Mechanistic roles of tyrosine phosphorylation in reversible amyloids,autoinhibition, and endosomal membrane association of ALIX

Human apoptosis-linked gene-2 interacting protein X (ALIX), a versatile adapter protein, regulates essential cellular processes by shuttling between late endosomal membranes and the cytosol, determined by its interactions with Src kinase. Here, we investigate the molecular basis of these transitions and the effects of tyrosine phosphorylation on the interplay between structure, assembly, and intramolecular and intermolecular interactions of ALIX. As evidenced by transmission electron microscopy, fluorescence and circular dichroism spectroscopy, the proline-rich domain of ALIX, which encodes binding epitopes of multiple cellular partners, formed rope-like β-sheet–rich reversible amyloid fibrils that dissolved upon Src-mediated phosphorylation and were restored on protein-tyrosine phosphatase 1B–mediated dephosphorylation of its conserved tyrosine residues. Analyses of the Bro1 domain of ALIX by solution NMR spectroscopy elucidated the conformational changes originating from its phosphorylation by Src and established that Bro1 binds to hyperphosphorylated proline-rich domain and to analogs of late endosomal membranes via its highly basic surface. These results uncover the autoinhibition mechanism that relocates ALIX to the cytosol and the diverse roles played by tyrosine phosphorylation in cellular and membrane functions of ALIX.     

Caenorhabditis elegans LET-413 Scribble is essential in the epidermis for growth,viability, and directional outgrowth of epithelial seam cells

The conserved adapter protein Scribble (Scrib) plays essential roles in a variety of cellular processes, including polarity establishment, proliferation, and directed cell migration. While the mechanisms through which Scrib promotes epithelial polarity are beginning to be unraveled, its roles in other cellular processes including cell migration remain enigmatic. In C. elegans, the Scrib ortholog LET-413 is essential for apical–basal polarization and junction formation in embryonic epithelia. However, whether LET-413 is required for postembryonic development or plays a role in migratory events is not known. Here, we use inducible protein degradation to investigate the functioning of LET-413 in larval epithelia. We find that LET-413 is essential in the epidermal epithelium for growth, viability, and junction maintenance. In addition, we identify a novel role for LET-413 in the polarized outgrowth of the epidermal seam cells. These stem cell-like epithelial cells extend anterior and posterior directed apical protrusions in each larval stage to reconnect to their neighbors. We show that the role of LET-413 in seam cell outgrowth is likely mediated largely by the junctional component DLG-1 discs large, which we demonstrate is also essential for directed outgrowth of the seam cells. Our data uncover multiple essential functions for LET-413 in larval development and show that the polarized outgrowth of the epithelial seam cells is controlled by LET-413 Scribble and DLG-1 Discs large.     

Purification of a 47-kDa calmodulin-binding polypeptide as an actin-binding protein from Neurospora crassa

We have enriched a 47-kDa polypeptide (p47) from Neurospora crassa on the basis of its affinity to calmodulin. The p47 was purified to homogeneity by chromatography on a Mono S cation exchange column and evidence is presented that the polypeptide co-sediments specifically with F-actin. The intracellular distribution of p47 and actin was also examined using indirect double immunofluorescence staining of cells at different stages of development. Our results suggest that by altering the conformation binding site of actin to p47, calmodulin could play a regulatory role in the polarized hyphal growth of N. crassa.     

The solution structure of small peptides: An IR CD study of aqueous solutions of (L-Ala)n [n = 3, 4, 5, 6] at different temperatures and ionic strengths

The solution conformation of a number of small, linear alanine oligomers was investigated via ir (or vibrational) CD (VCD). We find that these oligopeptides assume distinct solution conformations that depend primarily on chain lengths, and to a lesser degree on temperature, ionic strength, and pH. As expected, the longer chain oligomers exhibit more distinct VCD features and, presumably, more stable solution structures. At the level of the hexamer, however, aggregation of the peptide occurs. The fast time scale of VCD allows solution structures to be detected that may not be observable using slower techniques such as various forms of nmr spectroscopy. The VCD results reported here confirm that it is generally possible to obtain conformational information for small, linear homo- and heterooligopeptides via VCD spectroscopy. In this respect, the sensitivity of VCD is similar to that of electronic CD. Furthermore, the temperature dependence of the VCD results indicate that at elevated temperatures, the increasing number of conformational states results in a loss of discernible conformers, and consequently, a broadening and weakening of the VCD features. © 1998 John Wiley & Sons, Inc. Biopoly 46: 455–463, 1998     

A "stealth effect": adenocarcinoma cells engineered to express TRAIL elude tumor-specific and allogeneic T cell reactions.

BALB/c mammary adenocarcinoma cells engineered to express TNF-related apoptosis-inducing ligand (TRAIL)/APO-2 ligand (APO-2L) on their membrane (TSA-TRAIL) grow with kinetics similar to that of parental cells (TSA-pc) in vitro and in nu/nu mice. In contrast, TSA-TRAIL cells grow faster than TSA-pc in normal BALB/c mice. In DBA/2 mice, which differ from BALB/c mice at minor histocompatibility Ags, they also grow faster and display a higher percentage of tumor takes than TSA-pc. In fully histoincompatible C57BL/6 (B6) mice, TSA-TRAIL cells form evident tumors that are slowly rejected by most mice, but outgrow in a few. In contrast, TSA-pc cells are rejected at once by B6 mice. Since TRAIL/APO-2L induces apoptosis by interacting with a variety of specific receptors, this rapid growth in both syngeneic and allogeneic mice may be the result of an immunosuppressive mechanism. The following evidence supports this hypothesis: 1) TSA-TRAIL cells overcome the strong immunity against TSA-pc cells elicited in BALB/c mice by preimmunization with TSA cells engineered to release IL-4; 2) their rejection by B6 mice does not prime a CTL-mediated memory; 3) thymidine uptake by T lymphocytes unstimulated or stimulated by allogeneic cells is inhibited when TSA-TRAIL cells are added as third party cells; 4) CTL kill TSA-pc but not TSA-TRAIL cells in 48-h assays; and 5) activated lymphocytes interacting with TSA-TRAIL cells in vivo and in vitro undergo apoptosis.     

Food-Entrained Feeding and Locomotor Circadian Rhythms in Rats Under Different Lighting Conditions

It has been suggested that two endogenous timekeeping systems, a light-entrainable pacemaker (LEP) and a food-entrainable pacemaker (FEP), control circadian rhythms. To understand the function and interaction between these two mechanisms better, we studied two behavioral circadian rhythmicities, feeding and locomotor activity, in rats exposed to two conflicting zeitgebers, food restriction and light-dark cycles. For this, the food approaches and wheel-running activity of rats kept under light-dark (LD) 12:12, constant darkness (DD), or constant light (LL) conditions and subjected to different scheduled feeding patterns were continuously recorded. To facilitate comparison of the results obtained under the different lighting conditions, the period of the feeding cycles was set in all three cases about Ih less than the light-entrained or free-running circadian rhythms. The results showed that, depending on the lighting conditions, some components of the feeding and wheel-running circadian rhythms could be entrained by food pulses, while others retained their free-running or light-entrained state. Under LD, food pulses had little influence on the light-entrained feeding and loco-motor rhythms. Under DD, relative coordination between free-running and food-associated rhythms may appear. In both cases, the feeding activity associated with the food pulses could be divided into a prominent phase-dependent peak of activity within the period of food availability and another afterward. Wheel-running activity mainly followed the food pulses. Under LL conditions, the food-entrained activity consisted mainly of feeding and wheel-running anticipatory activity. The results provide new evidence that lighting conditions influence the establishment and persistence of food-entrained circadian rhythms in rats. The existence of two coupled pacemakers, LEP and FEP, or a multioscillatory LEP may both explain our experimental results.     

Protein phosphatase 2A holoenzyme assembly: identification of contacts between B-family regulatory and scaffolding A subunits

Protein serine/threonine phosphatase (PP) 2A is a ubiquitous enzyme with pleiotropic functions. Trimeric PP2A consists of a structural A subunit, a catalytic C subunit, and a variable regulatory subunit. Variable subunits (B, B', and B" families) dictate PP2A substrate specificity and subcellular localization. B-family subunits contain seven WD repeats predicted to fold into a beta-propeller structure. We carried out mutagenesis of Bgamma to identify domains important for association with A and C subunits in vivo. Several internal deletions in Bgamma abolished coimmunoprecipitation of A and C subunits expressed in COS-M6 cells. In contrast, small N- and C-terminal Bgamma deletions had no effect on incorporation into the PP2A heterotrimer. Thus, holoenzyme association of B-family subunits requires multiple, precisely aligned contacts within a core beta-propeller domain. Charge-reversal mutagenesis of Bgamma identified a cluster of conserved critical residues in Bgamma WD repeats 3 and 4. Acidic substitution of paired basic residues in Bgamma (RR165EE) abolished association with wild-type A and C subunits, while fostering incorporation of Bgamma into a PP2A heterotrimer containing an A subunit with an opposite charge-reversal mutation (EE100RR). Thus, binding of A and B subunits requires electrostatic interactions between conserved pairs of glutamates and arginines. By expressing complementary charge-reversal mutants in neuronal PC6-3 cells, we further show that holoenzyme incorporation protects Bgamma from rapid degradation by the ubiquitin/proteasome pathway.