Scission Sites Research Articles

The HEAT repeat protein HPO-27 is a lysosome fission factor.

Lysosomes are degradation and signalling centres crucial for homeostasis, development and ageing1. To meet diverse cellular demands, lysosomes remodel their morphology and function through constant fusion and fission2,3. Little is known about the molecular basis of fission. Here we identify HPO-27, a conserved HEAT repeat protein, as a lysosome scission factor in Caenorhabditis elegans. Loss of HPO-27 impairs lysosome fission and leads to an excessive tubular network that ultimately collapses. HPO-27 and its human homologue MROH1 are recruited to lysosomes by RAB-7 and enriched at scission sites. Super-resolution imaging, negative-staining electron microscopy and in vitro reconstitution assays reveal that HPO-27 and MROH1 self-assemble to mediate the constriction and scission of lysosomal tubules in worms and mammalian cells, respectively, and assemble to sever supported membrane tubes in vitro. Loss of HPO-27 affects lysosomal morphology, integrity and degradation activity, which impairs animal development and longevity. Thus, HPO-27 and MROH1 act as self-assembling scission factors to maintain lysosomal homeostasis and function.

Spatiotemporal Live-Cell Analysis of Photoreceptor Outer Segment Membrane Ingestion by the Retinal Pigment Epithelium Reveals Actin-Regulated Scission.

The photoreceptor outer segment (OS) is the phototransductive organelle in the vertebrate retina. OS tips are regularly ingested and degraded by the adjacent retinal pigment epithelium (RPE), offsetting the addition of new disk membrane at the base of the OS. This catabolic role of the RPE is essential for photoreceptor health, with defects in ingestion or degradation underlying different forms of retinal degeneration and blindness. Although proteins required for OS tip ingestion have been identified, spatiotemporal analysis of the ingestion process in live RPE cells is lacking; hence, the literature reflects no common understanding of the cellular mechanisms that affect ingestion. We imaged live RPE cells from mice (both sexes) to elucidate the ingestion events in real time. Our imaging revealed roles for f-actin dynamics and specific dynamic localizations of two BAR (Bin-Amphiphysin-Rvs) proteins, FBP17 and AMPH1-BAR, in shaping the RPE apical membrane as it surrounds the OS tip. Completion of ingestion was observed to occur by scission of the OS tip from the remainder of the OS, with a transient concentration of f-actin forming around the site of imminent scission. Actin dynamics were also required for regulating the size of the ingested OS tip, and the time course of the overall ingestion process. The size of the ingested tip is consistent with the term "phagocytosis." However, phagocytosis usually refers to engulfment of an entire particle or cell, whereas our observations of OS tip scission indicate a process that is more specifically described as "trogocytosis," in which one cell "nibbles" another cell.SIGNIFICANCE STATEMENT The ingestion of the photoreceptor outer segment (OS) tips by the retinal pigment epithelium (RPE) is a dynamic cellular process that has fascinated scientists for 60 years. Yet its molecular mechanisms had not been addressed in living cells. We developed a live-cell imaging approach to investigate OS tip ingestion, and focused on the dynamic participation of actin filaments and membrane-shaping BAR proteins. We observed scission of OS tips for the first time, and were able to monitor local changes in protein concentration preceding, during, and following scission. Our approach revealed that actin filaments were concentrated at the site of OS scission and were required for regulating the size of the ingested OS tip and the time course of the ingestion process.

Preparation and evaluation of effective thermal decomposition of tetraamminecopper (II) nitrate carried by graphene oxide

Graphene oxide (GO) was investigated in this paper as a promising catalyst for the effective thermal decomposition of tetraamminecopper (II) nitrate coordination complexes. CuC has been synthesized by doping GO nanoparticles in a one-step method and it demonstrates a uniform deposition of the produced nanocomposites onto the surface of the complex. The kinetics of decomposition and underlying mechanisms of GO on CuC have been studied using a Differential Scanning Calorimetry (DSC) technique and Thermal Gravimetric Analysis (TGA) coupled Fourier-Transform Infrared Spectroscopy (FTIR) technique and relevant theories. Under the effect of GO, the release of heat from the decomposition process is significantly increased from 97.1 J·g-1 to 69.2 J·g-1. Under the effect of GO, the activation energy of the CuC decreased from 94.3 to 82.2 ± 0.5 kJ·mol−1 (for endothermic reaction). The significance of this discovery is due to the reactive sites and great thermal conductivity of the GO. By FTIR analysis, GO affected by the formation of NH3 and N2O; and the decomposition of CuC complex in cooperation with the GO may follow a random scission model (A2) since it highly depends on the edge defects of GO as well as coordination center reactive sites (scission sites). In addition, the combustion behavior of CuC and CuC–GO in the physical mixture with B/KNO3 has been investigated. The results show that the pressure exponent “n” samples (CuC: n = 0.621 vs. CuC–GO: n = 0.703) can be stable and investigated composites are available for successful application as energetic materials.

Viral protease cleavage of MAVS in genetically modified mice with hepatitis A virus infection

Consistent with its relatively narrow host species range, hepatitis A virus (HAV) cannot infect C57BL/6 mice. However, in Mavs-/- mice with genetic deficiency of the innate immune signaling adaptor MAVS, HAV replicates robustly in the absence of disease. The HAV 3ABC protease cleaves MAVS in human cells, thereby disrupting virus-induced IFN responses, but it cannot cleave murine MAVS (mMAVS) due to sequence differences at the site of scission. Here, we sought to elucidate the role of 3ABC MAVS cleavage in determining HAV pathogenesis and host species range. Using CRISPR/Cas9 gene editing, we established two independent lineages of C57BL/6 mice with knock-in mutations altering two amino acids in mMAVS ('mMAVS-VS'), rendering it susceptible to 3ABC cleavage without loss of signaling function. We challenged homozygous Mavsvs/vs mice with HAV, and compared infection outcomes with C57BL/6 and genetically deficient Mavs-/- mice. The humanized murine mMAVS-VS protein was cleaved as efficiently as human MAVS when co-expressed with 3ABC in Huh-7 cells. In embyronic fibroblasts from Mavsvs/vs mice, mMAVS-VS was cleaved by ectopically expressed 3ABC, significantly disrupting Sendai virus-induced IFN responses. However, in contrast to Mavs-/- mice with genetic MAVS deficiency, HAV failed to establish infection in Mavsvs/vs mice, even with additional genetic knockout of Trif or Irf1. Nonetheless, when crossed with permissive Ifnar1-/- mice lacking type I IFN receptors, Mavsvs/vsIfnar1-/- mice demonstrated enhanced viral replication coupled with significant reductions in serum alanine aminotransferase, hepatocellular apoptosis, and intrahepatic inflammatory cell infiltrates compared with Ifnar1-/- mice. MAVS cleavage by 3ABC boosts viral replication and disrupts disease pathogenesis, but it is not by itself sufficient to break the host-species barrier to HAV infection in mice. The limited host range of human hepatitis viruses could be explained by species-specific viral strategies that disrupt innate immune responses. Both hepatitis A virus (HAV) and hepatitis C virus express viral proteases that cleave the innate immune adaptor protein MAVS, in human but not mouse cells. However, the impact of this immune evasion strategy has never been assessed invivo. Here we show that HAV 3ABC protease cleavage of MAVS enhances viral replication and lessens liver inflammation in mice lacking interferon receptors, but that it is insufficient by itself to overcome the cross-species barrier to infection in mice. These results enhance our understanding of how hepatitis viruses interact with the host and their impact on innate immune responses.

Conversion of low-density polyethylene into monocyclic aromatic hydrocarbons by catalytic pyrolysis: Comparison of HZSM-5, Hβ, HY and MCM-41

The catalytic properties of different catalytic materials on the pyrolysis of plastics have been extensively investigated. In order to better understand the relevant mechanism of catalytic pyrolysis of plastic waste, the effect of different acidity and pore size of catalytic material on its pyrolysis kinetic parameters and the distribution of pyrolysis products needs to be studied to provide a stronger theoretical basis for its resource utilization. This study aims to study the effects of HZSM-5, Hβ, HY and MCM-41 on the thermodynamic properties , kinetic parameters and pyrolysis characteristics of low-density polyethylene (LDPE), analyze the relationship between the specific catalytic pyrolysis products and the properties of the catalyst. Among the four catalysts, HZSM-5 obtained the most concentrated product distribution and the highest yield of monocyclic aromatic hydrocarbons , and it was found that the cracking of LDPE mainly occurred in its pores. Due to the large pore size and low concentration of acid sites, the main product type obtained by MCM-41 was olefins . In addition, it was found that HY reduced the pyrolysis temperature of LDPE and the activation energy of the reaction to the greatest extent. The different catalytic effects were mainly caused by different pore structures and acid site concentration. Moreover, it was found that the addition of catalyst changed the cracking mechanism of LDPE. Under the comprehensive consideration with the goal of producing monocyclic aromatic hydrocarbons, HZSM-5 is the most suitable catalyst among them. • The catalytic effects of HZSM-5, Hβ, HY and MCM-41 were studied. • HZSM-5 is the most suitable catalyst for the production of MAHs from LDPE. • Different pore structures and acid sites cause the changes of catalytic effects. • Partial LDPE scission sites changed from random-chain to end-chain due to catalysis. • The cracking reaction of LDPE occurs in the pores of HZSM-5.

Modeling the effect of dose rate and time on crosslinking and scission in irradiated polyethylene

The insulation around the electrical cabling in nuclear power plants is frequently made of ethylene-propylene rubber and crosslinked polyethylene that is subjected to low levels of environmental stressors and radiation over the duration of their decades long service life. For the purpose of maintenance and reactor recertification, it is necessary to develop a non-destructive approach to determine the degree of damage the insulation has sustained. Accelerated aging experiments are used to develop these methods, although it is unclear how to relate these specimens to in situ aged ones. Here a kinetic rate model is used to investigate the impact of radiation dose rate and total dose on the crosslinking and chain scission of polyethylene. Analytical expressions for the concentration of crosslinking and scission sites as a function of time and dose rate, both during and post irradiation, are presented. During irradiation the concentration of crosslinking sites increases linearly with time, and when the dosing ends the crosslinking reaction terminates. The scission reaction begins slower, due to the necessity of forming intermediate species, but increases in rate, and eventually the concentration of scission sites overtakes crosslinking. However, at low dose rates, less than 10 Gy/hr, scission damage is always the primary form of damage. Unlike the crosslinking reaction, scission continues for months and years due to the slow decomposition of the intermediate species.

Imaging Dynamin-Related Protein 1 (Drp1)-Mediated Mitochondrial Fission in Living Cells.

Mitochondria form highly dynamic networks that continuously undergo fission and fusion. Dynamin-related protein 1 (Drp1), a key regulator of mitochondrial division, self-assembles into a helical polymer around pre-marked scission sites and generates the constriction force necessary to sever the organelle. Live-cell fluorescence imaging of Drp1 oligomerization dynamics and mitochondrial fission can provide unprecedented insights into the spatiotemporal relationship between these coupled processes. The high-resolution images provided by the laser scanning confocal microscope facilitate the observation of the finer details of mitochondrial structure as well as Drp1 polymer dynamics in real time. We provide a detailed description of the confocal imaging methods used to characterize mitochondrial dynamics in living cells with an emphasis on Drp1-mediated mitochondrial fission.

Nematode Autotomy Requires Molting and Entails Tissue Healing without Obvious Regeneration.

Autotomy in C. elegans, which results in the severing of the body into two fragments, has been observed as a response to late larval worm-star formation after exposure to a bacterial surface pathogen. It was found that autotomy can occur in both hermaphroditic and gonochoristic nematode species, and during either the L3 or the L4 molt. Severing was hypothesized to be driven by a ‘balloon-twisting’ mechanism during molting but was found to be independent of lethargus-associated flipping. Extensive healing and apparent tissue fusion were seen at the site of scission. No obvious regeneration of lost body parts was seen in either L4 or adult truncated worms. A variety of mutants defective in processes of cell death, healing, regeneration, responses to damage, stress or pathogens were found to be competent to autotomize. Mutants specifically defective in autotomy have yet to be found. Autotomy may represent a modification of the essential normal process of molting.

Generalizing metallocene mechanochemistry to ruthenocene mechanophores.

Recent reports have shown that ferrocene displays an unexpected combination of force-free stability and mechanochemical activity, as it acts as the preferred site of chain scission along the backbone of highly extended polymer chains. This observation raises the tantalizing question as to whether similar mechanochemical activity might be present in other metallocenes, and, if so, what features of metallocenes dictate their relative ability to act as mechanophores. In this work, we elucidate polymerization methodologies towards main-chain ruthenocene-based polymers and explore the mechanochemistry of ruthenocene. We find that ruthenocene, in analogy to ferrocene, acts as a highly selective site of main chain scission despite the fact that it is even more inert. A comparison of ruthenocene and ferrocene reactivity provides insights as to the possible origins of metallocene mechanochemistry, including the relative importance of structural and thermodynamic parameters such as bond length and bond dissociation energy. These results suggest that metallocenes might be privileged mechanophores through which highly inert coordination complexes can be made dynamic in a stimuli-responsive fashion, offering potential opportunities in dynamic metallo-supramolecular materials and in mechanochemical routes to reactive intermediates that are otherwise difficult to obtain.

Tribo‐Induced Structural Transformation and Lubricant Dissociation at Amorphous Carbon–Alpha Olefin Interface

AbstractAmorphous carbon (a‐C) combined with a fluid lubricant is capable of providing an ultra‐low friction state and thus achieving long lifetime and reliable operation. However, the understanding of the atomistic process occurring at the sliding friction interfaces, especially the interfacial structure transformation and lubricant dissociation at different contact states, is still not well understood. Here, using reactive molecular dynamics simulation, the friction behavior of a self‐mated a‐C system composited with different alpha olefins (AOs) as lubricants is comparatively investigated, and the results present that due to the co‐existence of tribo‐induced thermal and shearing effects, AOs exhibit different physicochemical behaviors at the a‐C–a‐C interface compared to that at the a‐C surface. Although introducing AOs into a self‐mated a‐C system reduces the friction coefficient, its efficiency strongly relies on the AO variety and contact pressure. The pressure‐driven dissociation of AOs passivates the friction interface, resulting in the evolution of the primary friction mechanism from hydrodynamic lubrication to interfacial passivation that is not accessible by experimental characterization. The corresponding scission sites of different AOs are demonstrated, which enriches the fundamental understanding on sliding friction behavior and offers a comprehensive design criterion for lubricants (viscosity, chain length, and bond saturated states) and a‐C to achieve nearly frictionless sliding interface.

Quantitative and Mechanistic Mechanochemistry in Ferrocene Dissociation.

Ferrocene is classically regarded as being highly inert owing to the large dissociation energy of metal-cyclopentadienyl (Cp) bonds. We show that the Fe-Cp bond in ferrocene is the preferential site of mechanochemical scission in the pulsed ultrasonication of main-chain ferrocene-containing polybutadiene-derived polymers. Quantitative studies reveal that the Fe-Cp bond is similar in strength to the carbon-nitrogen bond of an azobisdialkylnitrile (bond dissociation energy < -0 kcal/mol), despite the significantly higher Fe-Cp bond dissociation energy (approximately 90 kcal/mol). Mechanistic studies are consistent with a predominately heterolytic mechanism of chain scission. DFT calculations provide insights into the origins of ferrocene's mechanical lability.

Development of a Molecular Probe Targeting Mitochondrial Fission Protein Fis1

Excessive mitochondrial fragmentation – caused by decreased mitochondrial fusion or increased fission – leads to mitochondrial dysfunction and has been implicated in ischemia‐reperfusion injury and diabetic cardiomyopathy. Genetic or pharmacological inhibition of fission is protective against hyperglycemic‐induced loss of mitochondrial connectivity and reactive oxygen species production. Dynamin‐related protein 1 (Drp1) is a cytosolic GTPase mechanoenzyme or “cutter” known to cause scission of the mitochondria, but what determines a site of scission is unknown. Drp1 is recruited to mitochondria by one of four mitochondrial anchored recruiters; one of which is the tetratricopeptide repeat protein, fission protein 1 (Fis1). Cell lines lacking individual, all, or a combination of Drp1 recruiter proteins demonstrate each recruiter supports Drp1 recruitment in unique pathways. Of the four Drp1 recruiters, Fis1 is the only recruiter conserved in all species with mitochondria, but the involvement of Fis1 in mammalian mitochondrial fission has been disputed. Recent data suggests mammalian Fis1 is specialized for stress‐induced fission but the molecular basis remains unknown. We aimed to develop a molecular probe targeting Fis1 to be used in testing the hypothesis that Fis1 recruits and subsequently activates Drp1 in stress‐induced mammalian mitochondrial fission. If successful, a molecular probe targeting Fis1 could be used to determine the ability of Fis1 to modulate Drp1 activity under a variety of cellular stresses. We are using two approaches to develop molecular probe(s): 1) fragment‐based screening by nuclear magnetic resonance (NMR) and 2) peptide design using molecular docking, molecular dynamics simulations, and NMR validation experiments. Fis1 was screened against ~ 2000 fragments using 1H‐15N SOFAST‐HMQC and 1H‐15N NUS‐HSQC NMR experiments. Fragments that bound Fis1 (hits) were identified using a novel scoring metric, Difference Intensity Analysis (DIA), which quantitates spectral changes in 2D difference spectra and functions regardless of NMR time scale. To determine binding modalities of Fis1‐peptide interactions, we used molecular docking followed by molecular dynamics simulations to dock and sample the conformational space of previously identified Fis1 interacting peptides. To obtain binding affinities and putative binding sites, NMR titration experiments of 15N‐Fis1 with individual fragment hits and synthesized peptides were performed. Our results to date on developing molecular probe(s) against Fis1 will be presented.Support or Funding InformationThis project was supported by the Clinical &amp; Translational Science Institute (CTSI) through the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant Number UL1TR001436. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. Project also supported by NIH grants NIH R01‐GM067180, and NIH R01‐HL128240.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Observing Frustrated Phagocytosis and Phagosome Formation and Closure Using Total Internal Reflection Fluorescence Microscopy (TIRFM).

Complementary methods to observe frustrated phagocytosis and phagosome closure using total internal reflection fluorescence microscopy (TIRFM) are described here. Frustrated phagocytosis occurs when phagocytic cells are exposed to an opsonized surface and spread as if trying to engulf it, allowing for the observation of phagocytic spreading and the biochemical events that directly precede it. Phagosome formation and closure is an inherently three-dimensional process though, and cannot be studied in the "frustrated" situation. Here we describe a method to visualize with unprecedented high-resolution phagosome formation and closure in three dimensions. It allows for observation of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

Recruitment of Drp1 in Mitochondrial Fission

Intracellular membranes are dynamically shaped by proteins including fission and fusion events important for vesicular transport, organelle trafficking, and cellular homeostasis. Yet how proteins interact with membranes to control these events is largely unknown. We are interested in addressing this question through mechanistic studies of a dynamin superfamily member central to the division, or fission, of mitochondria. Mitochondrial fission is driven by the GTPase mechanoenzyme, Dynamin‐related protein 1 (Drp1). At the mitochondrial surface, Drp1 is thought to assemble into rings around a future site of scission in a GTP‐dependent manner. Curiously, Drp1 is primarily localized to the cytoplasm and how it is recruited to the mitochondrial surface is unknown. Several proteins resident to the mitochondrial surface have been implicated in Drp1 recruitment and subsequent assembly, but the mechanism remains unclear. We are addressing these problems using biochemical, structural, and cell biological approaches. Our results to date will be presented and suggest a new model for Drp1 activity in mitochondrial fission.Support or Funding InformationThis project was supported by the Clinical &amp; Translational Science Institute (CTSI) through the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant Number UL1TR001436. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. Project also supported by NIH grants NIH R01‐GM067180, and NIH R01‐HL128240.

Correlation between free radical content and mechanical properties of ribose-protected irradiation sterilized cortical bone allografts

Event Abstract Back to Event Correlation between free radical content and mechanical properties of ribose-protected irradiation sterilized cortical bone allografts Tarik Attia1, 2*, Gagan Minhas2, Xing Ze Lu2*, Marc Grynpas1, 2* and Thomas Willett1, 2, 3* 1 University of Toronto, Institute of Biomaterials and Biomedical Engineering, Canada 2 Mount Sinai Hospital, Lunenfeld Research Institute, Canada 3 University of Toronto, Department of Surgery, Canada Introduction: Human bone transplants are used in the orthopaedic reconstruction of skeletal defects; however, bone allografts have insufficient mechanical properties[1]. In order to ensure the safety of the patient, bone transplants are sterilized to reduce the risk of pathogens. γ-irradiation is the gold standard, as it is an effective method to destroy pathogens. This method, however, causes damage to the bone collagen [2],[3]. Our lab has developed a novel method for improving static mechanical properties, tested, in 3-point-bending by using ribose to modify collagen network connectivity of bone prior/during the irradiation[4]. The purpose of this study is to understand the mechanism by which the ribose treatment protects the mechanical properties of bone from irradiation. We measured the free radical content of the bone and examined its correlation to the mechanical properties of different ribose concentration treatments. Method: Cortical bone beams were cut from human femurs with final dimensions of 2x4x50-60mm. The beams were separated into 5 different treatment groups: normal (N; not irradiated), irradiated (I), and ribose pre-treated + irradiation with a concentration of 0.9M, 1.2M and 2.0M. Subsequently, after irradiation each specimen was placed into an Electron Paramagnetic Resonance (EPR) cavity so that the entire cavity was filled. EPR spectra were collected running at -80oC to ensure stable free radical content. After EPR, 3-point bending tests to fracture were conducted. Results: 30kGy of γ-irradiation created large free radical contents in both the organic and mineral phases of the conventionally irradiated bone. Non-irradiated controls contained barely detectable amounts. The 0.9M ribose treatment resulted in 43%, 1.2M in 40% and the 2.0M in 44% less free radical content immediately after irradiation in comparison to the I-group (p<0.0001)(Fig.1A). No statistically difference between the R-groups were detected. Conventional irradiation greatly decreased the work-to-fracture of the bone by 45% (p<0.001). Ribose-treatment protected the work-to-fracture of the irradiated bone by 30% for 0,9M, 22% for 1.2M and 39% for 2.0M with no statistically difference between the R-groups (Fig.1B). Discussion: The formation of macromolecular free radicals in the organic phase of bone is a known result of scission sites in the collagen and defects in the mineral generated by γ–irradiation, which contributes to the loss of toughness. This study demonstrates that our ribose pre-treatment reduces the amount of free radicals formed and prevents some loss of toughness, independent of the ribose concentration. Significance: This study provides further evidence towards understanding the mechanism by which ribose pre-treatment protects the mechanical properties of irradiation sterilized cortical bone. Mount Sinai Allograft Technologies; Timothy Burrow at University of Toronto, Department of Chemistry; Funding: Canadian Institutes of Health Research (CIHR)

Cleavage Enhancement of Specific Chemical Bonds in DNA by Cisplatin Radiosensitization

X-ray photoelectron spectroscopy (XPS) is harnessed as an in situ efficient characterization technique for monitoring chemical bond transformation in DNA and cisplatin-DNA complexes under synergic X-ray irradiation. By analyzing the variation of relative peak area of core elements of DNA as a function of irradiation time, we find that the most vulnerable scission sites in DNA are those containing phosphate and glycosidic bonds. Compared to DNA, the effective rate constants of the corresponding phosphodiester and glycosidic bond cleavages for cisplatin-DNA complexes are 1.8 and 1.9 folds larger. These damages and their enhancements are similar to those induced by low energy electrons (LEE). Consistently, the magnitude of the secondary electron distribution produced by the X-rays on the cisplatin-DNA complexes is considerably increased compared to that of pristine DNA. The data suggest that DNA radiosensization by cisplatin results not only from the sensitization of DNA to the action of LEE, but also from an increase the production of LEE at the site of binding of the cisplatin. The results provide new insights into the mechanisms of cisplatin-induced sensitization of DNA under X-ray irradiation, which could be helpful in the design of new cisplatin-based antitumor drugs.

Partial Blocking of Mouse DSPP Processing by Substitution of Gly451–Asp452 Bond Suggests the Presence of Secondary Cleavage Site(s)

Dentin sialophosphoprotein (DSPP) in the extracellular matrix of dentin is cleaved into dentin sialoprotein and dentin phosphoprotein, which originate from the NH2-terminal and COOH-terminal regions of DSPP, respectively. In the proteolytic processing of mouse DSPP, the peptide bond at Gly451–Asp452 has been shown to be cleaved by bone morphogenetic protein 1 (BMP1)/Tolloid-like metalloproteinases. In this study, we generated transgenic mice expressing a mutant DSPP in which Asp452 was substituted by Ala452. Protein chemistry analyses of extracts from the long bone of these transgenic mice showed that the D452A substitution partially blocked DSPP processing in vivo. When the full-length form of mutant DSPP (designated “D452A-DSPP”) isolated from the transgenic mice was treated with BMP1 in vitro, a portion of the D452A-DSPP was cleaved, suggesting the presence of secondary peptide bond(s) that can be broken by BMP1. To identify the potential secondary DSPP cleavage site(s), site-directed mutagenesis was performed to generate nine DNA constructs expressing DSPP-bearing substitutions at potential scission sites. These different types of mutant DSPP made in eukaryotic cell lines were treated with BMP1 and the digestion products were assessed by Western immunoblotting. All of the mutant DSPP molecular species were partially cleaved by BMP1, giving rise to a protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis similar to that of normal dentin sialoprotein. Taken together, we concluded that in addition to the peptide bond Gly451–Asp452, there must be a cryptic cleavage site or sites close to Asp452 in the mouse DSPP that can be cleaved by BMP1.

Dynamics of ESCRT protein recruitment during retroviral assembly

The ESCRT (Endosomal Sorting Complex Required for Transport) complexes and associated proteins mediate membrane scission reactions, such as multi-vesicular body formation, the terminal stages of cytokinesis and retroviral particle release. These proteins are believed to be sequentially recruited to the site of membrane scission, and then complexes are disassembled by the ATPase Vps4A. However these events have never been observed in living cells and their dynamics are unknown. By quantifying the recruitment of several ESCRT and associated proteins during the assembly of two retroviruses, we show that Alix progressively accumulated at viral assembly sites, coincident with the accumulation of the major viral structural protein, Gag, and was not recycled after assembly. In contrast, ESCRT-III and Vps4A were only transiently recruited when the accumulation of Gag was complete. These data suggest that the rapid and transient recruitment of proteins that act late in the ESCRT pathway and carry out membrane fission is triggered by prior and progressive accumulation of proteins that bridge viral proteins and the late-acting ESCRT proteins.

Conserved sequence in the aggrecan interglobular domain modulates cleavage by ADAMTS-4 and ADAMTS-5

Background Cleavage of aggrecan by ADAMTS proteinases at specific sites within highly conserved regions may be important to normal physiological enzyme functions, as well as pathological degradation. Methods To examine ADAMTS selectivity, we assayed ADAMTS-4 and -5 cleavage of recombinant bovine aggrecan mutated at amino acids N-terminal or C-terminal to the interglobular domain cleavage site. Results Mutations of conserved amino acids from P18 to P12 to increase hydrophilicity resulted in ADAMTS-4 cleavage inhibition. Mutation of Thr, but not Asn within the conserved N-glycosylation motif Asn-Ile-Thr from P6 to P4 enhanced cleavage. Mutation of conserved Thr residues from P22 to P17 to increase hydrophobicity enhanced ADAMTS-4 cleavage. A P4′ Ser377Gln mutant inhibited cleavage by ADAMTS-4 and -5, while a neutral Ser377Ala mutant and species mimicking mutants Ser377Thr, Ser377Asn, and Arg375Leu were cleaved normally by ADAMTS-4. The Ser377Thr mutant, however, was resistant to cleavage by ADAMTS-5. Conclusion We have identified multiple conserved amino acids within regions N- and C-terminal to the site of scission that may influence enzyme–substrate recognition, and may interact with exosites on ADAMTS-4 and ADAMTS-5. General significance Inhibition of the binding of ADAMTS-4 and ADAMTS-5 exosites to aggrecan should be explored as a therapeutic intervention for osteoarthritis.

Contributions to Bax insertion and oligomerization of lipids of the mitochondrial outer membrane

Under many apoptotic conditions, Bax undergoes conformational rearrangements, leading to its insertion in the mitochondrial outer membrane as a transmembrane oligomer. At the same time, mitochondria undergo fragmentation and activated Bax was reported to localize to fission sites. We studied how lipid composition and membrane curvature regulate Bax activation. When isolated mitochondria were incubated with phospholipase A2, which led to phosphatidylethanolamine and cardiolipin hydrolysis, tBid and Bax insertion were hindered. We thus studied in liposomes how phosphatidylethanolamine, cardiolipin, and its hydrolysis products affect Bax activation. Whereas phosphatidylethanolamine, a lipid with negative curvature, did not affect Bax insertion, it inhibited Bax oligomerization. Conversely, Bax insertion required cardiolipin, and was not blocked by cardiolipin hydrolysis products. These experiments support a direct role for cardiolipin in the recruitment and activation of Bax. To examine if the increase in membrane curvature that accompanies mitochondrial fission participates in Bax activation, we studied how liposome size affects the process, and observed that it was inhibited in small liposomes (<or=200 nm diameter). Therefore, the localization of Bax to mitochondrial scission sites does not result from a preference for curved bilayers. Our experiments show that membrane properties can control the process of Bax activation, providing an additional level to the mechanisms of regulation of mitochondrial permeability.