p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death

Autophagic degradation of ubiquitinated protein aggregates is important for cell survival, but it is not known how the autophagic machinery recognizes such aggregates. In this study, we report that polymerization of the polyubiquitin-binding protein p62/SQSTM1 yields protein bodies that either reside free in the cytosol and nucleus or occur within autophagosomes and lysosomal structures. Inhibition of autophagy led to an increase in the size and number of p62 bodies and p62 protein levels. The autophagic marker light chain 3 (LC3) colocalized with p62 bodies and co-immunoprecipitated with p62, suggesting that these two proteins participate in the same complexes. The depletion of p62 inhibited recruitment of LC3 to autophagosomes under starvation conditions. Strikingly, p62 and LC3 formed a shell surrounding aggregates of mutant huntingtin. Reduction of p62 protein levels or interference with p62 function significantly increased cell death that was induced by the expression of mutant huntingtin. We suggest that p62 may, via LC3, be involved in linking polyubiquitinated protein aggregates to the autophagy machinery.     

The ESCRT-III subunit hVps24 is required for degradation but not silencing of the epidermal growth factor receptor

The endosomal sorting complexes required for transport, ESCRT-I, -II, and -III, are thought to mediate the biogenesis of multivesicular endosomes (MVEs) and endosomal sorting of ubiquitinated membrane proteins. Here, we have compared the importance of the ESCRT-I subunit tumor susceptibility gene 101 (Tsg101) and the ESCRT-III subunit hVps24/CHMP3 for endosomal functions and receptor signaling. Like Tsg101, endogenous hVps24 localized mainly to late endosomes. Depletion of hVps24 by siRNA showed that this ESCRT subunit, like Tsg101, is important for degradation of the epidermal growth factor (EGF) receptor (EGFR) and for transport of the receptor from early endosomes to lysosomes. Surprisingly, however, whereas depletion of Tsg101 caused sustained EGF activation of the mitogen-activated protein kinase pathway, depletion of hVps24 had no such effect. Moreover, depletion of Tsg101 but not of hVps24 caused a major fraction of internalized EGF to accumulate in nonacidified endosomes. Electron microscopy of hVps24-depleted cells showed an accumulation of EGFRs in MVEs that were significantly smaller than those in control cells, probably because of an impaired fusion with lyso-bisphosphatidic acid-positive late endosomes/lysosomes. Together, our results reveal functional differences between ESCRT-I and ESCRT-III in degradative protein trafficking and indicate that degradation of the EGFR is not required for termination of its signaling.     

Validation of a pressure diameter method for determining modulus and strain of collagen engagement for long branches of bovine pulmonary arteries

Recent studies have shown that capacitance measurements of large arteries provide better prognosis and diagnosis than tests of resistance alone in pulmonary hypertension (Mahapatra et al., 2006, "Relationship of Pulmonary Arterial Capacitance and Mortality in Idiopathic Pulmonary Arterial Hypertension," J. Am. Coll. Cardiol., 47(4), pp. 799-803; Reuben, 1971, "Compliance of the Human Pulmonary Arterial System in Disease," Circ. Res., 29, pp. 40-50]. Decreased arterial capacitance causes increased load to the heart and is the direct result of increased stiffness and elastic modulus of the arterial wall. Here, we validate a pressure-diameter (PD) method for comparing the elastic modulus and collagen engagement for post-hilar pulmonary arteries with a large range of arterial diameter. The tissue mechanics of the post-hilar arteries are not well-characterized in pulmonary hypertension. It is believed that future studies with this method will provide useful insight into the role of passive tissue mechanics of these arteries in the pathophysiology of pulmonary hypertension, eventually improving clinical diagnosis, prognosis, and treatment. Post-hilar pulmonary arteries, excised from healthy and hypertensive calves and healthy cows, were inflated over a range of 0 [mm Hg] to 110 [mm Hg] in an isolated tissue bath. Internal pressure was recorded with an electric pressure catheter. Artery diameter and longitudinal stretch were recorded photographically. Stress-strain data curves were extracted using Lame's law of thick-walled tubes. Radial strips were removed from each section and tested in a uniaxial (MTS) tester for validation. Both the elastic modulus and collagen engagement strain were similar to results obtained by more traditional means. The average difference between measured values of the two methods for collagen engagement strain was 3.3% of the average value of the engagement strain. The average difference between the measured values of the two methods for modulus of elasticity was 7.4% of the average value of the modulus. The maximum, theoretical, relative error for the stress determined with the PD method was calculated at 20.3%. The PD method proved to be a suitable replacement for uniaxial strain tests in comparing collagen engagement strains. The method allowed faster testing of tissues of multiple diameters, while removing the effect of end conditions. The PD method will be of further utility in continued study of tissue mechanics in pulmonary hypertension studies.     

Nedd4-dependent lysine-11-linked polyubiquitination of the tumour suppressor Beclin 1

Beclin 1, a subunit of the class III phosphatidylinositol 3-kinase complex, is a tumour suppressor with a central role in endocytic trafficking, cytokinesis and the cross-regulation between autophagy and apoptosis. Interestingly, not only reduced expression but also overexpression of Beclin 1 is correlated with cancer development and metastasis. Thus it seems necessary for the cell to balance the protein levels of Beclin 1. In the present study we describe a regulatory link between Beclin 1 and the ubiquitin ligase Nedd4 (neural-precursor-cell-expressed developmentally down-regulated 4). We establish Nedd4 as a novel binding partner of Beclin 1 and demonstrate that Nedd4 polyubiquitinates Beclin 1 with Lys11- and Lys63-linked chains. Importantly, Nedd4 expression controls the stability of Beclin 1, and depletion of the Beclin 1-interacting protein VPS34 causes Nedd4-mediated proteasomal degradation of Beclin 1 via Lys11-linked polyubiquitin chains. Beclin 1 is thus the first tumour suppressor reported to be controlled by Lys11-linked polyubiquitination.     

Mammalian SEPT9 isoforms direct microtubule-dependent arrangements of septin core heteromers

Septin-family proteins assemble into rod-shaped heteromeric complexes that form higher-order arrangements at the cell cortex, where they serve apparently conserved functions as diffusion barriers and molecular scaffolds. There are 13 confirmed septin paralogues in mammals, which may be ubiquitous or tissue specific. Septin hetero-oligomerization appears homology subgroup directed, which in turn determines the subunit arrangement of six- to eight-subunit core heteromers. Here we address functional properties of human SEPT9, which, due to variable mRNA splicing, exists as multiple isoforms that differ between tissues. Myeloid K562 cells express three SEPT9 isoforms, all of which have an equal propensity to hetero-oligomerize with SEPT7-containing hexamers to generate octameric heteromers. However, due to limiting amounts of SEPT9, K562 cells contain both hexameric and octameric heteromers. To generate cell lines with controllable hexamer-to-octamer ratios and that express single SEPT9 isoforms, we developed a gene product replacement strategy. By this means we identified SEPT9 isoform–specific properties that either facilitate septin heteromer polymerization along microtubules or modulate the size range of submembranous septin disks—a prevalent septin structure in nonadhered cells. Our findings show that the SEPT9 expression level directs the hexamer-to-octamer ratio, and that the isoform composition and expression level together determine higher-order arrangements of septins.     

Changes in the structure-function relationship of elastin and its impact on the proximal pulmonary arterial mechanics of hypertensive calves

Extracellular matrix remodeling has been proposed as one mechanism by which proximal pulmonary arteries stiffen during pulmonary arterial hypertension (PAH). Although some attention has been paid to the role of collagen and metallomatrix proteins in affecting vascular stiffness, much less work has been performed on changes in elastin structure-function relationships in PAH. Such work is warranted, given the importance of elastin as the structural protein primarily responsible for the passive elastic behavior of these conduit arteries. Here, we study structure-function relationships of fresh arterial tissue and purified arterial elastin from the main, left, and right pulmonary artery branches of normotensive and hypoxia-induced pulmonary hypertensive neonatal calves. PAH resulted in an average 81 and 72% increase in stiffness of fresh and digested tissue, respectively. Increase in stiffness appears most attributable to elevated elastic modulus, which increased 46 and 65%, respectively, for fresh and digested tissue. Comparison between fresh and digested tissues shows that, at 35% strain, a minimum of 48% of the arterial load is carried by elastin, and a minimum of 43% of the change in stiffness of arterial tissue is due to the change in elastin stiffness. Analysis of the stress-strain behavior revealed that PAH causes an increase in the strains associated with the physiological pressure range but had no effect on the strain of transition from elastin-dominant to collagen-dominant behavior. These results indicate that mechanobiological adaptations of the continuum and geometric properties of elastin, in response to PAH, significantly elevate the circumferential stiffness of proximal pulmonary arterial tissue.     

Phosphorylation of Hrs downstream of the epidermal growth factor receptor.

The hepatocyte growth factor-regulated tyrosine kinase substrate Hrs is an early endosomal protein that is thought to play a regulatory role in the trafficking of growth factor/receptor complexes through early endosomes. Stimulation of cells with epidermal growth factor (EGF) rapidly leads to phosphorylation of Hrs, raising the question whether the receptor tyrosine kinase phosphorylates Hrs directly. Here, we present evidence that a downstream kinase, rather than the active receptor kinase is responsible. We show that the nonreceptor tyrosine kinase Src is able to phosphorylate Hrs in vitro and in vivo, but that Hrs is nevertheless phosphorylated in Src-, Yes- and Fyn-negative cells. Moreover, we show that only 10-20% of Hrs is phosphorylated following EGF stimulation, and that phosphorylation occurs at multiple tyrosines located in different parts of Hrs. These results suggest that Hrs is a substrate for several kinases downstream of the EGF receptor.     

Production of Diamino propionic acid ammonia lyase by a new strain of Salmonella typhimurium PU011

Background  

Seeds of the legume plant Lathyrus sativus, which is grown in arid and semi arid tropical regions, contain Diamino Propionic acid (DAP). DAP is a neurotoxin, which, when consumed, causes a disease called Lathyrism. Lathryrism may manifest as Neurolathyrism or Osteolathyrism, in which the nervous system, and bone formation respectively, are affected. DAP ammonia lyase is produced by a few microorganisms such as Salmonella typhi, Salmonella typhimurium and Pseudomonas, and is capable of detoxifying DAP.