The study of cerebellar development has been at the forefront of neuroscience since the pioneering work of Wilhelm His Sr., Santiago Ramón y Cajal and many others since the 19th century. They laid the foundation to identify the circuitry of the cerebellum, already revealing its stereotypic three-layered cortex and discerning several of its neuronal components. Their work was fundamental in the acceptance of the neuron doctrine, which acknowledges the key role of individual neurons in forming the basic units of the nervous system. Increasing evidence shows that the cerebellum performs a variety of homeostatic and higher order neuronal functions beyond the mere control of motor behaviour. Over the last three decades, many studies have revealed the molecular machinery that regulates distinct aspects of cerebellar development, from the establishment of a cerebellar anlage in the posterior brain to the identification of cerebellar neuron diversity at the single cell level. In this review, we focus on summarizing our current knowledge on early cerebellar development with a particular emphasis on the molecular determinants that secure neuron specification and contribute to the diversity of cerebellar neurons.

Cell-cell communication is an essential aspect of multicellular life, key for coordinating cell proliferation, growth, and death in response to environmental changes. Whilst caspases are well-known for facilitating apoptotic and pyroptotic cell death, several recent investigations are uncovering new roles for these enzymes in biological scenarios requiring long-range intercellular signalling mediated by extracellular vesicles (EVs). EVs are small membrane-bound nanoparticles released from cells that may carry and deliver cargo between distant cells, thus helping to coordinate their behaviour. Intriguingly, there is emerging evidence indicating a key contribution of caspases in the biogenesis of EVs, the selection of their cargo content, and EV uptake/function in recipient cells. Here, we discuss the latest findings supporting the interplay between caspases and EVs, and the biological relevance of this molecular convergence for cellular signalling, principally in non-apoptotic scenarios.

Flaviviruses comprise a number of mosquito- or tick-transmitted human pathogens of global public health importance. Advances in structural biology techniques have contributed substantially to our current understanding of the life cycle of these small enveloped RNA viruses and led to deep insights into details of virus assembly, maturation and cell entry. In addition to large-scale conformational changes and oligomeric rearrangements of envelope proteins during these processes, there is increasing evidence that smaller-scale protein dynamics (referred to as virus "breathing") can confer extra flexibility to these viruses for the fine-tuning of their interactions with the immune system and possibly with cellular factors they encounter in their complex ecological cycles in arthropod and vertebrate hosts. In this review, we discuss how work with tick-borne encephalitis virus has extended our view on flavivirus breathing, leading to the identification of a novel mechanism of antibody-mediated infection enhancement and demonstrating breathing intermediates of the envelope protein in the process of membrane fusion. These data are discussed in the context of other flaviviruses and the perspective of a potential role of virus breathing to cope with the requirements of adaptation and replication in evolutionarily very different hosts.

Cytokines are soluble factors vital for mammalian physiology. Cytokines elicit highly pleiotropic activities, characterized by their ability to induce a wide spectrum of functional responses in a diverse range of cell subsets, which makes their study very challenging. Cytokines activate signalling via receptor dimerization/oligomerization, triggering activation of the JAK (Janus kinase)/STAT (signal transducer and activator of transcription) signalling pathway. Given the strong crosstalk and shared usage of key components of cytokine signalling pathways, a long-standing question in the field pertains to how functional diversity is achieved by cytokines. Here, we discuss how biophysical - for example, ligand-receptor binding affinity and topology - and cellular - for example, receptor, JAK and STAT protein levels, endosomal compartment - parameters contribute to the modulation and diversification of cytokine responses. We review how these parameters ultimately converge into a common mechanism to fine-tune cytokine signalling that involves the control of the number of Tyr residues phosphorylated in the receptor intracellular domain upon cytokine stimulation. This results in different kinetics of STAT activation, and induction of specific gene expression programs, ensuring the generation of functional diversity by cytokines using a limited set of signalling intermediaries. We describe how these first principles of cytokine signalling have been exploited using protein engineering to design cytokine variants with more specific and less toxic responses for immunotherapy.

Post-transcriptional regulation of messenger RNAs (mRNAs) (i.e., mechanisms that control translation, stability and localization) is a critical focal point in spatiotemporal regulation of gene expression in response to changes in environmental conditions. The human genome encodes ~ 2000 microRNAs (miRNAs), each of which could control the expression of hundreds of protein-coding mRNAs by inducing translational repression and/or promoting mRNA decay. While mRNA degradation is a terminal event, translational repression is reversible and can be employed for rapid response to internal or external cues. Recent years have seen significant progress in our understanding of how miRNAs induce degradation or translational repression of the target mRNAs. Here, we review the recent findings that illustrate the cellular machinery that contributes to miRNA-induced silencing, with a focus on the factors that could influence translational repression vs. decay.

Kinases are key regulatory signalling proteins governing numerous essential biological processes and cellular functions. Dysregulation of many protein kinases is associated with cancer initiation and progression. Given their crucial roles, there has been increasing interest in harnessing kinases as prospective drug targets for cancer. In recent decades, numerous small-molecule kinase inhibitors have been developed and revolutionized the cancer treatment landscape. Despite their great potential, challenges remain in developing highly selective and effective kinase inhibitors, with toxicity and resistance issues frequently arising. In this review, we first provide an overview of the role of kinases in carcinogenesis and describe the current progress with small-molecule kinase inhibitors that have been approved for clinical use. We then discuss the application of mass spectrometry (MS)-based proteomics strategies to help in the design of kinase inhibitors. Finally, we discuss the challenges and outlook concerning MS-based proteomics techniques for kinase drug research.

Hedgehog signalling is essential for development, crucial for normal anatomical arrangement and activated during tissue damage repair. Dysregulation of hedgehog signalling is associated with cancer, developmental disorders and other diseases including osteoarthritis (OA). The hedgehog gene was first discovered in Drosophila melanogaster, and the pathway is evolutionarily conserved in most animals. Although there are several hedgehog ligands with different protein expression patterns, they share a common plasma membrane receptor, Patched1 and hedgehog signalling pathway activation is transduced through the G-protein-coupled receptor-like protein Smoothened (SMO) and downstream effectors. Functional assays revealed that activation of SMO is dependent on sterol binding, and cholesterol was observed bound to SMO in crystallography experiments. In vertebrates, hedgehog signalling coordinates endochondral ossification and balances osteoblast and osteoclast activation to maintain homeostasis. A recently discovered mutation of SMO in humans (SMOR173C ) is predicted to alter cholesterol binding and is associated with a higher risk of hip OA. Functional studies in mice and human tissue analysis provide evidence that hedgehog signalling is pathologically activated in chondrocytes of osteoarthritic cartilage.

Histone deacetylases (HDACs) catalyse removal of acetyl groups from lysine residues on both histone and non-histone proteins to control numerous cellular processes. Of the 11 zinc-dependent classical HDACs, HDAC4, 5, 7 and 9 are class IIa HDAC enzymes that regulate cellular and developmental processes through both enzymatic and non-enzymatic mechanisms. Over the last two decades, HDAC7 has been associated with key roles in numerous physiological and pathological processes. Molecular, cellular, in vivo and disease association studies have revealed that HDAC7 acts through multiple mechanisms to control biological processes in immune cells, osteoclasts, muscle, the endothelium and epithelium. This HDAC protein regulates gene expression, cell proliferation, cell differentiation and cell survival and consequently controls development, angiogenesis, immune functions, inflammation and metabolism. This review focuses on the cell biology of HDAC7, including the regulation of its cellular localisation and molecular mechanisms of action, as well as its associative and causal links with cancer and inflammatory, metabolic and fibrotic diseases. We also review the development status of small molecule inhibitors targeting HDAC7 and their potential for intervention in different disease contexts.

Mammalian metabolism comprises a series of interlinking pathways that include two major cycles: the folate and methionine cycles. The folate-mediated metabolic cycle uses several oxidation states of tetrahydrofolate to carry activated one-carbon units to be readily used and interconverted within the cell. They are required for nucleotide synthesis, methylation and metabolism, and particularly for proliferation of cancer cells. Based on the latest progress in genome-wide CRISPR loss-of-function viability screening of 789 cell lines, we focus on the most cancer-dependent enzymes in this pathway, especially those that are hyperactivated in cancer, to provide new insight into the chemical basis for cancer drug development. Since the complete 3D structure of several of these enzymes of the one-carbon pathway in their active form are not available, we used homology modelling integrated with the interpretation of the reaction mechanism. In addition, have reconstructed the most likely scenario for the reactions taking place paired with their catalytic competence that provides a testable framework for this pathway.

Over the past two decades, there has been an explosion in the numbers of clinical trials using mesenchymal stem cells (MSCs). While the safety profile of MSC therapy has been excellent, therapeutic success has not been as robust as expected. In addition to variabilities inherent in all live-cell products because of donor-specific differences and manufacturing practices, MSCs may have an additional layer of complexity due to the availability of many tissues/organ sources for isolation. Since first isolation from the bone marrow (BM) over 50 years ago, human MSCs have been robustly found in multiple tissues/organs. The increased variety of MSC sources is reflected in clinical trials: while BMMSCs was used in nearly all trials prior to 2008, they are used in less than 50% of clinical trials in recent years. While the majority of single-source MSC preclinical data accumulated over the past several decades do reveal biological differences between tissue-specific sources of MSCs, studies directly comparing different MSC sources are relatively rare. In this Review, we summarise these past findings and also specifically focus on studies comparing MSCs isolated from the most commonly utilised sources of BM, adipose tissue and post-partum discarded extraembryonic tissue. The MSC functions discussed here include paraxial mesodermal trilineage differentiation capacity, and also other well-studied and translationally relevant MSC functions of haematopoietic support, immunomodulation and paracrine capacities. Finally, we will discuss the implications of tissue-specific MSC functional differences on future research avenues, manufacturing practices, as well as clinical implementation.

Integrative structure modeling is increasingly used for determining the architectures of biological assemblies, especially those that are structurally heterogeneous. Recently, we reported on how to convert in vivo genetic interaction measurements into spatial restraints for structural modeling: first, phenotypic profiles are generated for each point mutation and thousands of gene deletions or environmental perturbations. Following, the phenotypic profile similarities are converted into distance restraints on the pairs of mutated residues. We illustrate the approach by determining the structure of the histone H3-H4 complex. The method is implemented in our open-source IMP program, expanding the structural biology toolbox by allowing structural characterization based on in vivo data without the need to purify the target system. We compare genetic interaction measurements to other sources of structural information, such as residue coevolution and deep-learning structure prediction of complex subunits. We also suggest that determining genetic interactions could benefit from new technologies, such as CRISPR-Cas9 approaches to gene editing, especially for mammalian cells. Finally, we highlight the opportunity for using genetic interactions to determine recalcitrant biomolecular structures, such as those of disordered proteins, transient protein assemblies, and host-pathogen protein complexes.

Dipeptidyl peptidase 3 (DPP3), a zinc-dependent aminopeptidase, is a highly conserved enzyme among higher animals. The enzyme cleaves dipeptides from the N-terminus of tetra- to decapeptides, thereby taking part in activation as well as degradation of signalling peptides critical in physiological and pathological processes such as blood pressure regulation, nociception, inflammation and cancer. Besides its catalytic activity, DPP3 moonlights as a regulator of the cellular oxidative stress response pathway, e.g., the Keap1-Nrf2 mediated antioxidative response. The enzyme is also recognized as a key modulator of the renin-angiotensin system. Recently, DPP3 has been attracting growing attention within the scientific community, which has significantly augmented our knowledge of its physiological relevance. Herein, we review recent advances in our understanding of the structure and catalytic activity of DPP3, with a focus on attributing its molecular architecture and catalytic mechanism to its wide-ranging biological functions. We further highlight recent intriguing reports that implicate a broader role for DPP3 as a valuable biomarker in cardiovascular and renal pathologies and furthermore discuss its potential as a promising drug target.

Astrocytes mediate many important aspects of neural homeostasis, but until recently, their role in sleep was largely unknown. The situation has dramatically changed in the last decade. The use of transgenic animals, optogenetics, chemogenetics, brain imaging and sophisticated molecular assays has led to exciting discoveries. Astrocytes dynamically change their activity across the sleep-wake cycle and may encode sleep need via changes in intracellular signalling pathways. Astrocytes also exocytose/secrete sleep-inducing molecules which modulate brain activity, sleep architecture and sleep regulation. Many of these observations have been made in mice and Drosophila melanogaster, indicating that astroglial sleep mechanisms are evolutionarily conserved. We review recent findings and discuss future directions.

Heritable loss-of-function mutations in genes encoding key regulators of DNA repair and genome stability can result in degenerative progeroid and/or cancer predisposition syndromes; however, such mutations have never been found to affect the Chk1 protein kinase, despite its central role in DNA damage signalling and checkpoint activation. Remarkably, two recent reports now demonstrate that heritable, gain-of-function mutations within the Chk1 C-terminal regulatory domain can cause female infertility in humans. In vitro, oocytes from individuals heterozygous for such mutant Chk1 alleles fail to undergo the first mitotic division after fertilization owing to arrest in G2 phase of the cell cycle. This arrest results from inhibition of the master regulator of mitosis, the cyclin-dependent kinase CDK1, through the same molecular mechanisms that are engaged by activated Chk1 to impose G2 checkpoint arrest in somatic cells bearing DNA damage. Remarkably, the failure of this first zygotic division in heterozygotes in vitro can be rescued through treatment with selective Chk1 inhibitor drugs, allowing development of apparently normal blastocysts and offering hope that a pharmacological solution to this cause of infertility may be possible.