The key roles of the lysine acetyltransferases KAT6A and KAT6B in physiology and pathology.

Histone modifications and more specifically ε-lysine acylations are key epigenetic regulators that control chromatin structure and gene transcription, thereby impacting on various important cellular processes and phenotypes. Furthermore, lysine acetylation of many non-histone proteins is involved in key cellular processes including transcription, DNA damage repair, metabolism, cellular proliferation, mitosis, signal transduction, protein folding, and autophagy. Acetylation affects protein functions through multiple mechanisms including regulation of protein stability, enzymatic activity, subcellular localization, crosstalk with other post-translational modifications as well as regulation of protein-protein and protein-DNA interactions. The paralogous lysine acetyltransferases KAT6A and KAT6B which belong to the MYST family of acetyltransferases, were first discovered approximately 25 years ago. KAT6 acetyltransferases acylate both histone H3 and non-histone proteins. In this respect, KAT6 acetyltransferases play key roles in regulation of transcription, various developmental processes, maintenance of hematopoietic and neural stem cells, regulation of hematopoietic cell differentiation, cell cycle progression as well as mitosis. In the current review, we discuss the physiological functions of the acetyltransferases KAT6A and KAT6B as well as their functions under pathological conditions of aberrant expression, leading to several developmental syndromes and cancer. Importantly, both upregulation and downregulation of KAT6 proteins was shown to play a role in cancer formation, progression, and therapy resistance, suggesting that they can act as oncogenes or tumor suppressors. We also describe reciprocal regulation of expression between KAT6 proteins and several microRNAs as well as their involvement in cancer formation, progression and resistance to therapy.

Overexpression and purification of human myosins from transiently and stably transfected suspension adapted HEK293SF-3F6 cells.

The myosin family of motor proteins is an attractive target of therapeutic small-molecule protein inhibitors and modulators. Milligrams of protein quantities are required to conduct proper biophysical and biochemical studies to understand myosin functions. Myosin protein expression and purification represent a critical starting point towards this goal. Established utilization of Dictyostelium discoideum, Drosophila melanogaster, insect and mouse cells for myosin expression and purification is limited, cost, labor and time inefficient particularly for (full-length) human myosins. Here we are presenting detailed protocols for production of several difficult-to-purify recombinant human myosins in efficient quantities up to 1 mg of protein per liter of cell culture. This is the first time that myosins have been purified in large scales from suspension adapted transiently and stably expressing human cells. The method is also useful for expressing other human proteins in quantities sufficient to perform extensive biochemical and biophysical characterization.

Nuclear lamins: key regulators of nuclear structure and activities.

The nuclear lamina is a proteinaceous structure located underneath the inner nuclear membrane (INM), where it associates with the peripheral chromatin. It contains lamins and lamin-associated proteins, including many integral proteins of the INM, chromatin modifying proteins, transcriptional repressors and structural proteins. A fraction of lamins is also present in the nucleoplasm, where it forms stable complexes and is associated with specific nucleoplasmic proteins. The lamins and their associated proteins are required for most nuclear activities, mitosis and for linking the nucleoplasm to all major cytoskeletal networks in the cytoplasm. Mutations in nuclear lamins and their associated proteins cause about 20 different diseases that are collectively called laminopathies'. This review concentrates mainly on lamins, their structure and their roles in DNA replication, chromatin organization, adult stem cell differentiation, aging, tumorogenesis and the lamin mutations leading to laminopathic diseases.

ROS induced distribution of mitochondria to filopodia by Myo19 depends on a class specific tryptophan in the motor domain.

The role of the actin cytoskeleton in relation to mitochondria function and dynamics is only recently beginning to be recognized. Myo19 is an actin-based motor that is bound to the outer mitochondrial membrane and promotes the localization of mitochondria to filopodia in response to glucose starvation. However, how glucose starvation induces mitochondria localization to filopodia, what are the dynamics of this process and which enzymatic adaptation allows the translocation of mitochondria to filopodia are not known. Here we show that reactive oxygen species (ROS) mimic and mediate the glucose starvation induced phenotype. In addition, time-lapse fluorescent microscopy reveals that ROS-induced Myo19 motility is a highly dynamic process which is coupled to filopodia elongation and retraction. Interestingly, Myo19 motility is inhibited by back-to-consensus-mutation of a unique residue of class XIX myosins in the motor domain. Kinetic analysis of the purified mutant Myo19 motor domain reveals that the duty ratio (time spent strongly bound to actin) is highly compromised in comparison to that of the WT motor domain, indicating that Myo19 unique motor properties are necessary to propel mitochondria to filopodia tips. In summary, our study demonstrates the contribution of actin-based motility to the mitochondrial localization to filopodia by specific cellular cues.

The effect of endothelial cells on hESC-derived pancreatic progenitors in a 3D environment.

Generating transplantable ß-like-cells from human embryonic stem cells (hESC) could serve as an ideal cell-based therapy for treatment of type 1 diabetes, which is characterized by the destruction of insulin-secreting pancreatic ß-cells. There are several protocols for differentiating hESCs into pancreatic or endocrine precursors. However, so far, production of mature, functional ß-like-cells has been achieved mainly by transplanting hESC derived pancreatic progenitors (PPs) and allowing several months for maturation to occur in vivo. One approach, believed to have potential in promoting differentiation into ß-like-cells prior to transplantation, is culturing PPs alongside blood vessels. Endothelium and blood vessels have been shown to direct pancreatic development during embryogenesis and also induce endocrine differentiation in vitro. Here we designed a three-dimensional (3D) construct utilizing highly porous polymeric scaffolds that mimic natural conditions and provide cells with mechanical support, and used it in the differentiation protocol. Clusters of hESC derived pancreatic precursor cells were embedded within the scaffolds along with human endothelial cells (ECs) and fibroblasts forming vessel-like networks. Culturing these clusters with ECs for one week significantly increased the population of PPs, characterized by co-expression of the pancreatic markers Pdx1 and Nkx6.1 and also highly induced Ngn3 expression which indicates commitment to endocrine fate. The presence of fibroblasts, however, reduced this cell population. Three months upon implantation of constructs containing clusters and ECs or clusters alone, implanted mice retained normal blood glucose levels after treatment with STZ, while un-implanted mice became diabetic. These findings may lay the foundation for creating an optimal tissue-construct that will support PPs' maturation in vitro and enhance graft function upon implantation.

A laminopathic mutation disrupting lamin filament assembly causes disease-like phenotypes in Caenorhabditis elegans.

Mutations in the human LMNA gene underlie many laminopathic diseases, including Emery-Dreifuss muscular dystrophy (EDMD); however, a mechanistic link between the effect of mutations on lamin filament assembly and disease phenotypes has not been established. We studied the ΔK46 Caenorhabditis elegans lamin mutant, corresponding to EDMD-linked ΔK32 in human lamins A and C. Cryo-electron tomography of lamin ΔK46 filaments in vitro revealed alterations in the lateral assembly of dimeric head-to-tail polymers, which causes abnormal organization of tetrameric protofilaments. Green fluorescent protein (GFP):ΔK46 lamin expressed in C. elegans was found in nuclear aggregates in postembryonic stages along with LEM-2. GFP:ΔK46 also caused mislocalization of emerin away from the nuclear periphery, consistent with a decreased ability of purified emerin to associate with lamin ΔK46 filaments in vitro. GFP:ΔK46 animals had motility defects and muscle structure abnormalities. These results show that changes in lamin filament structure can translate into disease-like phenotypes via altering the localization of nuclear lamina proteins, and suggest a model for how the ΔK32 lamin mutation may cause EDMD in humans.