Evaluation of Therapeutics for Advanced-Stage Heart Failure and Other Severely-Debilitating or Life-Threatening Diseases.

Severely-debilitating or life-threatening (SDLT) diseases include conditions in which life expectancy is short or quality of life is greatly diminished despite available therapies. As such, the medical context for SDLT diseases is comparable to advanced cancer and the benefit vs. risk assessment and development of SDLT disease therapeutics should be similar to that of advanced cancer therapeutics. A streamlined development approach would allow patients with SDLT conditions earlier access to therapeutics and increase the speed of progression through development. In addition, this will likely increase the SDLT disease therapeutic pipeline, directly benefiting patients and reducing the economic and societal burden of SDLT conditions. Using advanced-stage heart failure (HF) as an example that illustrates the concepts applicable to other SDLT indications, this article proposes a streamlined development paradigm for SDLT disease therapeutics and recommends development of aligned global regulatory guidance.

Germline BRCA1-2 mutations in non-Ashkenazi families with double primary breast and ovarian cancer.

OBJECTIVE: Ashkenazi women with double primary breast and ovarian cancer have a high prevalence (57%) of germline Jewish founder mutations in the BRCA1 (185delAG, 5382insC) and BRCA2 (6174delT) genes. The purpose of this study was to determine the frequency and type of BRCA1-2 mutations in non-Ashkenazi families with at least one member having double primary breast and ovarian cancer. METHODS: Women at increased risk for cancer based upon their family history were enrolled at the University of Texas Southwestern Familial Cancer Registry between 1992 and 2000. Blood samples from patients desiring genetic testing were sent for complete DNA sequencing of the BRCA1 and BRCA2 genes. Families with a member having both breast and ovarian cancer were identified and clinical data were obtained. RESULTS: Sixty-two (7%) of 900 enrolled families were non-Ashkenazi and had at least one member with double primary breast and ovarian cancer. Twenty-one families had members who underwent genetic testing; 41 did not. Thirteen (62%) families had a germline BRCA1 (n = 11) or BRCA2 (n = 2) mutation; only one Jewish founder mutation (185delAG) was detected. Eight (38%) families tested negative. Six (86%) of seven women undergoing genetic testing who themselves had double primary breast and ovarian cancer were BRCA1-2 mutation carriers. CONCLUSIONS: Germline BRCA1-2 mutations are common in non-Ashkenazi families with a member having double primary breast and ovarian cancer. These mutations occurred throughout both genes, emphasizing the need for comprehensive sequencing. One family had the BRCA2 6985delCT mutation, which lies beyond the "ovarian cancer cluster" region.

In vivo importance of actin nucleotide exchange catalyzed by profilin.

The actin monomer-binding protein, profilin, influences the dynamics of actin filaments in vitro by suppressing nucleation, enhancing nucleotide exchange on actin, and promoting barbed-end assembly. Profilin may also link signaling pathways to actin cytoskeleton organization by binding to the phosphoinositide PIP(2) and to polyproline stretches on several proteins. Although activities of profilin have been studied extensively in vitro, the significance of each of these activities in vivo needs to be tested. To study profilin function, we extensively mutagenized the Saccharomyces cerevisiae profilin gene (PFY1) and examined the consequences of specific point mutations on growth and actin organization. The actin-binding region of profilin was shown to be critical in vivo. act1-157, an actin mutant with an increased intrinsic rate of nucleotide exchange, suppressed defects in actin organization, cell growth, and fluid-phase endocytosis of pfy1-4, a profilin mutant defective in actin binding. In reactions containing actin, profilin, and cofilin, profilin was required for fast rates of actin filament turnover. However, Act1-157p circumvented the requirement for profilin. Based on the results of these studies, we conclude that in living cells profilin promotes rapid actin dynamics by regenerating ATP actin from ADP actin-cofilin generated during filament disassembly.

Profilin is required for optimal actin-dependent transcription of respiratory syncytial virus genome RNA.

Transcription of human respiratory syncytial virus (RSV) genome RNA exhibited an obligatory need for the host cytoskeletal protein actin. Optimal transcription, however, required the participation of another cellular protein that was characterized as profilin by a number of criteria. The amino acid sequence of the protein, purified on the basis of its transcription-optimizing activity in vitro, exactly matched that of profilin. RSV transcription was inhibited 60 to 80% by antiprofilin antibody or poly-L-proline, molecules that specifically bind profilin. Native profilin, purified from extracts of lung epithelial cells by affinity binding to a poly-L-proline matrix, stimulated the actin-saturated RSV transcription by 2.5- to 3-fold. Recombinant profilin, expressed in bacteria, stimulated viral transcription as effectively as the native protein and was also inhibited by poly-L-proline. Profilin alone, in the absence of actin, did not activate viral transcription. It is estimated that at optimal levels of transcription, every molecule of viral genomic RNA associates with approximately the following number of protein molecules: 30 molecules of L, 120 molecules of phosphoprotein P, and 60 molecules each of actin and profilin. Together, these results demonstrated for the first time a cardinal role for profilin, an actin-modulatory protein, in the transcription of a paramyxovirus RNA genome.

Profilin binds proline-rich ligands in two distinct amide backbone orientations.

The actin regulatory protein profilin is targeted to specific cellular regions through interactions with highly proline-rich motifs embedded within its binding partners. New X-ray crystallographic results demonstrate that profilin, like SH3 domains, can bind proline-rich ligands in two distinct amide backbone orientations. By further analogy with SH3 domains, these data suggest that non-proline residues in profilin ligands may dictate the polarity and register of binding, and the detailed organization of the assemblies involving profilin. This degeneracy may be a general feature of modules that bind proline-rich ligands, including WW and EVH1 domains, and has implications for the assembly and activity of macromolecular complexes involved in signaling and the regulation of the actin cytoskeleton.

Crystallization and preliminary X-ray analysis of human platelet profilin complexed with an oligo proline peptide.

Profilin is an actin-monomer binding protein that regulates the distribution and dynamics of the actin cytoskeleton. Profilin binds poly-L-proline and proline-rich peptides in vitro and co-localizes with proline-rich proteins in focal adhesions and at the site of actin tail assembly on the surface of intracellular parasites such as Listeria monocytogenes. The crystallization of the complex between human platelet profilin (HPP) and an L-proline decamer [(Pro)10] is reported here. Diffraction from these crystals is consistent with the space group P21212 with unit-cell constants a = 68.25, b = 97.64, c = 39.10 A. The crystals contain two HPP molecules per asymmetric unit and diffract to 2.2 A.

Structure determination and characterization of Saccharomyces cerevisiae profilin.

The structure of profilin from the budding yeast Saccharomyces cerevisiae has been determined by X-ray crystallography at 2.3 A resolution. The overall fold of yeast profilin is similar to the fold observed for other profilin structures. The interactions of yeast and human platelet profilins with rabbit skeletal muscle actin were characterized by titration microcalorimetry, fluorescence titrations, and nucleotide exchange kinetics. The affinity of yeast profilin for rabbit actin (2.9 microM) is approximately 30-fold weaker than the affinity of human platelet profilin for rabbit actin (0.1 microM), and the relative contributions of entropic and enthalpic terms to the overall free energy of binding are different for the two profilins. The titration of pyrene-labeled rabbit skeletal actin with human profilin yielded a Kd of 2.8 microM, similar to the Kd of 2.0 microM for the interaction between yeast profilin and pyrene-labeled yeast actin. The binding data are discussed in the context of the known crystal structures of profilin and actin, and the residues present at the actin-profilin interface. The affinity of yeast profilin for poly-L-proline was determined from fluorescence measurements and is similar to the reported affinity of Acanthamoeba profilin for poly-L-proline. Yeast profilin was shown to catalyze adenine nucleotide exchange from yeast actin almost 2 orders of magnitude less efficiently than human profilin and rabbit skeletal muscle actin. The in vivo and in vitro properties of yeast profilin mutants with altered poly-L-proline and actin binding sites are discussed in the context of the crystal structure.

The modular structure of actin-regulatory proteins.

Filamentous actin structures possess unique biophysical and biochemical properties and are required for cell locomotion, cell division, compartmentalization and morphological processes. The site-specific assembly and disassembly of these structures are directed by actin-regulatory proteins. This article reviews how structural studies are now defining the atomic details of small modular domains present in actin-regulatory proteins responsible for crosslinking, severing and capping of actin filaments, as well as for localization of actin filament assembly. These studies have identified three modular strategies for the design of proteins that regulate the actin cytoskeleton.

Structure of the profilin-poly-L-proline complex involved in morphogenesis and cytoskeletal regulation.

Profilin, a ubiquitous low molecular weight (13,000-15,000 M(r)) actin binding protein, regulates the formation of F-actin structures in vivo, and is localized to specific cellular regions through interaction with proline-rich sequences. Here we report the 2.2 A X-ray structure of the complex between human platelet profilin (HPP) and a decamer of L-proline (L-Pro10). The L-Pro10 peptide adopts a left-handed type II poly-L-proline helix (PPII) and binds to a highly conserved patch of aromatic amino acids on the surface of profilin. The peptide and actin binding sites reside on orthogonal surfaces, and L-Pro10 binding does not result in a conformational rearrangement of HPP. This structure suggests a mechanism for the localization of profilin and its actin-related activities to sites of actin filament assembly in vivo.

The molecular basis for allergen cross-reactivity: crystal structure and IgE-epitope mapping of birch pollen profilin.

BACKGROUND: The profilins are a group of ubiquitous actin monomer binding proteins that are responsible for regulating the normal distribution of filamentous actin networks in eukaryotic cells. Profilins also bind polyphosphoinositides, which can disrupt the profilin-action complex, and proline-rich ligands which localize profilin to sites requiring extensive actin filament accumulation. Profilins represent cross-reactive allergens for almost 20 % of all pollen allergic patients. RESULTS: We report the X-ray crystal structure of birch pollen profilin (BPP) at 2.4 resolution. The major IgE-reactive epitopes have been mapped and were found to cluster on the N- and C-terminal alpha helices and a segment of the protein containing two strands of the beta sheet. The overall fold of this protein is similar to that of the mammalian and amoeba profilins, however, there is a significant change in the orientation of the N-terminal alpha helix in BPP. This change in orientation alters the topography of a hydrophobic patch on the surface of the molecule, which is thought to be involved in the binding of proline-rich ligands. CONCLUSIONS: Profilin has been identified as an important cross-reactive allergen for patients suffering from multivalent type I allergy. The prevalent epitopic areas are located in regions with conserved sequence and secondary structure and overlap the binding sites for natural profilin ligands, indicating that the native ligand-free profilin acts as the original cross-sensitizing agent. Structural homology indicates that the basic features of the G actin-profilin interaction are conserved in all eukaryotic organisms, but suggests that mechanistic differences in the binding of proline-rich ligands may exist. The structure of BPP provides a molecular basis for understanding allergen cross-reactivity.