Structure and function analysis of the aminophospholipid translocase TAT-1.

The aminophospholipid translocase TAT-1 maintains phosphatidylserine (PS) asymmetry in the plasma membrane and regulates endocytic transport. Despite these important functions, the structure-function relationship of this protein is poorly understood. Taking advantage of the mutations identified by the million mutation project, we investigated the effects of 16 single amino acid substitutions on the two functions of the TAT-1 protein.

Regulation of CED-3 caspase localization and activation by C. elegans nuclear-membrane protein NPP-14.

Caspases are cysteine proteases with critical roles in apoptosis. The Caenorhabditis elegans caspase CED-3 is activated by autocatalytic cleavage, a process enhanced by CED-4. Here we report that the CED-3 zymogen localizes to the perinuclear region in C.

Mitochondrial endonuclease G mediates breakdown of paternal mitochondria upon fertilization.

Mitochondria are inherited maternally in most animals, but the mechanisms of selective paternal mitochondrial elimination (PME) are unknown. While examining fertilization in Caenorhabditis elegans, we observed that paternal mitochondria rapidly lose their inner membrane integrity. CPS-6, a mitochondrial endonuclease G, serves as a paternal mitochondrial factor that is critical for PME.

EFF-1-mediated regenerative axonal fusion requires components of the apoptotic pathway.

Functional regeneration after nervous system injury requires transected axons to reconnect with their original target tissue. Axonal fusion, a spontaneous regenerative mechanism identified in several species, provides an efficient means of achieving target reconnection as a regrowing axon is able to contact and fuse with its own separated axon fragment, thereby re-establishing the original axonal tract. Here we report a molecular characterization of this process in Caenorhabditis elegans, revealing dynamic changes in the subcellular localization of the EFF-1 fusogen after axotomy, and establishing phosphatidylserine (PS) and the PS receptor (PSR-1) as critical components for axonal fusion.

CED-3 caspase acts with miRNAs to regulate non-apoptotic gene expression dynamics for robust development in C. elegans.

Genetic redundancy and pleiotropism have limited the discovery of functions associated with miRNAs and other regulatory mechanisms. To overcome this, we performed an enhancer screen for developmental defects caused by compromising both global miRISC function and individual genes in Caenorhabditis elegans. Among 126 interactors with miRNAs, we surprisingly found the CED-3 caspase that has only been well studied for its role in promoting apoptosis, mostly through protein activation.

Caspase protocols in Caenorhabditis elegans.

Caenorhabditis elegans genome has four genes (ced-3, csp-1, csp-2, and csp-3) encoding caspase-like proteins. Among these four proteins, CED-3 is the most well-known cell-killing caspase. Elucidation of the role of CED-3 as a central component of the apoptotic pathway in C.

Caspase-mediated activation of Caenorhabditis elegans CED-8 promotes apoptosis and phosphatidylserine externalization.

During apoptosis, phosphatidylserine (PS), normally restricted to the inner leaflet of the plasma membrane, is exposed on the surface of apoptotic cells and serves as an 'eat-me' signal to trigger phagocytosis. It is poorly understood how PS exposure is activated in apoptotic cells. Here we report that CED-8, a Caenorhabditis elegans protein implicated in controlling the kinetics of apoptosis and a homologue of the XK family proteins, is a substrate of the CED-3 caspase.

Hepatitis B virus X protein targets the Bcl-2 protein CED-9 to induce intracellular Ca2+ increase and cell death in Caenorhabditis elegans.

HBx is a multifunctional hepatitis B virus (HBV) protein that is crucial for HBV infection and pathogenesis and a contributing cause of hepatocyte carcinogenesis. However, the host targets and mechanisms of action of HBx are poorly characterized. We show here that expression of HBx in Caenorhabditis elegans induces both necrotic and apoptotic cell death, mimicking an early event of liver infection by HBV.

Diverse functions of glycosaminoglycans in infectious diseases.

Glycosaminoglycans (GAGs) are complex carbohydrates that are expressed ubiquitously and abundantly on the cell surface and in the extracellular matrix (ECM). The extraordinary structural diversity of GAGs enables them to interact with a wide variety of biological molecules. Through these interactions, GAGs modulate various biological processes, such as cell adhesion, proliferation and migration, ECM assembly, tissue repair, coagulation, and immune responses, among many others.

Structure-activity relationships of heteroaromatic esters as human rhinovirus 3C protease inhibitors.

Human rhinovirus 3C protease (HRV 3C(pro)) is known to be a promising target for development of therapeutic agents against the common cold because of the importance of the protease in viral replication as well as its expression in a large number of serotypes. To explore non-peptidic inhibitors of HRV 3C(pro), a series of novel heteroaromatic esters was synthesized and evaluated for inhibitory activity against HRV 3C(pro), to determine the structure-activity relationships. The most potent inhibitor, 7, with a 5-bromopyridinyl group, had an IC(50) value of 80nM.

Engineering of protease variants exhibiting altered substrate specificity.

By using an improved genetic screening system, variants of the HAV 3CP protease which exhibit altered P2 specificity were obtained. We randomly mutated the His145, Lys146, Lys147, and Leu155 residues that constitute the S2 pocket of 3CP and then isolated variants that preferred substrates with Gln over the original Thr at the P2 position using a yeast-based screening method. One of the isolated variants cleaved the Gln-containing peptide substrate more efficiently in vitro, proving the efficiency of our method in isolating engineered proteases with desired substrate selectivity.

Development of potent inhibitors of the coxsackievirus 3C protease.

Coxsackievirus B3 (CVB3) 3C protease (3CP) plays essential roles in the viral replication cycle, and therefore, provides an attractive therapeutic target for treatment of human diseases caused by CVB3 infection. CVB3 3CP and human rhinovirus (HRV) 3CP have a high degree of amino acid sequence similarity. Comparative modeling of these two 3CPs revealed one prominent distinction; an Asn residue delineating the S2' pocket in HRV 3CP is replaced by a Tyr residue in CVB3 3CP.

Structure of the CED-4-CED-9 complex provides insights into programmed cell death in Caenorhabditis elegans.

Interplay among four genes--egl-1, ced-9, ced-4 and ced-3--controls the onset of programmed cell death in the nematode Caenorhabditis elegans. Activation of the cell-killing protease CED-3 requires CED-4. However, CED-4 is constitutively inhibited by CED-9 until its release by EGL-1.

Identification of CED-3 substrates by a yeast-based screening method.

Identifying cellular substrates repertoire of individual proteases will facilitate our understanding of their physiological and pathological roles. In this article, we employed a yeast-based screening method to isolate CED-3 substrates. This method uses a transcription factor anchored to the plasma membrane by fusion to a library of cellular protein sequences.

Oxygen-evolving enhancer protein 2 is phosphorylated by glycine-rich protein 3/wall-associated kinase 1 in Arabidopsis.

The Arabidopsis wall-associated receptor kinase, WAK1, is a member of WAK family that links the plasma membrane to the extracellular matrix. A glycine-rich secreted protein, AtGRP-3, was previously shown to regulate WAK1 functions through binding to the extracellular domain of WAK1. In this study, we sought to determine the downstream molecules of the AtGRP-3/WAK1 signaling pathway, by using two-dimensional gel electrophoresis combined with Edman sequencing and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS).