http://www.maths.qmul.ac.uk/%7Elatora/report_06.pdf

Complex networks: Structure and dynamics

Autophagy in the Pathogenesis of Disease

Autophagy, or cellular self-digestion, is a cellular pathway involved in protein and organelle degradation, with an astonishing number of connections to human disease and physiology. For example, autophagic dysfunction is associated with cancer, neurodegeneration, microbial infection and ageing. Paradoxically, although autophagy is primarily a protective process for the cell, it can also play a role in cell death. Understanding autophagy may ultimately allow scientists and clinicians to harness this process for the purpose of improving human health.

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Autophagy fights disease through cellular self-digestion

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes.

For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.

Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

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Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

The most highly connected proteins in the cell are the most important for its survival. Proteins are traditionally identified on the basis of their individual actions as catalysts, signalling molecules, or building blocks in cells and microorganisms. But our post-genomic view is expanding the protein's role into an element in a network of protein–protein interactions as well, in which it has a contextual or cellular function within functional modules1,2. Here we provide quantitative support for this idea by demonstrating that the phenotypic consequence of a single gene deletion in the yeast Saccharomyces cerevisiae is affected to a large extent by the topological position of its protein product in the complex hierarchical web of molecular interactions.

Lethality and centrality in protein networks

Skin aspartic acid protease (SASPase) is believed to be a key enzyme involved in filaggrin processing during epidermal terminal differentiation. Since little is known about the regulation of SASPase function, the aim of this study was to identify involved protein partners in the process. Yeast two hybrid analyses using SASPase as bait against a human reconstructed skin library identified that the N-terminal domain of filaggrin 2 binds to the N-terminal fragment of SASPase. This interaction was confirmed in reciprocal yeast two hybrid screens and by Surface Plasmon Resonance analyses. Immunohistochemical studies in human skin, using specific antibodies to SASPase and the N-terminal domain of filaggrin 2, showed that the two proteins partially co-localized to the stratum granulosum. In vitro enzymatic assays showed that the N-terminal domain of filaggrin 2 enhanced the autoactivation of SASPase to its 14 kDa active form. Taken together, the data suggest that the N-terminal domain of filaggrin 2 regulates the activation of SASPase that may be a key event upstream of filaggrin processing to natural moisturizing factors in the human epidermis.

/pdf/filaggrin-and-filaggrin-2-processing-are-linked-together-4min59l8xa.pdf

Filaggrin and filaggrin 2 processing are linked together through skin aspartic acid protease activation.

The invention relates to substrates and methods suitable for assaying deubiquitinating enzyme activity, and in particular for screening inhibitors of enzymes involved in removal of mono or poly-ubiquitin chains from a target protein as well as inhibitors of ubiquitin precursors. Such inhibitors may have utility in the treatment of disease states associated with ubiquitin processing as well as proteolysis.

Substrates and methods for assaying deubiquitinating enzymes

The invention relates to a method of screening and identifying modulators of the protein interaction between new peptides and anti-apoptotic members of the Bcl-2 protein family. The modulators identified on the basis of this method are administered to patients with cancer in order to bring about apoptotic-type and/or autophagic-type programmed cell death in those patients.

Peptides which interact with anti-apoptotic members of the Bcl-2 protein family, and uses

Disclosed are polypeptides named HP1122, Cj1464 and PA3351 which are the anti-σ 28 factor of Helicobacter pylori, Campylobacter jejuni and Pseudomonas aeruginosa , respectively and fragments and variants thereof. Also disclosed is a polypeptide named SID1122 which is the domain of Helicobacter pylori's HP1122 polypeptide involved in a specific interaction with Helicobacter pylori σ 28 (HP1032) and which has an anti-σ 28 factor activity. Further disclosed are a SID1122 polypeptide that interacts with HP1032, identification of the HP1032 interacting domain (SID1032) that is specifically involved in the interaction with HP1122, complexes of two polypeptides such as HP1122-HP1032, or SID1122-SID1032, fragments and variants of the SID1122 and SID1032 polypeptides, antibodies to the SID1122 and SID1032 polypeptides, methods for screening drugs or agents which modulate the interaction of Helicobacter pylori's polypeptides encoded by HP1122 and HP1032, and pharmaceutical compositions for treating or preventing Gram negative flagellated bacteria infection in a human or mammal, more specifically Helicobacter sp. or Campylobacter jejuni or Pseudomonas aeruginosa infection, in particular Helicobacter pylori infection in a human or a mammal.

Anti-sigma28 factors in Helicobacter pylori, Campylobacter jejuni and Pseudomonas aeruginosa and applications thereof

The invention relates to a method of identifying modulators of programmed cell death, comprising an interaction between a motif of Beclin protein and an anti-apoptotic member of the family of Bcl-2 proteins and the detection of said interaction be means of fluorescence polarisation. The modulators identified on the basis of said method are administered to cancer patients in order to induce apoptotic- and/or autophagic-type programmed cell death. The invention also relates to a motif of the Beclin protein which can interact with an anti-apoptic member of the family of Bcl-2 proteins and to the use thereof in order to induce programmed cell death in a cancer patient.

Jean-Christophe Rain

Papers

Filaggrin and filaggrin 2 processing are linked together through skin aspartic acid protease activation.

Substrates and methods for assaying deubiquitinating enzymes

Peptides which interact with anti-apoptotic members of the Bcl-2 protein family, and uses

Anti-sigma28 factors in Helicobacter pylori, Campylobacter jejuni and Pseudomonas aeruginosa and applications thereof

Motif of beclin protein which interacts with anti-apoptotic members of the family of bcl-2 proteins and use thereof