domain-containing proteins. Here, we have determined using solution nuclear magnetic resonance spectroscopy the structure of a 36-residue peptide fragment of MBP (murine 18.5 kDa residues S72-S107, denoted the α2-peptide) comprising these two structural motifs, in association with dodecylphosphocholine (DPC) micelles. The structure was calculated using CS-ROSETTA (version 1.01) because the nuclear Overhauser effect restraints were insufficient for this protein. The experimental studies were complemented by molecular dynamics simulations of a corresponding 24-residue peptide fragment (murine 18.5 kDa residues E80-G103, denoted the MD-peptide), also in association with a DPC micelle in silico. The experimental and theoretical results agreed well with one another, despite the independence of the starting structures and analyses, both showing membrane association via the amphipathic α-helix, and a sharp bend in the vicinity of the Pro93 residue (murine 18.5 kDa sequence numbering). Overall, the conformations elucidated here show how the SH3 ligand is presented to the cytoplasm for interaction with SH3 domain-containing proteins such as Fyn and contribute to our understanding of myelin architecture at the molecular level.

Inhibiting the Nucleation of Amyloid Structure in a Huntingtin Fragment by Targeting α-Helix-Rich Oligomeric Intermediates

Although oligomeric intermediates are transiently formed in almost all known amyloid assembly reactions, their mechanistic roles are poorly understood. Recently, we demonstrated a critical role for the 17-amino-acid N-terminus (httNT segment) of huntingtin (htt) in the oligomer-mediated amyloid assembly of htt N-terminal fragments. In this mechanism, the httNT segment forms the α-helix-rich core of the oligomers, leaving much of the polyglutamine (polyQ) segment disordered and solvent-exposed. Nucleation of amyloid structure occurs within this local high concentration of disordered polyQ. Here we demonstrate the kinetic importance of httNT self-assembly by describing inhibitory httNT-containing peptides that appear to work by targeting nucleation within the oligomer fraction. These molecules inhibit amyloid nucleation by forming mixed oligomers with the httNT domains of polyQ-containing htt N-terminal fragments. In one class of inhibitors, nucleation is passively suppressed due to the reduced local concentration of polyQ within the mixed oligomer. In the other class, nucleation is actively suppressed by a proline-rich polyQ segment covalently attached to httNT. Studies with d-amino acid and scrambled sequence versions of httNT suggest that inhibition activity is strongly linked to the propensity of inhibitory peptides to make amphipathic α-helices. HttNT derivatives with C-terminal cell-penetrating peptide segments also exhibit excellent inhibitory activity. The httNT-based peptides described here, especially those with protease-resistant d-amino acids and/or with cell-penetrating sequences, may prove useful as lead therapeutics for inhibiting the nucleation of amyloid formation in Huntington's disease.

Novel p65 Binding Glucocorticoid-induced Leucine Zipper Peptide Suppresses Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is a neurological disease characterized by inflammatory demyelination in the brain and spinal cord. The immune-mediated inflammation involves well orchestrated intermolecular interactions that exhibit rapid binding kinetics. The binding interfaces of transient interactions frequently include proline residues that favor an extended conformation for molecular recognition. Linear interface peptides are excellent lead inhibitors of specific protein-protein interactions because only small segments of the interface contribute to the binding. Glucocorticoid-induced leucine zipper (GILZ), a recently identified molecule exhibits potent anti-inflammatory properties. Mechanistically, a proline-rich segment in the carboxyl terminus of GILZ physically binds the p65 subunit of nuclear factor-κB and inhibits the transactivation of inflammatory cytokines. Integrating knowledge derived from the mechanism of action of GILZ with in silico structure prediction identified an immunomodulatory peptide, the GILZ-P. Treatment with GILZ-P exhibited therapeutic efficacy in experimental autoimmune encephalomyelitis, a model for human MS.

GPR56 Regulates VEGF Production and Angiogenesis during Melanoma Progression

Angiogenesis is a critical step during cancer progression. The VEGF is a major stimulator for angiogenesis and is predominantly contributed by cancer cells in tumors. Inhibition of the VEGF signaling pathway has shown promising therapeutic benefits for cancer patients, but adaptive tumor responses are often observed, indicating the need for further understanding of VEGF regulation. We report that a novel G protein-coupled receptor, GPR56, inhibits VEGF production from the melanoma cell lines and impedes melanoma angiogenesis and growth, through the serine threonine proline-rich segment in its N-terminus and a signaling pathway involving protein kinase Cα. We also present evidence that the two fragments of GPR56, which are generated by autocatalyzed cleavage, played distinct roles in regulating VEGF production and melanoma progression. Finally, consistent with its suppressive roles in melanoma progression, the expression levels of GPR56 are inversely correlated with the malignancy of melanomas in human subjects. We propose that components of the GPR56-mediated signaling pathway may serve as new targets for antiangiogenic treatment of melanoma.

Conformational choreography of a molecular switch region in myelin basic protein—Molecular dynamics shows induced folding and secondary structure type conversion upon threonyl phosphorylation in both aqueous and membrane-associated environments

The 18.5kDa isoform of myelin basic protein is essential to maintaining the close apposition of myelin membranes in central nervous system myelin, but its intrinsic disorder (conformational dependence on environment), a variety of post-translational modifications, and a diversity of protein ligands (e.g., actin and tubulin) all indicate it to be multifunctional. We have performed molecular dynamics simulations of a conserved central segment of 18.5kDa myelin basic protein (residues Glu80–Gly103, murine sequence numbering) in aqueous and membrane-associated environments to ascertain the stability of constituent secondary structure elements (α-helix from Glu80–Val91 and extended poly-proline type II from Thr92–Gly103) and the effects of phosphorylation of residues Thr92 and Thr95, individually and together. In aqueous solution, all four forms of the peptide bent in the middle to form a hydrophobic cluster. The phosphorylated variants were stabilized further by electrostatic interactions and formation of β-structures, in agreement with previous spectroscopic data. In simulations performed with the peptide in association with a dimyristoylphosphatidylcholine bilayer, the amphipathic α-helical segment remained stable and membrane-associated, although the degree of penetration was less in the phosphorylated variants, and the tilt of the α-helix with respect to the plane of the membrane also changed significantly with the modifications. The extended segment adjacent to this α-helix represents a putative SH3-ligand and remained exposed to the cytoplasm (and thus accessible to binding partners). The results of these simulations demonstrate how this segment of the protein can act as a molecular switch: an amphipathic α-helical segment of the protein is membrane-associated and presents a subsequent proline-rich segment to the cytoplasm for interaction with other proteins. Phosphorylation of threonyl residues alters the degree of membrane penetration of the α-helix and the accessibility of the proline-rich ligand and can stabilize a β-bend. A bend in this region of 18.5kDa myelin basic protein suggests that the N- and C-termini of the proteins can interact with different leaflets of the myelin membrane and explain how a single protein can bring them close together.

Isolation, characterization and cDNA sequencing of acrosin from turkey spermatozoa

Acrosin (EC 3.4.21.10) is serine proteinase localized in the sperm acrosome and considered to play an essential role in fertilization. In contrast to mammalian, there are only limited data concerning avian acrosin, mostly focused on the characterization of mature enzyme. In the present study, acrosin was isolated from turkey spermatozoa using gel filtration in the presence of 4 M urea at acidic pH. N-terminal Edman sequencing allowed the identification of the first 26 N-terminal amino acids: VVGGTEALHG SWPWIVSIQNPRFAGT. This sequence was used to construct primers and obtain a cDNA sequence from the testis. The amino acid sequence deduced from the cDNA shows that turkey acrosin is initially synthesized as prepro-protein with 19-residue signal peptide. This signal sequence is followed by a 327-residue sequence corresponding to the acrosin zymogen. Turkey proacrosin does not contain a proline-rich segment at the C-terminal portion. Mature turkey acrosin is a two-chain molecule consisting of light and heavy chains and was found to be glycoprotein. The proacrosin/acrosin system exists in turkey spermatozoa and this system can be activated similarly to that of mammals. The high value of association constant strongly suggests that acrosin activity in turkeys can be controlled by a seminal plasma Kazal inhibitor under physiological conditions.

Structure and Function of a G-actin Sequestering Protein with a Vital Role in Malaria Oocyst Development inside the Mosquito Vector

Cyclase-associated proteins (CAPs) are evolutionary conserved G-actin-binding proteins that regulate microfilament turnover. CAPs have a modular structure consisting of an N-terminal adenylate cyclase binding domain, a central proline-rich segment, and a C-terminal actin binding domain. Protozoan parasites of the phylum Apicomplexa, such as Cryptosporidium and the malaria parasite Plasmodium, express small CAP orthologs with homology to the C-terminal actin binding domain (C-CAP). Here, we demonstrate by reverse genetics that C-CAP is dispensable for the pathogenic Plasmodium blood stages. However, c-cap(-) parasites display a complete defect in oocyst development in the insect vector. By trans-species complementation we show that the Cryptosporidium parvum ortholog complements the Plasmodium gene functions. Purified recombinant C. parvum C-CAP protein binds actin monomers and prevents actin polymerization. The crystal structure of C. parvum C-CAP shows two monomers with a right-handed beta-helical fold intercalated at their C termini to form the putative physiological dimer. Our results reveal a specific vital role for an apicomplexan G-actin-binding protein during sporogony, the parasite replication phase that precedes formation of malaria transmission stages. This study also exemplifies how Plasmodium reverse genetics combined with biochemical and structural analyses of orthologous proteins can offer a fast track toward systematic gene characterization in apicomplexan parasites.