Optimising the function of a protein of length N amino acids by directed evolution involves navigating a ‘search space’ of possible sequences of some 20N. Optimising the expression levels of P proteins that materially affect host performance, each of which might also take 20 (logarithmically spaced) values, implies a similar search space of 20P. In this combinatorial sense, then, the problems of directed

Sterol Regulatory Element Binding Protein-1c is a transcription factor that controls the synthesis of lipids from glucose in the liver, a process which is of utmost importance for the storage of energy. Discovered in the early nineties by B. Spiegelman and by M. Brown and J. Goldstein, it has generated more than 5000 studies in order to elucidate its mechanism of activation and its role in physiology

The cystic fibrosis transmembrane conductance regulator (CFTR) gene lies within a topologically associated domain (TAD) in which multiple cis-regulatory elements (CREs) and transcription factors (TFs) regulate its cell-specific expression. The CREs are recruited to the gene promoter by a looping mechanism that depends upon both architectural proteins and specific TFs. An siRNA screen to identify TFs

Sphingolipid-mediated regulation in cancer development and treatment is largely ceramide-centered with the complex sphingolipid metabolic pathways unfolding as attractive targets for anticancer drug discovery. The dynamic interconversion of sphingolipids is tightly controlled at the level of enzymes and cellular compartments in response to endogenous or exogenous stimuli, such as anticancer drugs,

The spatial distribution of proteins in cell membranes is crucial for signal transduction, cell communication and membrane trafficking. Members of the Tetraspanin family organize functional protein clusters within the plasma membrane into so-called Tetraspanin-enriched microdomains (TEMs). Direct interactions between Tetraspanins are believed to be important for this organization. However, studies

Several Schistosoma species cause Schistosomiasis, an endemic disease in 78 countries that is ranked second amongst the parasitic diseases in terms of its socioeconomic impact and human health importance. The drug recommended for treatment by the WHO is praziquantel (PZQ), but there are concerns associated with PZQ, such as the lack of information about its exact mechanism of action, its high price

COVID-19, the clinical syndrome caused by the SARS-CoV-2 virus, has rapidly spread globally causing hundreds of millions of infections and over two million deaths. The potential animal reservoirs for SARS-CoV-2 are currently unknown, however sequence analysis has provided plausible potential candidate species. SARS-CoV-2 binds to the angiotensin I converting enzyme 2 (ACE2) to enable its entry into

Histidine phosphorylation is an important and ubiquitous post-translational modification. Histidine undergoes phosphorylation on either of the nitrogens in its imidazole side chain, giving rise to 1- and 3- phosphohistidine (pHis) isomers, each having a phosphoramidate linkage that is labile at high temperatures and low pH, in contrast with stable phosphomonoester protein modifications. While all organisms

The hetero-oligomeric retinoid oxidoreductase complex (ROC) catalyzes the interconversion of all-trans-retinol and all-trans-retinaldehyde to maintain the steady-state output of retinaldehyde, the precursor of all-trans-retinoic acid that regulates the transcription of numerous genes. The interconversion is catalyzed by two distinct components of the ROC: the NAD(H)-dependent retinol dehydrogenase

Methylation of lysine residues plays crucial roles in a wide variety of cell signaling processes. While the biological importance of recognition of methylated histones by reader domains in the cell nucleus is well established, the processes associated with methylation of non-histone proteins, particularly in the cytoplasm of the cell, are not well understood. Here, we describe a search for potential

Hydrogen sulfide (H2S) modulates many biological processes, including ageing. Initially considered a hazardous toxic gas, it is now recognised that H2S is produced endogenously across taxa and is a key mediator of processes that promote longevity and improve late-life health. In this review, we consider the key developments in our understanding of this gaseous signalling molecule in the context of

DJ-1 is known to play neuroprotective roles by eliminating reactive oxygen species (ROS) as an antioxidant protein. However, the molecular mechanism of DJ-1 function has not been well elucidated. This study explored the structural and functional changes of DJ-1 in response to oxidative stress. Human DJ-1 has three cysteine residues (Cys46, Cys53 and Cys106). We found that, in addition to Cys106, Cys46

We have been developing bacterial expression systems for human mucin-type O-glycosylation on therapeutic proteins, which is initiated by the addition of α-linked GalNAc to serine or threonine residues by enzymes in the GT-27 family of glycosyltransferases. Substrate preference across different isoforms of this enzyme is influenced by isoform-specific amino acid sequences at the site of glycosylation

Calpain proteolysis contributes to the pathogenesis of heart failure but the calpain isoforms responsible and their substrate specificities have not been rigorously defined. One substrate, Junctophilin-2 (JP2), is essential for maintaining junctional cardiac dyads and excitation-contraction coupling. We previously demonstrated that mouse JP2 is cleaved by calpain-1 (CAPN1) between Arginine 565 (R565)

Much effort has been devoted to the development of selective inhibitors of the LRRK2 as a potential treatment for LRRK2 driven Parkinson's disease. In this study, we first compare the properties of Type I (GSK3357679A and MLi-2) and Type II (GZD-824, Rebastinib and Ponatinib) kinase inhibitors that bind to the closed or open conformations of the LRRK2 kinase domain, respectively. We show that Type

Mutations in breast cancer type 1 susceptibility protein (BRCA1) and its heterodimeric binding partner BARD1 confer a high risk for the development of breast and ovarian cancers. The sole enzymatic function of the BRCA1/BARD1 complex is as a RING-type E3 ubiquitin (Ub) ligase, leading to the deposition of Ub signals onto a variety of substrate proteins. Distinct types of Ub signals deposited by BRCA1/BARD1

The ubiquitin-proteasome pathway (UPP) and autophagy play integral roles in cellular homeostasis. As part of their normal life cycle, most proteins undergo ubiquitination for some form of redistribution, localization and/or functional modulation. However, ubiquitination is also important to the UPP and several autophagic processes. The UPP is initiated after specific lysine residues of short-lived

Nitroreductases catalyse the NAD(P)H-dependent nitro reduction in nitrofuran antibiotics, which activates them into cytotoxic molecules leading to cell death. The design of new effective nitrofuran antibiotics relies on knowledge of the kinetic mechanism and nitrofuran binding mode of microbial nitroreductases NfsA and NfsB. This has been hampered by multiple co-crystallisation studies revealing ligand

Phospholipid synthesis is crucial for membrane proliferation in malaria parasites during the entire cycle in the host cell. The major phospholipid of parasite membranes, phosphatidylcholine (PC), is mainly synthesized through the Kennedy pathway. The phosphocholine required for this synthetic pathway is generated by phosphorylation of choline derived from the catabolism of the lyso-phosphatidylcholine

OTU proteases antagonize the cellular defense in the host cells and involve in pathogenesis. Intriguingly, P. falciparum, P. vivax, and P. yoelii have an uncharacterized and highly conserved viral OTU-like proteins. However, their structure, function or inhibitors have not been previously reported. To this end, we have performed structural modeling, small molecule screening, deconjugation assays to

Cancer metastasis remains a major clinical challenge for cancer treatment. It is therefore crucial to understand how cancer cells establish and maintain their metastatic traits. However, metastasis-specific genetic mutations have not been identified in most exome or genome sequencing studies. Emerging evidence suggests that key steps of metastasis are controlled by reversible epigenetic mechanisms

Alzheimer's Disease (AD) and Type 2 Diabetes (T2D) share a common hallmark of insulin resistance. Reportedly, two non-canonical Receptor Tyrosine Kinases (RTKs), ALK and RYK, both targets of the same micro RNA miR-1271, exhibit significant and consistent functional down-regulation in post-mortem AD and T2D tissues. Incidentally, both have Grb2 as a common downstream adapter and NOX4 as a common ROS

Angiotensinogen fine-tunes the tightly controlled activity of the renin-angiotensin system by modulating the release of angiotensin peptides that control blood pressure. One mechanism by which this modulation is achieved is via angiotensinogen's Cys18–Cys138 disulfide bond that acts as a redox switch. Molecular dynamics simulations of each redox state of angiotensinogen reveal subtle dynamic differences

Co-signaling receptors for the T cell receptor (TCR) are important therapeutic targets, with blockade of co-inhibitory receptors such as PD-1 now central in immuno-oncology. Advancing additional therapeutic immune modulation approaches requires understanding ligand regulation of other co-signaling receptors. One poorly understood potential therapeutic target is TIM-3 (T cell immunoglobulin and mucin

EphB6 and EphA10 are two poorly characterised pseudokinase members of the Eph receptor family, which collectively serves as mediators of contact-dependent cell–cell communication to transmit extracellular cues into intracellular signals. As per their active counterparts, EphB6 and EphA10 deregulation is strongly linked to proliferative diseases. However, unlike active Eph receptors, whose catalytic

Lactate is the main product generated at the end of anaerobic glycolysis or during the Warburg effect and its role as an active signalling molecule is increasingly recognised. Lactate can be released and used by host cells, by pathogens and commensal organisms, thus being essential for the homeostasis of host–microbe interactions. Infection can alter this intricate balance, and the presence of lactate

Apoptosis is a cell death program that is executed by the caspases, a family of cysteine proteases that typically cleave after aspartate residues during a proteolytic cascade that systematically dismantles the dying cell. Extensive signaling crosstalk occurs between caspase-mediated proteolysis and kinase-mediated phosphorylation, enabling integration of signals from multiple pathways into the decision

p97 protein is a highly conserved, abundant, functionally diverse, structurally dynamic homohexameric AAA enzyme-containing N, D1, and D2 domains. A truncated p97 protein containing the N and D1 domains and the D1–D2 linker (ND1L) exhibits 79% of wild-type (WT) ATPase activity whereas the ND1 domain alone without the linker only has 2% of WT activity. To investigate the relationship between the D1–D2

Recent advances in genome sequencing have led to the identification of new ion and metabolite transporters, many of which have not been characterized. Due to the variety of subcellular localizations, cargo and transport mechanisms, such characterization is a daunting task, and predictive approaches focused on the functional context of transporters are very much needed. Here we present a case for identifying

The lysosomal degradation of heparan sulfate is mediated by the concerted action of nine different enzymes. Within this degradation pathway, Arylsulfatase G (ARSG) is critical for removing 3-O-sulfate from glucosamine, and mutations in ARSG are causative for Usher syndrome type IV. We developed a specific ARSG enzyme assay using sulfated monosaccharide substrates, which reflect derivatives of its natural

Ferrochelatase catalyzes the insertion of ferrous iron into a porphyrin macrocycle to produce the essential cofactor, heme. In humans this enzyme not only catalyzes the terminal step, but also serves a regulatory step in the heme synthesis pathway. Over a dozen crystal structures of human ferrochelatase have been solved and many variants have been characterized kinetically. In addition, hydrogen deuterium

The reaction centre light-harvesting 1 (RC–LH1) complex is the core functional component of bacterial photosynthesis. We determined the cryo-electron microscopy (cryo-EM) structure of the RC–LH1 complex from Rhodospirillum rubrum at 2.5 Å resolution, which reveals a unique monomeric bacteriochlorophyll with a phospholipid ligand in the gap between the RC and LH1 complexes. The LH1 complex comprises

Plant and fungal THI4 thiazole synthases produce the thiamin thiazole moiety in aerobic conditions via a single-turnover suicide reaction that uses an active-site Cys residue as sulfur donor. Multiple-turnover (i.e. catalytic) THI4s lacking an active-site Cys (non-Cys THI4s) that use sulfide as sulfur donor have been biochemically characterized —– but only from archaeal methanogens that are anaerobic

The [4Fe-4S] cluster containing scaffold complex HypCD is the central construction site for the assembly of the [Fe](CN)2CO cofactor precursor of [NiFe]-hydrogenase. While the importance of the HypCD complex is well established, not much is known about the mechanism by which the CN− and CO ligands are transferred and attached to the iron ion. We report an efficient expression and purification system

Autophagy receptor p62/SQSTM1 signals a complex network that links autophagy-lysosomal system to proteasome. Phosphorylation of p62 on Serine 349 (P-Ser349 p62) is involved in a cell protective, antioxidant pathway. We have shown previously that P-Ser349 p62 occurs and is rapidly degraded during human synovial fibroblasts autophagy. In this work we observed that fingolimod (FTY720), used as a medication

Clathrin is best known for its contribution to clathrin-mediated endocytosis yet it also participates to a diverse range of cellular functions. Key to this is clathrin's ability to assemble into polyhedral lattices that include curved football or basket shapes, flat lattices or even tubular structures. In this review, we discuss clathrin structure and coated vesicle formation, how clathrin is utilised

Mitochondria import about 1000 proteins that are produced as precursors on cytosolic ribosomes. Defects in mitochondrial protein import result in the accumulation of non-imported precursor proteins and proteotoxic stress. The cell is equipped with different quality control mechanisms to monitor protein transport into mitochondria. First, molecular chaperones guide unfolded proteins to mitochondria

Plant cell walls are highly dynamic structures that are composed predominately of polysaccharides. As such, endogenous carbohydrate active enzymes (CAZymes) are central to the synthesis and subsequent modification of plant cells during morphogenesis. The endo-glucanase 16 (EG16) members constitute a distinct group of plant CAZymes, angiosperm orthologs of which were recently shown to have dual β-glucan/xyloglucan

DevR/DosR response regulator is believed to participate in virulence, dormancy adaptation and antibiotic tolerance mechanisms of Mycobacterium tuberculosis by regulating the expression of the dormancy regulon. We have previously shown that the interaction of DevR with RNA polymerase is essential for the expression of DevR-regulated genes. Here, we developed a M. tuberculosis-specific in vivo transcription

Many protein misfolding diseases (e.g. type II diabetes and Alzheimer's disease) are characterised by amyloid deposition. Human islet amyloid polypeptide (hIAPP, involved in type II diabetes) spontaneously undergoes liquid–liquid phase separation (LLPS) and a kinetically complex hydrogelation, both catalysed by hydrophobic–hydrophilic interfaces (e.g. air–water interface and/or phospholipids–water

Activation of enzymes by monovalent cations (M+) is a widespread phenomenon in biology. Despite this, there are few structure-based studies describing the underlying molecular details. Thiolases are a ubiquitous and highly conserved family of enzymes containing both K+-activated and K+-independent members. Guided by structures of naturally occurring K+-activated thiolases, we have used a structure-based

The Unfolded Protein response is an adaptive pathway triggered upon alteration of endoplasmic reticulum (ER) homeostasis. It is transduced by three major ER stress sensors, among which the Inositol Requiring Enzyme 1 (IRE1) is the most evolutionarily conserved. IRE1 is an ER-resident type I transmembrane protein exhibiting an ER luminal domain that senses the protein folding status and a catalytic

SBI-0206965, originally identified as an inhibitor of the autophagy initiator kinase ULK1, has recently been reported as a more potent and selective AMP-activated protein kinase (AMPK) inhibitor relative to the widely used, but promiscuous inhibitor Compound C/Dorsomorphin. Here, we studied the effects of SBI-0206965 on AMPK signalling and metabolic readouts in multiple cell types, including hepatocytes

We recently described a signal transduction pathway that contributes to androgen receptor (AR) regulation based on site-specific ADP-ribosylation by PARP7, a mono-ADP-ribosyltransferase implicated in several human cancers. ADP-ribosylated AR is recognized by PARP9/DTX3L, a heterodimeric complex that contains an ADP-ribose reader (PARP9) and a ubiquitin E3 ligase (DTX3L). Here, we have characterized

This perspective article describes some of the key points of my personal journey through the intriguing world of intrinsically disordered proteins (IDPs). It also shows the evolution of my perception of functional proteins from a standard lock-and-key theory, where a unique function is defined by a unique 3D structure, to the structure–function continuum model, where the structural heterogeneity and

Since the discovery of mutations in leucine-rich repeat kinase 2 (LRRK2) as an underlying genetic cause for the development of Parkinson's disease (PD) in 2004 (Neuron 44, 601–607; Neuron 44, 595–600), and subsequent efforts to develop LRRK2 kinase inhibitors as a therapy for Parkinson's (Expert Opin. Ther. Targets 21, 751–753), elucidating the atomic resolution structure of LRRK2 has been a major

The sum total of life course exposures creates an exposome that has a significant impact on age-related health. Understanding the interplay between exposome factors and the (epi) genome, offers pertinent insights into the ageing process and its relationship with the accumulation of allostatic load. We propose to exploit this to develop a biomimetic approach that will provide insight into how evolution

Cancer stem cells (CSCs) are involved in the initiation and progression of human malignancies by enabling cancer tissue self-renewal capacity and constituting the therapy-resistant population of tumor cells. However, despite the exhausting characterization of CSC genetics, epigenetics, and kinase signaling, eradication of CSCs remains an unattainable goal in most human malignancies. While phosphatases

Secretion of misfolded tau, a microtubule-binding protein enriched in nerve cells, is linked to the progression of tau pathology. However, the molecular mechanisms underlying tau secretion are poorly understood. Recent work by Lee et al. [Biochemical J. (2021) 478: 1471–1484] demonstrated that the transmembrane domains of syntaxin6 and syntaxin8 could be exploited for tau release, setting a stage for

The release of glucose from lignocellulosic waste for subsequent fermentation into biofuels holds promise for securing humankind's future energy needs. The discovery of a set of copper-dependent enzymes known as lytic polysaccharide monooxygenases (LPMOs) has galvanised new research in this area. LPMOs act by oxidatively introducing chain breaks into cellulose and other polysaccharides, boosting the

Neurodegenerative diseases such as Alzheimer's and Parkinson's remain highly prevalent and incurable disorders. A major challenge in fully understanding and combating the progression of these diseases is the complexity of the network of processes that lead to progressive neuronal dysfunction and death. An ideal therapeutic avenue is conceivably one that could address many if not all of these multiple

Reversible protein phosphorylation is a widespread post-translational modification fundamental for signaling across all domains of life. Tyrosine (Tyr) phosphorylation has recently emerged as being important for plant receptor kinase (RK)-mediated signaling, particularly during plant immunity. How Tyr phosphorylation regulates RK function is however largely unknown. Notably, the expansion of protein

Plants are surrounded by a vast diversity of microorganisms. Limiting pathogenic microorganisms is crucial for plant survival. On the other hand, the interaction of plants with beneficial microorganisms promotes their growth or allows them to overcome nutrient deficiencies. Balancing the number and nature of these interactions is crucial for plant growth and development, and thus, for crop productivity

Post-translational modifications (PTMs) on histone proteins are known as epigenetic marks that demarcate the status of chromatin. These modifications are ‘read' by specific reader proteins, which in turn recruit additional factors to modulate chromatin accessibility and the activity of the underlying DNA. Accumulating evidence suggests that these modifications are not restricted solely to histones

Growth factor receptor-bound protein 2 (GRB2) is a trivalent adaptor protein and a key element in signal transduction. It interacts via its flanking nSH3 and cSH3 domains with the proline-rich domain (PRD) of the RAS activator SOS1 and via its central SH2 domain with phosphorylated tyrosine residues of receptor tyrosine kinases (RTKs; e.g. HER2). The elucidation of structural organization and mechanistic

The human protein kinase ULK3 regulates the timing of membrane abscission, thus being involved in exosome budding and cytokinesis. Herein, we present the first high-resolution structures of the ULK3 kinase domain. Its unique features are explored against the background of other ULK kinases. An inhibitor fingerprint indicates that ULK3 is highly druggable and capable of adopting a wide range of conformations

Cullin ubiquitin ligases drive replisome disassembly during DNA replication termination. In worm, frog and mouse cells, CUL2LRR1 is required to ubiquitylate the MCM7 subunit of the CMG helicase. Here, we show that cullin ligases also drive CMG-MCM7 ubiquitylation in human cells, thereby making the helicase into a substrate for the p97 unfoldase. Using purified human proteins, including a panel of E2

The interaction of insect-selective scorpion depressant β-toxins (LqhIT2 and Lqh-dprIT3 from Leiurus quinquestriatus hebraeus) with the Blattella germanica sodium channel, BgNav1-1a, was investigated using site-directed mutagenesis, electrophysiological analyses, and structural modeling. Focusing on the pharmacologically defined binding site-4 of scorpion β-toxins at the voltage-sensing domain II (VSD-II)

The redox potential values of cytochromes can be modulated by the protonation/deprotonation of neighbor groups (redox-Bohr effect), a mechanism that permits the proteins to couple electron/proton transfer. In the respiratory chains, this effect is particularly relevant if observed in the physiological pH range, as it may contribute to the electrochemical gradient for ATP synthesis. A constitutively

Cells must adapt to changes in their environment to maintain cell, tissue and organismal integrity in the face of mechanical, chemical or microbiological stress. Nuclear factor-κB (NF-κB) is one of the most important transcription factors that controls inducible gene expression as cells attempt to restore homeostasis. It plays critical roles in the immune system, from acute inflammation to the development