Showing papers in "Biochemical Society Transactions in 2016"

The Warburg effect: 80 years on.

Abstract: Influential research by Warburg and Cori in the 1920s ignited interest in how cancer cells' energy generation is different from that of normal cells. They observed high glucose consumption and large amounts of lactate excretion from cancer cells compared with normal cells, which oxidised glucose using mitochondria. It was therefore assumed that cancer cells were generating energy using glycolysis rather than mitochondrial oxidative phosphorylation, and that the mitochondria were dysfunctional. Advances in research techniques since then have shown the mitochondria in cancer cells to be functional across a range of tumour types. However, different tumour populations have different bioenergetic alterations in order to meet their high energy requirement; the Warburg effect is not consistent across all cancer types. This review will discuss the metabolic reprogramming of cancer, possible explanations for the high glucose consumption in cancer cells observed by Warburg, and suggest key experimental practices we should consider when studying the metabolism of cancer.

The contribution of intrinsically disordered regions to protein function, cellular complexity, and human disease

Abstract: In the 1960s, Christian Anfinsen postulated that the unique three-dimensional structure of a protein is determined by its amino acid sequence. This work laid the foundation for the sequence–structure–function paradigm, which states that the sequence of a protein determines its structure, and structure determines function. However, a class of polypeptide segments called intrinsically disordered regions does not conform to this postulate. In this review, I will first describe established and emerging ideas about how disordered regions contribute to protein function. I will then discuss molecular principles by which regulatory mechanisms, such as alternative splicing and asymmetric localization of transcripts that encode disordered regions, can increase the functional versatility of proteins. Finally, I will discuss how disordered regions contribute to human disease and the emergence of cellular complexity during organismal evolution.

Armored CAR T-cells: utilizing cytokines and pro-inflammatory ligands to enhance CAR T-cell anti-tumour efficacy

Abstract: Chimaeric antigen receptor (CAR) T-cells are T-cells that have been genetically modified to express an artificial construct consisting of a synthetic T-cell receptor (TCR) targeted to a predetermined antigen expressed on a tumour. Coupling the T-cell receptor to a CD3ζ signalling domain paved the way for first generation CAR T-cells that were efficacious against cluster of differentiation (CD)19-expressing B-cell malignancies. Optimization with additional signalling domains such as CD28 or 4-1BB in addition to CD3ζ provided T-cell activation signal 2 and further improved the efficacy and persistence of these second generation CAR T-cells. Third generation CAR T-cells which utilize two tandem costimulatory domains have also been reported. In this review, we discuss a different approach to optimization of CAR T-cells. Through additional genetic modifications, these resultant armored CAR T-cells are typically modified second generation CAR T-cells that have been further optimized to inducibly or constitutively secrete active cytokines or express ligands that further armor CAR T-cells to improve efficacy and persistence. The choice of the ‘armor’ agent is based on knowledge of the tumour microenvironment and the roles of other elements of the innate and adaptive immune system. Although there are several variants of armored CAR T-cells under investigation, here we focus on three unique approaches using interleukin-12 (IL-12), CD40L and 4-1BBL. These agents have been shown to further enhance CAR T-cell efficacy and persistence in the face of a hostile tumour microenvironment via different mechanisms.

Discovery and industrial applications of lytic polysaccharide mono-oxygenases.

Abstract: The recent discovery of copper-dependent lytic polysaccharide mono-oxygenases (LPMOs) has opened up a vast area of research covering several fields of application. The biotech company Novozymes A/S holds patents on the use of these enzymes for the conversion of steam-pre-treated plant residues such as straw to free sugars. These patents predate the correct classification of LPMOs and the striking synergistic effect of fungal LPMOs when combined with canonical cellulases was discovered when fractions of fungal secretomes were evaluated in industrially relevant enzyme performance assays. Today, LPMOs are a central component in the Cellic CTec enzyme products which are used in several large-scale plants for the industrial production of lignocellulosic ethanol. LPMOs are characterized by an N-terminal histidine residue which, together with an internal histidine and a tyrosine residue, co-ordinates a single copper atom in a so-called histidine brace. The mechanism by which oxygen binds to the reduced copper atom has been reported and the general mechanism of copper-oxygen-mediated activation of carbon is being investigated in the light of these discoveries. LPMOs are widespread in both the fungal and the bacterial kingdoms, although the range of action of these enzymes remains to be elucidated. However, based on the high abundance of LPMOs expressed by microbes involved in the decomposition of organic matter, the importance of LPMOs in the natural carbon-cycle is predicted to be significant. In addition, it has been suggested that LPMOs play a role in the pathology of infectious diseases such as cholera and to thus be relevant in the field of medicine.

Mitochondrial fission and fusion.

Abstract: Mitochondrial fission and fusion have been recognized as critical processes in the health of mitochondria and cells. Two decades of studies have generated a great deal of information about mitochondrial fission and fusion; however, still much needs to be understood for the basic molecular mechanisms of these important cellular processes. The core protein factors for mitochondrial fission and fusion are dynamin proteins that possess membrane-remodeling properties. This short review covers a recent development and understanding of the mechanisms by which these mechanochemical enzymes mediate mitochondrial fission and fusion.

Understanding and preventing mitochondrial oxidative damage

Abstract: Mitochondrial oxidative damage has long been known to contribute to damage in conditions such as ischaemia–reperfusion (IR) injury in heart attack. Over the past years, we have developed a series of mitochondria-targeted compounds designed to ameliorate or determine how this damage occurs. I will outline some of this work, from MitoQ to the mitochondria-targeted S -nitrosating agent, called MitoSNO, that we showed was effective in preventing reactive oxygen species (ROS) formation in IR injury with therapeutic implications. In addition, the protection by this compound suggested that ROS production in IR injury was mainly coming from complex I. This led us to investigate the mechanism of the ROS production and using a metabolomic approach, we found that the ROS production in IR injury came from the accumulation of succinate during ischaemia that then drove mitochondrial ROS production by reverse electron transport at complex I during reperfusion. This surprising mechanism led us to develop further new therapeutic approaches to have an impact on the damage that mitochondrial ROS do in pathology and also to explore how mitochondrial ROS can act as redox signals. I will discuss how these approaches have led to a better understanding of mitochondrial oxidative damage in pathology and also to the development of new therapeutic strategies.

The PLK4-STIL-SAS-6 module at the core of centriole duplication.

Abstract: Centrioles are microtubule-based core components of centrosomes and cilia. They are duplicated exactly once during S-phase progression. Central to formation of each new (daughter) centriole is the formation of a nine-fold symmetrical cartwheel structure onto which microtubule triplets are deposited. In recent years, a module comprising the protein kinase polo-like kinase 4 (PLK4) and the two proteins STIL and SAS-6 have been shown to stay at the core of centriole duplication. Depletion of any one of these three proteins blocks centriole duplication and, conversely, overexpression causes centriole amplification. In this short review article, we summarize recent insights into how PLK4, STIL and SAS-6 co-operate in space and time to form a new centriole. These advances begin to shed light on the very first steps of centriole biogenesis.

Sialidases from gut bacteria: a mini-review

Abstract: Sialidases are a large group of enzymes, the majority of which catalyses the cleavage of terminal sialic acids from complex carbohydrates on glycoproteins or glycolipids. In the gastrointestinal (GI) tract, sialic acid residues are mostly found in terminal location of mucins via α2-3/6 glycosidic linkages. Many enteric commensal and pathogenic bacteria can utilize sialic acids as a nutrient source, but not all express the sialidases that are required to release free sialic acid. Sialidases encoded by gut bacteria vary in terms of their substrate specificity and their enzymatic reaction. Most are hydrolytic sialidases, which release free sialic acid from sialylated substrates. However, there are also examples with transglycosylation activities. Recently, a third class of sialidases, intramolecular trans-sialidase (IT-sialidase), has been discovered in gut microbiota, releasing (2,7-anhydro-Neu5Ac) 2,7-anydro-N-acetylneuraminic acid instead of sialic acid. Reaction specificity varies, with hydrolytic sialidases demonstrating broad activity against α2,3-, α2,6- and α2,8-linked substrates, whereas IT-sialidases tend to be specific for α2,3-linked substrates. In this mini-review, we summarize the current knowledge on the structural and biochemical properties of sialidases involved in the interaction between gut bacteria and epithelial surfaces.

Treatment of metastatic renal cell carcinoma (mRCC) with CAIX CAR-engineered T-cells-a completed study overview.

Abstract: We studied safety and proof of concept of a phase I/II trial with chimeric antigen receptor (CAR) T-cells in patients with metastatic renal cell carcinoma (mRCC). The CAR was based on the G250 mAb that recognized an epitope of carboxy-anhydrase-IX (CAIX). Twelve patients with CAIX+ mRCC were treated in three cohorts with a maximum of 10 daily infusions of 2×10 7 to 2×10 9 CAR T-cells. Circulating CAR T-cells were transiently detectable in all patients and maintained antigen-specific immune functions following their isolation post-treatment. Blood cytokine profiles mirrored CAR T-cell presence and in vivo activity. Unfortunately, patients developed anti-CAR T-cell antibodies and cellular immune responses. Moreover, CAR T-cell infusions induced liver enzyme disturbances reaching CTC grades 2–4, which necessitated cessation of treatment in four out of eight patients (cohort 1+2). Examination of liver biopsies revealed T-cell infiltration around bile ducts and CAIX expression on bile duct epithelium, adding to the notion of on-target toxicity. No such toxicities were observed in four patients that were pretreated with G250 mAb (cohort 3). The study was stopped due to the advent of competing treatments before reaching therapeutic or maximum tolerated dose in cohort 3. No clinical responses have been recorded. Despite that, from this trial numerous recommendations for future trials and their immune monitoring could be formulated, such as choice of the target antigen, format and immunogenicity of receptor and how the latter relates to peripheral T-cell persistence.

Emerging evidence of signalling roles for PI(3,4)P2 in Class I and II PI3K-regulated pathways

Abstract: There are eight members of the phosphoinositide family of phospholipids in eukaryotes; PI, PI3P, PI4P, PI5P, PI(4,5) P 2 , PI(3,4) P 2 , PI(3,5) P 2 and PI(3,4,5) P 3 . Receptor activation of Class I PI3Ks stimulates the phosphorylation of PI(4,5) P 2 to form PI(3,4,5) P 3 . PI(3,4,5) P 3 is an important messenger molecule that is part of a complex signalling network controlling cell growth and division. PI(3,4,5) P 3 can be dephosphorylated by both 3- and 5-phosphatases, producing PI(4,5) P 2 and PI(3,4) P 2 , respectively. There is now strong evidence that PI(3,4) P 2 generated by this route does not merely represent another pathway for removal of PI(3,4,5) P 3 , but can act as a signalling molecule in its own right, regulating macropinocytosis, fast endophilin-mediated endocytosis (FEME), membrane ruffling, lamellipodia and invadopodia. PI(3,4) P 2 can also be synthesized directly from PI4P by Class II PI3Ks and this is important for the maturation of clathrin-coated pits [clathrin-mediated endocytosis (CME)] and signalling in early endosomes. Thus PI(3,4) P 2 is emerging as an important signalling molecule involved in the coordination of several specific membrane and cytoskeletal responses. Further, its inappropriate accumulation contributes to pathology caused by mutations in genes encoding enzymes responsible for its degradation, e.g. Inpp4B.

Transcription by RNA polymerase III: insights into mechanism and regulation

Abstract: The highly abundant, small stable RNAs that are synthesized by RNA polymerase III (RNAPIII) have key functional roles, particularly in the protein synthesis apparatus. Their expression is metabolically demanding, and is therefore coupled to changing demands for protein synthesis during cell growth and division. Here, we review the regulatory mechanisms that control the levels of RNAPIII transcripts and discuss their potential physiological relevance. Recent analyses have revealed differential regulation of tRNA expression at all steps on its biogenesis, with significant deregulation of mature tRNAs in cancer cells.

Against the tide: the role of bacterial adhesion in host colonization.

Abstract: Evolving under the constant exposure to an abundance of diverse microbial life, the human body has developed many ways of defining the boundaries between self and non-self. Many physical and immunological barriers to microbial invasion exist, and yet bacteria have found a multitude of ways to overcome these, initiate interactions with and colonize the human host. Adhesion to host cells and tissues is a key feature allowing bacteria to persist in an environment under constant flux and to initiate transient or permanent symbioses with the host. This review discusses reasons why adhesion is such a seemingly indispensable requirement for bacteria–host interactions, and whether bacteria can bypass the need to adhere and still persist. It further outlines open questions about the role of adhesion in bacterial colonization and persistence within the host.

Dichotomous roles of TGF-β in human cancer

Abstract: Transforming growth factor-β (TGF-β) mediates numerous biological processes, including embryonic development and the maintenance of cellular homeostasis in a context-dependent manner. Consistent with its central role in maintaining cellular homeostasis, inhibition of TGF-β signaling results in disruption of normal homeostatic processes and subsequent carcinogenesis, defining the TGF-β signaling pathway as a tumor suppressor. However, once carcinogenesis is initiated, the TGF-β signaling pathway promotes cancer progression. This dichotomous function of the TGF-β signaling pathway is mediated through altering effects on both the cancer cells, by inducing apoptosis and inhibiting proliferation, and the tumor microenvironment, by promoting angiogenesis and inhibiting immunosurveillance. Current studies support inhibition of TGF-β signaling either alone, or in conjunction with anti-angiogenic therapy or immunotherapy as a promising strategy for the treatment of human cancers.

SHP-1: the next checkpoint target for cancer immunotherapy?

Abstract: The immense power of the immune system is harnessed in healthy individuals by a range of negative regulatory signals and checkpoints. Manipulating these checkpoints through inhibition has resulted in striking immune-mediated clearance of otherwise untreatable tumours and metastases; unfortunately, not all patients respond to treatment with the currently available inhibitors of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1). Combinatorial studies using both anti-CTLA-4 and anti-PD-1 demonstrate synergistic effects of targeting multiple checkpoints, paving the way for other immune checkpoints to be targeted. Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1) is a widely expressed inhibitory protein tyrosine phosphatase (PTP). In T-cells, it is a negative regulator of antigen-dependent activation and proliferation. It is a cytosolic protein, and therefore not amenable to antibody-mediated therapies, but its role in activation and proliferation makes it an attractive target for genetic manipulation in adoptive transfer strategies, such as chimeric antigen receptor (CAR) T-cells. This review will discuss the potential value of SHP-1 inhibition in future tumour immunotherapy.

From endolysins to Artilysin®s: novel enzyme-based approaches to kill drug-resistant bacteria

Abstract: One of the last untapped reservoirs in nature for the identification of new anti-microbials is bacteriophages, the natural killers of bacteria. Lytic bacteriophages encode peptidoglycan (PG) lytic enzymes able to degrade the PG layer in different steps of their infection cycle. Endolysins degrade the bacterial cell wall at the end of the infection cycle, causing lysis of the host to release the viral progeny. Recombinant endolysins have been successfully applied as anti-bacterial agent against antibiotic-resistant Gram-positive pathogens. This has boosted the study of these enzymes as new anti-microbials in different fields (e.g. medical, food technology). A key example is the recent development of endolysin-based anti-bacterials against Gram-negative pathogens in which the exogenous application of endolysins is hindered by the outer membrane (OM). These novel anti-microbials, termed Artilysin®s, are able to pass through the OM and reach the PG where they exert their action. In addition, mycobacteria whose cell wall is structurally different from both Gram-positive and Gram-negative bacteria have also been reported to be inhibited by mycobacteriophage-encoded endolysins. Endolysins and endolysin-based anti-microbials can be considered as ideal candidates for an alternative to antibiotics for several reasons: (1) their unique mode of action and activity against bacterial persisters (independent of an active host metabolism), (2) their selective activity against both Gram-positive and Gram-negative pathogens (including antibiotic resistant strains) and mycobacteria, (3) the limited resistance development reported so far. The present review summarizes and discusses the potential applications of endolysins as new anti-microbials.

The SLC28 (CNT) and SLC29 (ENT) nucleoside transporter families: a 30-year collaborative odyssey

Abstract: Specialized nucleoside transporter (NT) proteins are required for passage of nucleosides and hydrophilic nucleoside analogues across biological membranes. Physiologic nucleosides serve as central salvage metabolites in nucleotide biosynthesis, and nucleoside analogues are used as chemotherapeutic agents in the treatment of cancer and antiviral diseases. The nucleoside adenosine modulates numerous cellular events via purino-receptor cell signalling pathways. Human NTs are divided into two structurally unrelated protein families: the SLC28 concentrative nucleoside transporter (CNT) family and the SLC29 equilibrative nucleoside transporter (ENT) family. Human CNTs are inwardly directed Na + -dependent nucleoside transporters found predominantly in intestinal and renal epithelial and other specialized cell types. Human ENTs mediate bidirectional fluxes of purine and pyrimidine nucleosides down their concentration gradients and are ubiquitously found in most, possibly all, cell types. Both protein families are evolutionarily old: CNTs are present in both eukaryotes and prokaryotes; ENTs are widely distributed in mammalian, lower vertebrate and other eukaryote species. This mini-review describes a 30-year collaboration with Professor Stephen Baldwin to identify and understand the structures and functions of these physiologically and clinically important transport proteins.

Role of the colony-stimulating factor (CSF)/CSF-1 receptor axis in cancer.

Abstract: Cancer cells employ a variety of mechanisms to evade apoptosis and senescence. Pre-eminent among these is the aberrant co-expression of growth factors and their ligands, forming an autocrine growth loop that promotes tumour formation and progression. One growth loop whose transforming potential has been repeatedly demonstrated is the CSF-1/CSF-1R axis. Expression of CSF-1 and/or CSF-1R has been documented in a number of human malignancies, including breast, prostate and ovarian cancer and classical Hodgkin9s lymphoma (cHL). This review summarizes the large body of work undertaken to study the role of this cytokine receptor system in malignant transformation. These studies have attributed a key role to the CSF-1/CSF-1R axis in supporting tumour cell survival, proliferation and enhanced motility. Moreover, increasing evidence implicates paracrine interactions between CSF-1 and its receptor in defining a tumour-permissive and immunosuppressive tumour-associated stroma. Against this background, we briefly consider the prospects for therapeutic targeting of this system in malignant disease.

'Nothing of chemistry disappears in biology': the Top 30 damage-prone endogenous metabolites.

Abstract: Many common metabolites are intrinsically unstable and reactive, and hence prone to chemical (i.e. non-enzymatic) damage in vivo Although this fact is widely recognized, the purely chemical side-reactions of metabolic intermediates can be surprisingly hard to track down in the literature and are often treated in an unprioritized case-by-case way. Moreover, spontaneous chemical side-reactions tend to be overshadowed today by side-reactions mediated by promiscuous ('sloppy') enzymes even though chemical damage to metabolites may be even more prevalent than damage from enzyme sloppiness, has similar outcomes, and is held in check by similar biochemical repair or pre-emption mechanisms. To address these limitations and imbalances, here we draw together and systematically integrate information from the (bio)chemical literature, from cheminformatics, and from genome-scale metabolic models to objectively define a 'Top 30' list of damage-prone metabolites. A foundational part of this process was to derive general reaction rules for the damage chemistries involved. The criteria for a 'Top 30' metabolite included predicted chemical reactivity, essentiality, and occurrence in diverse organisms. We also explain how the damage chemistry reaction rules ('operators') are implemented in the Chemical-Damage-MINE (CD-MINE) database (minedatabase.mcs.anl.gov/#/top30) to provide a predictive tool for many additional potential metabolite damage products. Lastly, we illustrate how defining a 'Top 30' list can drive genomics-enabled discovery of the enzymes of previously unrecognized damage-control systems, and how applying chemical damage reaction rules can help identify previously unknown peaks in metabolomics profiles.

The multidrug transporter ABCG2: still more questions than answers.

Abstract: ABCG2 is one of at least three human ATP binding cassette (ABC) transporters which can facilitate the export from cells of a wide range of chemically unrelated drug molecules. This capacity for multidrug transport is not only a confounding factor in chemotherapy, but is also one of the more perplexing phenomena in transporter biochemistry. Since its discovery in the last decade of the 20th century much has been revealed about ABCG2's localization, physiological function and its broad substrate range. There have also been many investigations of its structure and molecular mechanism. In this mini review article we take a Rumsfeldian approach to ABCG2 and essentially ask what we do know about this transporter, and what we will need to know about this transporter if we wish to use modulation of ABCG2 activity as a therapeutic approach.

PHLPPing through history: a decade in the life of PHLPP phosphatases

Abstract: In the decade since their discovery, the PH domain leucine-rich repeat protein phosphatases (PHLPP) have emerged as critical regulators of cellular homeostasis, and their dysregulation is associated with various pathophysiologies, ranging from cancer to degenerative diseases, such as diabetes and heart disease. The two PHLPP isozymes, PHLPP1 and PHLPP2, were identified in a search for phosphatases that dephosphorylate Akt, and thus suppress growth factor signaling. However, given that there are over 200 000 phosphorylated residues in a single cell, and fewer than 50 Ser/Thr protein phosphatases, it is not surprising that PHLPP has many other cellular functions yet to be discovered, including a recently identified role in regulating the epigenome. Both PHLPP1 and PHLPP2 are commonly deleted in human cancers, supporting a tumor suppressive role. Conversely, the levels of one isozyme, PHLPP1, are elevated in diabetes. Thus, mechanisms to correctly control PHLPP activity in cells are critical for normal cellular homeostasis. This review summarizes the known functions of PHLPP and its role in disease.

Learning from microbial strategies for polysaccharide degradation.

Abstract: Complex carbohydrates are ubiquitous in all kingdoms of life. As major components of the plant cell wall they constitute both a rich renewable carbon source for biotechnological transformation into fuels, chemicals and materials, and also form an important energy source as part of a healthy human diet. In both contexts, there has been significant, sustained interest in understanding how microbes transform these substrates. Classical perspectives of microbial polysaccharide degradation are currently being augmented by recent advances in the discovery of lytic polysaccharide monooxygenases (LPMOs) and polysaccharide utilization loci (PULs). Fundamental discoveries in carbohydrate enzymology are both advancing biological understanding, as well as informing applications in industrial biomass conversion and modulation of the human gut microbiota to mediate health benefits.

Structure and function of bacterial H-NS protein.

Abstract: The histone-like nucleoid structuring (H-NS) protein is a major component of the folded chromosome in Escherichia coli and related bacteria. Functions attributed to H-NS include management of genome evolution, DNA condensation, and transcription. The wide-ranging influence of H-NS is remarkable given the simplicity of the protein, a small peptide, possessing rudimentary determinants for self-association, hetero-oligomerisation and DNA binding. In this review, I will discuss our understanding of H-NS with a focus on these structural elements. In particular, I will consider how these interaction surfaces allow H-NS to exert its different effects.

The importance of ribosome production, and the 5S RNP–MDM2 pathway, in health and disease

Abstract: Ribosomes are abundant, large RNA–protein complexes that are the source of all protein synthesis in the cell. The production of ribosomes is an extremely energetically expensive cellular process that has long been linked to human health and disease. More recently, it has been shown that ribosome biogenesis is intimately linked to multiple cellular signalling pathways and that defects in ribosome production can lead to a wide variety of human diseases. Furthermore, changes in ribosome production in response to nutrient levels in the diet lead to metabolic re-programming of the liver. Reduced or abnormal ribosome production in response to cellular stress or mutations in genes encoding factors critical for ribosome biogenesis causes the activation of the tumour suppressor p53, which leads to re-programming of cellular transcription. The ribosomal assembly intermediate 5S RNP (ribonucleoprotein particle), containing RPL5, RPL11 and the 5S rRNA, accumulates when ribosome biogenesis is blocked. The excess 5S RNP binds to murine double minute 2 (MDM2), the main p53-suppressor in the cell, inhibiting its function and leading to p53 activation. Here, we discuss the involvement of ribosome biogenesis in the homoeostasis of p53 in the cell and in human health and disease.

Selection platforms for directed evolution in synthetic biology

Abstract: Life on Earth is incredibly diverse. Yet, underneath that diversity, there are a number of constants and highly conserved processes: all life is based on DNA and RNA; the genetic code is universal; biology is limited to a small subset of potential chemistries. A vast amount of knowledge has been accrued through describing and characterizing enzymes, biological processes and organisms. Nevertheless, much remains to be understood about the natural world. One of the goals in Synthetic Biology is to recapitulate biological complexity from simple systems made from biological molecules–gaining a deeper understanding of life in the process. Directed evolution is a powerful tool in Synthetic Biology, able to bypass gaps in knowledge and capable of engineering even the most highly conserved biological processes. It encompasses a range of methodologies to create variation in a population and to select individual variants with the desired function–be it a ligand, enzyme, pathway or even whole organisms. Here, we present some of the basic frameworks that underpin all evolution platforms and review some of the recent contributions from directed evolution to synthetic biology, in particular methods that have been used to engineer the Central Dogma and the genetic code.

Phosphatidylinositol 3,5-bisphosphate: regulation of cellular events in space and time

Abstract: Phosphorylated phosphatidylinositol lipids are crucial for most eukaryotes and have diverse cellular functions. The low-abundance signalling lipid phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] is critical for cellular homoeostasis and adaptation to stimuli. A large complex of proteins that includes the lipid kinase Fab1-PIKfyve, dynamically regulates the levels of PI(3,5)P2. Deficiencies in PI(3,5)P2 are linked to some human diseases, especially those of the nervous system. Future studies will probably determine new, undiscovered regulatory roles of PI(3,5)P2, as well as uncover mechanistic insights into how PI(3,5)P2 contributes to normal human physiology.

The control of inflammation via the phosphorylation and dephosphorylation of tristetraprolin: a tale of two phosphatases

Abstract: Twenty years ago, the first description of a tristetraprolin (TTP) knockout mouse highlighted the fundamental role of TTP in the restraint of inflammation. Since then, work from several groups has generated a detailed picture of the expression and function of TTP. It is a sequence-specific RNA-binding protein that orchestrates the deadenylation and degradation of several mRNAs encoding inflammatory mediators. It is very extensively post-translationally modified, with more than 30 phosphorylations that are supported by at least two independent lines of evidence. The phosphorylation of two particular residues, serines 52 and 178 of mouse TTP (serines 60 and 186 of the human orthologue), has profound effects on the expression, function and localisation of TTP. Here, we discuss the control of TTP biology via its phosphorylation and dephosphorylation, with a particular focus on recent advances and on questions that remain unanswered.

Construction of membrane-bound artificial cells using microfluidics: a new frontier in bottom-up synthetic biology

Abstract: The quest to construct artificial cells from the bottom-up using simple building blocks has received much attention over recent decades and is one of the grand challenges in synthetic biology. Cell mimics that are encapsulated by lipid membranes are a particularly powerful class of artificial cells due to their biocompatibility and the ability to reconstitute biological machinery within them. One of the key obstacles in the field centres on the following: how can membrane-based artificial cells be generated in a controlled way and in high-throughput? In particular, how can they be constructed to have precisely defined parameters including size, biomolecular composition and spatial organization? Microfluidic generation strategies have proved instrumental in addressing these questions. This article will outline some of the major principles underpinning membrane-based artificial cells and their construction using microfluidics, and will detail some recent landmarks that have been achieved.

Xylan decoration patterns and the plant secondary cell wall molecular architecture.

Abstract: The molecular architecture of plant secondary cell walls is still not resolved. There are several proposed structures for cellulose fibrils, the main component of plant cell walls and the conformation of other molecules is even less well known. Glucuronic acid (GlcA) substitution of xylan (GUX) enzymes, in CAZy family glycosyl transferase (GT)8, decorate the xylan backbone with various specific patterns of GlcA. It was recently discovered that dicot xylan has a domain with the side chain decorations distributed on every second unit of the backbone (xylose). If the xylan backbone folds in a similar way to glucan chains in cellulose (2-fold helix), this kind of arrangement may allow the undecorated side of the xylan chain to hydrogen bond with the hydrophilic surface of cellulose microfibrils. MD simulations suggest that such interactions are energetically stable. We discuss the possible role of this xylan decoration pattern in building of the plant cell wall.

Non-coding RNAs and disease: the classical ncRNAs make a comeback.

Abstract: Many human diseases have been attributed to mutation in the protein coding regions of the human genome. The protein coding portion of the human genome, however, is very small compared with the non-coding portion of the genome. As such, there are a disproportionate number of diseases attributed to the coding compared with the non-coding portion of the genome. It is now clear that the non-coding portion of the genome produces many functional non-coding RNAs and these RNAs are slowly being linked to human diseases. Here we discuss examples where mutation in classical non-coding RNAs have been attributed to human disease and identify the future potential for the non-coding portion of the genome in disease biology.

Synthetic biology meets bioprinting: enabling technologies for humans on Mars (and Earth)

Abstract: Human exploration off planet is severely limited by the cost of launching materials into space and by re-supply. Thus materials brought from Earth must be light, stable and reliable at destination. Using traditional approaches, a lunar or Mars base would require either transporting a hefty store of metals or heavy manufacturing equipment and construction materials for in situ extraction; both would severely limit any other mission objectives. Long-term human space presence requires periodic replenishment, adding a massive cost overhead. Even robotic missions often sacrifice science goals for heavy radiation and thermal protection. Biology has the potential to solve these problems because life can replicate and repair itself, and perform a wide variety of chemical reactions including making food, fuel and materials. Synthetic biology enhances and expands life's evolved repertoire. Using organisms as feedstock, additive manufacturing through bioprinting will make possible the dream of producing bespoke tools, food, smart fabrics and even replacement organs on demand. This new approach and the resulting novel products will enable human exploration and settlement on Mars, while providing new manufacturing approaches for life on Earth.