Structural Biology: Parkin’s Serpentine Shape Revealed in the Year of the Snake

SummaryParkin is an E3 ubiquitin ligase, mutations in which are responsible for autosomal recessive juvenile parkinsonism. Recently, several structures of Parkin have been solved, revealing its serpentine shape and modes of auto-inhibition

The utilisation of hydrogels for iPSC-cardiomyocyte research

Cardiac fibroblasts' (FBs) and cardiomyocytes' (CMs) behaviour and morphology are influenced by their environment such as remodelling of the myocardium, thus highlighting the importance of biomaterial substrates in cell culture. Biomaterials have emerged as important tools for the development of physiological models, due to the range of adaptable properties of these materials, such as degradability and biocompatibility. Biomaterial hydrogels can act as alternative substrates for cellular studies, which have been particularly key to the progression of the cardiovascular field. This review will focus on the role of hydrogels in cardiac research, specifically the use of natural and synthetic biomaterials such as hyaluronic acid, polydimethylsiloxane and polyethylene glycol for culturing induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). The ability to fine-tune mechanical properties such as stiffness and the versatility of biomaterials is assessed, alongside applications of hydrogels with iPSC-CMs. Natural hydrogels often display higher biocompatibility with iPSC-CMs but often degrade quicker, whereas synthetic hydrogels can be modified to facilitate cell attachment and decrease degradation rates. iPSC-CM structure and electrophysiology can be assessed on natural and synthetic hydrogels, often resolving issues such as immaturity of iPSC-CMs. Biomaterial hydrogels can thus provide a more physiological model of the cardiac extracellular matrix compared to traditional 2D models, with the cardiac field expansively utilising hydrogels to recapitulate disease conditions such as stiffness, encourage alignment of iPSC-CMs and facilitate further model development such as engineered heart tissues (EHTs)

The Utilisation of Hydrogels for iPSC-Cardiomyocyte Research

Cardiac fibroblasts’ (FBs) and cardiomyocytes’ (CMs) behaviour and morphology are influenced by their environment such as remodelling of the myocardium, thus highlighting the importance of biomaterial substrates in cell culture. Biomaterials have emerged as important tools for the development of physiological models, due to the range of adaptable properties of these materials, such as degradability and biocompatibility. Biomaterial hydrogels can act as alternative substrates for cellular studies, which have been particularly key to the progression of the cardiovascular field. This review will focus on the role of hydrogels in cardiac research, specifically the use of natural and synthetic biomaterials such as hyaluronic acid, polydimethylsiloxane and polyethylene glycol for culturing induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). The ability to fine-tune mechanical properties such as stiffness and the versatility of biomaterials is assessed, alongside applications of hydrogels with iPSC-CMs. Natural hydrogels often display higher biocompatibility with iPSC-CMs but often degrade quicker, whereas synthetic hydrogels can be modified to facilitate cell attachment and decrease degradation rates. iPSC-CM structure and electrophysiology can be assessed on natural and synthetic hydrogels, often resolving issues such as immaturity of iPSC-CMs. Biomaterial hydrogels can thus provide a more physiological model of the cardiac extracellular matrix compared to traditional 2D models, with the cardiac field expansively utilising hydrogels to recapitulate disease conditions such as stiffness, encourage alignment of iPSC-CMs and facilitate further model development such as engineered heart tissues (EHTs)

Molecular insights into RBR E3 ligase ubiquitin transfer mechanisms

National Institute of General Medical Sciences R01 GM0880555T32 GM007270, Francis Crick Institute FCI01, Cancer Research UK, Medical Research Council U117565398, Wellcome Trus

Between Hope and Hype: Traditional Knowledge(s) Held by Marginal Communities

Traditional Knowledge (TK) systems have always been integral to the survival and adaptation of human societies. Yet, they enjoy a fairly recent recognition and popularization by scientists, the media, politicians, corporates and the wider public. In this paper we present a typology of key driving forces behind the popularization of TK held by marginal communities: an equality preference motive, a value motive, a compliance motive, a scarcity motive and a strategic motive. Secondly, through the use of a simple model, we discuss the hype's impact on marginal communities. Moreover, we critically assess the outcome of a number of policy instruments that intend, in part, to protect traditional knowledge bases of such communities. Our analysis primarily draws upon secondary literature; policy documents and case studies within economics, the social sciences, conservation biology and legal studies. We argue that whilst the public and institutional hype around TK may have resulted in its prioritization within international conventions and frameworks, its institutionalization may have adversely impacted marginalized communities, and in particular contexts, unintentionally led to the creation of 'new' marginals. We purport that the traditional innovation incentive motive does not hold for protecting TK within a private property regime. Instead we identify a conservation incentive motive and a distribution motive that justify deriving policy instruments that focus on TK to protect marginal communities

Molecular Insights into the Ubiquitin Transfer Mechanism of RING-in-between-RING Ubiquitin ligases

Thesis (Ph.D.)--University of Washington, 2016-12Ubiquitination is a posttranslational modification that regulates virtually every aspect of cellular function in eukaryotes including cell cycle progression, endocytosis, cell signaling, transcription, translation, DNA damage and even autophagy. Substrate modification with ubiquitin (Ub) requires the coordination of two types of enzymes: Ub-conjugating enzymes (E2s) and Ub ligases (E3s). While E3s are generally thought to bind substrates, substrate ubiquitination can be performed by either an E2 or an E3, depending on the type of E3. There are three classes of eukaryotic E3s: the RING (Really Interesting New Gene) E3s, do not contain an active site. They bind E2~Ub and activate Ub transfer directly onto a substrate. RBR (RING-in-between-RING) and HECT (Homologous to E6AP C-Terminus) E3s contain an active site Cys residue to which Ub is transferred from the E2~Ub to generate a covalent E3~Ub thioester that transfers Ub to a substrate. It was only six years ago that a landmark study discovered that RBR E3 are indeed not RING-type Es3 as had been assumed for many years. In 2011, when I began my studies, very little was known about how RBR E3s function. In fact, RBR E3s were originally termed RING-type E3s based on their primary sequence analysis of the two domains: RING1 (binds E2) and RING2 (contains active site). Based on structural work by other groups, we know today that RING2 does not contain a typical RING fold whereas RING1 domains are structurally similar to canonical RINGs. Nevertheless, in my graduate work I demonstrate that RING1 domains perform opposing functions to canonical RING E3s: instead of promoting closed E2~Ub conformations, RING1s actively favor open E2~Ub conformations. This strategy ensures that the transfer of Ub proceeds via the active site Cys on RING2 and therefore that the type of product generated is determined by the RBR E3 and not by the E2 (as done in the context of canonical RING domains). That RBR RING1 domains have opposing functions to canonical RING domains can be explained functionally, yet a structural explanation for this observation is not immediately apparent. I found that a two-residue extension of the second Zn2+-loop - unique to RING1 domains - is largely responsible for promoting open E2~Ubs. Three years ago, an initial study proposed that the RBR E3 HHARI is able to bind to neddylated cullins (N8-CUL) which are RING-type E3s; and that this interaction results in activation of the normally auto-inhibited HHARI. The biological significance of this complex formation was perplexing. In conjunction with others, my work shows that two types of E3s, the RBR E3 HHARI and the RING E3 N8-CUL-1, work together with their respective E2s to coordinately ubiquitinate a common substrate and that this plays an essential role in a developmental pathway in C. elegans. This work was based on the combination of structural, biochemical and organismal studies that led to a deeper understanding of how RBR E3s work. Ubiquitination is a posttranslational modification that regulates virtually every aspect of cellular function in eukaryotes including cell cycle progression, endocytosis, cell signaling, transcription, translation, DNA damage and even autophagy. Substrate modification with ubiquitin (Ub) requires the coordination of two types of enzymes: Ub-conjugating enzymes (E2s) and Ub ligases (E3s). While E3s are generally thought to bind substrates, substrate ubiquitination can be performed by either an E2 or an E3, depending on the type of E3. There are three classes of eukaryotic E3s: the RING (Really Interesting New Gene) E3s, do not contain an active site. They bind E2~Ub and activate Ub transfer directly onto a substrate. RBR (RING-in-between-RING) and HECT (Homologous to E6AP C-Terminus) E3s contain an active site Cys residue to which Ub is transferred from the E2~Ub to generate a covalent E3~Ub thioester that transfers Ub to a substrate. It was only six years ago that a landmark study discovered that RBR E3 are indeed not RING-type Es3 as had been assumed for many years. In 2011, when I began my studies, very little was known about how RBR E3s function. In fact, RBR E3s were originally termed RING-type E3s based on their primary sequence analysis of the two domains: RING1 (binds E2) and RING2 (contains active site). Based on structural work by other groups, we know today that RING2 does not contain a typical RING fold whereas RING1 domains are structurally similar to canonical RINGs. Nevertheless, in my graduate work I demonstrate that RING1 domains perform opposing functions to canonical RING E3s: instead of promoting closed E2~Ub conformations, RING1s actively favor open E2~Ub conformations. This strategy ensures that the transfer of Ub proceeds via the active site Cys on RING2 and therefore that the type of product generated is determined by the RBR E3 and not by the E2 (as done in the context of canonical RING domains). That RBR RING1 domains have opposing functions to canonical RING domains can be explained functionally, yet a structural explanation for this observation is not immediately apparent. I found that a two-residue extension of the second Zn2+-loop - unique to RING1 domains - is largely responsible for promoting open E2~Ubs. Three years ago, an initial study proposed that the RBR E3 HHARI is able to bind to neddylated cullins (N8-CUL) which are RING-type E3s; and that this interaction results in activation of the normally auto-inhibited HHARI. The biological significance of this complex formation was perplexing. In conjunction with others, my work shows that two types of E3s, the RBR E3 HHARI and the RING E3 N8-CUL-1, work together with their respective E2s to coordinately ubiquitinate a common substrate and that this plays an essential role in a developmental pathway in C. elegans. This work was based on the combination of structural, biochemical and organismal studies that led to a deeper understanding of how RBR E3s work

Phosphate starvation signaling increases mitochondrial membrane potential through respiration-independent mechanisms.

Mitochondrial membrane potential directly powers many critical functions of mitochondria, including ATP production, mitochondrial protein import, and metabolite transport. Its loss is a cardinal feature of aging and mitochondrial diseases, and cells closely monitor membrane potential as an indicator of mitochondrial health. Given its central importance, it is logical that cells would modulate mitochondrial membrane potential in response to demand and environmental cues, but there has been little exploration of this question. We report that loss of the Sit4 protein phosphatase in yeast increases mitochondrial membrane potential, both by inducing the electron transport chain and the phosphate starvation response. Indeed, a similarly elevated mitochondrial membrane potential is also elicited simply by phosphate starvation or by abrogation of the Pho85-dependent phosphate sensing pathway. This enhanced membrane potential is primarily driven by an unexpected activity of the ADP/ATP carrier. We also demonstrate that this connection between phosphate limitation and enhancement of mitochondrial membrane potential is observed in primary and immortalized mammalian cells as well as in Drosophila. These data suggest that mitochondrial membrane potential is subject to environmental stimuli and intracellular signaling regulation and raise the possibility for therapeutic enhancement of mitochondrial function even in defective mitochondria