Cannon, Barbara, and Jan Nedergaard. Brown Adipose Tissue: Function and Physiological Significance. Physiol Rev 84: 277–359, 2004; 10.1152/physrev.00015.2003.—The function of brown adipose tissue i...

Brown Adipose Tissue: Function and Physiological Significance

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)

Conserved signaling pathways that activate the mitogen-activated protein kinases (MAPKs) are involved in relaying extracellular stimulations to intracellular responses. The MAPKs coordinately regulate cell proliferation, differentiation, motility, and survival, which are functions also known to be mediated by members of a growing family of MAPK-activated protein kinases (MKs; formerly known as MAPKAP kinases). The MKs are related serine/threonine kinases that respond to mitogenic and stress stimuli through proline-directed phosphorylation and activation of the kinase domain by extracellular signal-regulated kinases 1 and 2 and p38 MAPKs. There are currently 11 vertebrate MKs in five subfamilies based on primary sequence homology: the ribosomal S6 kinases, the mitogen- and stress-activated kinases, the MAPK-interacting kinases, MAPK-activated protein kinases 2 and 3, and MK5. In the last 5 years, several MK substrates have been identified, which has helped tremendously to identify the biological role of the members of this family. Together with data from the study of MK-knockout mice, the identities of the MK substrates indicate that they play important roles in diverse biological processes, including mRNA translation, cell proliferation and survival, and the nuclear genomic response to mitogens and cellular stresses. In this article, we review the existing data on the MKs and discuss their physiological functions based on recent discoveries.

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Erk and p38 mapk-activated protein kinases: a family of protein kinases with diverse biological functions

SUMMARY The mitogen-activated protein kinases (MAPKs) regulate diverse cellular programs by relaying extracellular signals to intracellular responses. In mammals, there are more than a dozen MAPK enzymes that coordinately regulate cell proliferation, differentiation, motility, and survival. The best known are the conventional MAPKs, which include the extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun amino-terminal kinases 1 to 3 (JNK1 to -3), p38 (α, β, γ, and δ), and ERK5 families. There are additional, atypical MAPK enzymes, including ERK3/4, ERK7/8, and Nemo-like kinase (NLK), which have distinct regulation and functions. Together, the MAPKs regulate a large number of substrates, including members of a family of protein Ser/Thr kinases termed MAPK-activated protein kinases (MAPKAPKs). The MAPKAPKs are related enzymes that respond to extracellular stimulation through direct MAPK-dependent activation loop phosphorylation and kinase activation. There are five MAPKAPK subfamilies: the p90 ribosomal S6 kinase (RSK), the mitogen- and stress-activated kinase (MSK), the MAPK-interacting kinase (MNK), the MAPK-activated protein kinase 2/3 (MK2/3), and MK5 (also known as p38-regulated/activated protein kinase [PRAK]). These enzymes have diverse biological functions, including regulation of nucleosome and gene expression, mRNA stability and translation, and cell proliferation and survival. Here we review the mechanisms of MAPKAPK activation by the different MAPKs and discuss their physiological roles based on established substrates and recent discoveries.

Activation and Function of the MAPKs and Their Substrates, the MAPK-Activated Protein Kinases

Post-translational modifications modulate the activity of most eukaryote proteins. Analysis of these modifications presents formidable challenges but their determination generates indispensable insight into biological function. Strategies developed to characterize individual proteins are now systematically applied to protein populations. The combination of function- or structure-based purification of modified 'subproteomes', such as phosphorylated proteins or modified membrane proteins, with mass spectrometry is proving particularly successful. To map modification sites in molecular detail, novel mass spectrometric peptide sequencing and analysis technologies hold tremendous potential. Finally, stable isotope labeling strategies in combination with mass spectrometry have been applied successfully to study the dynamics of modifications.

Proteomic analysis of post-translational modifications.

Selektive Inhibierung von Rezeptor-Tyrosinkinasen mit Aeroplysinin-Analoga†

Congenital dyserythropoietic anaemia (CDA) type I is a rare blood disorder characterised by moderate to severe macrocytic anaemia and hepatomegaly, with spongy heterochromatin and inter-nuclear bridges seen in bone marrow erythroblasts. The vast majority of cases of CDA type I are caused by mutations in the CDAN1 gene. The product of CDAN1 is Codanin-1, which interacts the histone chaperone ASF1 in the cytoplasm. Codanin-1 is a negative regulator of chromatin replication, sequestering ASF1 in the cytoplasm, restraining histone deposition and thereby limiting DNA replication. The remainder of CDA-I cases are caused by mutations in the C15ORF41 gene, but very little is known about the product of this gene. Here, we report that C15ORF41 forms a tight, near-stoichiometric complex with Codanin1 in human cells, interacting with the C-terminal region of Codanin-1. We present the characterisation of the C15ORF41-Codanin-1 complex in humans in cells and in vitro, and demonstrate that Codanin-1 appears to sequester C15ORF41 in the cytoplasm as previously shown for ASF1. The findings in this study have major implications for understanding the functions of C15ORF41 and Codanin-1, and the aetiology of CDA-I.

A complex comprising C15ORF41 and Codanin-1: the products of two genes mutated in congenital dyserythropoietic anaemia type I (CDA-I).

LRRK2 serine/threonine kinase is associated with inherited Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases within their switch 2 motif to control their interactions with effectors. Recent work has shown that the metal-dependent protein phosphatase PPM1H counteracts LRRK2 by dephosphorylating Rabs. PPM1H is highly selective for LRRK2 phosphorylated Rabs, and closely related PPM1J exhibits no activity towards substrates such as Rab8a phosphorylated at Thr72 (pThr72). Here, we have identified the molecular determinant of PPM1H specificity for Rabs. The crystal structure of PPM1H reveals a structurally conserved phosphatase fold that strikingly has evolved a 110-residue flap domain adjacent to the active site. The flap domain distantly resembles tudor domains that interact with histones in the context of epigenetics. Cellular assays, crosslinking and 3-D modelling suggest that the flap domain encodes the docking motif for phosphorylated Rabs. Consistent with this hypothesis, a PPM1J chimaera with the PPM1H flap domain dephosphorylates pThr72 of Rab8a both in vitro and in cellular assays. Therefore, PPM1H has acquired a Rab-specific interaction domain within a conserved phosphatase fold.

Structural basis for the specificity of PPM1H phosphatase for Rab GTPases.

Branched ubiquitin (Ub) chains make up a significant proportion of Ub polymers in human cells and are formed when two or more sites on a single Ub molecule are modified with Ub creating bifurcated architectures. Despite their abundance, we have a poor understanding of the cellular functions of branched Ub signals that stems from a lack of facile tools and methods to study them. Here we develop a comprehensive pipeline to define branched Ub function, using K48-K63-branched chains as a case study. We discover branch-specific binders and, by developing a method that monitors cleavage of linkages within complex polyUb, we discover the VCP/p97-associated ATXN3, and MINDY family deubiquitinases to act as debranching enzymes. By engineering and utilizing a branched K48-K63-Ub chain-specific nanobody, we reveal roles for these chains in VCP/p97-related processes. In summary, we provide a blueprint to investigate branched Ub function that can be readily applied to study other branched chain types. Highlights Assembly of defined branched ubiquitin chains enables identification of specific binding proteins Development of quantitative DUB assay monitoring cleavage of individual Ub linkages within complex ubiquitin chains identifies debranching enzymes Engineering specific, high-affinity nanobody against branched K48-K63 ubiquitin reveals roles in VCP/p97 related processes and DNA damage responses General blueprint of new methods and tools for in-depth characterization of branched ubiquitin chains and their underlying biology

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Comprehensive approach to study branched ubiquitin chains reveals roles for K48-K63 branches in VCP/p97-related processes

The expression of genes is predominantly determined by conditions of the microenvironment of cells. Prime examples of such regulation are found in embryonic development of all multicellular organisms and also in the adult when various cytokines and hormones exert highly inducer-specific influences on genes. The naturally occurring regulating agents interact with specific receptors: e.g., the retinoids, vitamin D3, thyroid hormones and the steroid hormones with appropriate nuclear receptors (RARs, RXRs, VDR and the specific steroid hormone receptors), or the members of the large TGFβ and FGF families with their respective cell surface receptors. While nuclear receptors act as transcription factors themselves and select their genes by receptor-specific recognition elements, the growth factors induce, through their cell surface receptors, a complex process of signal transduction to the nucleus (for reviews see Beato, 1989; Karin, 1994; Gilbert, 1994; Angel and Herrlich, 1994; McCormick, 1995; Howe and Weiss, 1995; Ullrich and Simon, 1995).

Axel Knebel

Papers

Selektive Inhibierung von Rezeptor-Tyrosinkinasen mit Aeroplysinin-Analoga†

A complex comprising C15ORF41 and Codanin-1: the products of two genes mutated in congenital dyserythropoietic anaemia type I (CDA-I).

Structural basis for the specificity of PPM1H phosphatase for Rab GTPases.

Comprehensive approach to study branched ubiquitin chains reveals roles for K48-K63 branches in VCP/p97-related processes

An Oxidant Sensor at the Plasma Membrane