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The synthesis of fatty acids from acetyl-coenzyme A (AcCoA) is deregulated in diverse pathologies, including cancer. Here, we report that fatty acid accumulation is negatively regulated by nucleoside diphosphate kinases 1 and 2 (NME1/2), housekeeping enzymes involved in nucleotide homeostasis that were recently found to bind CoA. We show that NME1 additionally binds AcCoA and that ligand recognition involves a unique binding mode dependent on the CoA/AcCoA 3' phosphate. We report that Nme2 knockout mice fed a high-fat diet (HFD) exhibit excessive triglyceride synthesis and liver steatosis. In liver cells, NME2 mediates a gene transcriptional response to HFD leading to the repression of fatty acid accumulation and activation of a protective gene expression program via targeted histone acetylation. Our findings implicate NME1/2 in the epigenetic regulation of a protective liver response to HFD and suggest a potential role in controlling AcCoA usage between the competing paths of histone acetylation and fatty acid synthesis.

Self-assembling dipeptides have emerged in the last two decades as promising building blocks for the development of novel biomaterials. Among the various classes of dipeptides, aromatic dipeptides and especially diphenylalanine (Phe-Phe), which forms hexagonal nanotubes, have been the most extensively studied. However, aliphatic peptides or mixed aromatic-aliphatic dipeptides seem just as promising, exhibiting various structures ranging from amyloid fibrils to microtubes. Herein we report the single-crystal structure of an aliphatic dipeptide, alanine-isoleucine (Ala-Ile), C 17 H 24 N 2 O 5 , protected with a benzyloxycarbonyl (Z) group at the N-terminus. The protected dipeptide crystallizes in the orthorhombic space group P2 1 2 1 2 1 and forms hollow microtubes with orthorhombic symmetry upon evaporation on glass surfaces, as shown by field emission scanning electron microscopy (FESEM). These findings provide an increased understanding of the correlation between the single-crystal structure of the peptide building block and its self-assembly mechanism, and expand the library of available building blocks for microtechnological applications., (open access.)

The mitotic kinesin Eg5 has emerged as a potential anti-mitotic target for the purposes of cancer chemotherapy. Whether clinical resistance to these inhibitors can arise is unclear. We exploited HCT116 cancer cell line to select resistant clones to S-trityl-L-cysteine (STLC), an extensively studied Eg5 loop-L5 binding inhibitor. The STLC resistant clones differed in their resistance to other loop-L5 binding inhibitors but remained sensitive to the ATP class of competitive Eg5 specific inhibitors. Eg5 is still necessary for bipolar spindle formation in the resistant clones since the cells were sensitive to RNAi mediated depletion of Eg5. One clone expressing Eg5(T107N), a dominant point mutation in the P-loop of the ATP binding domain of the motor, appeared to be not only resistant but also dependent on the presence of STLC. Eg5(T107N) expression was associated also with resistance to the clinical relevant loop-L5 Eg5 inhibitors, Arry-520 and ispinesib. Ectopic expression of the Eg5(T107N) mutant in the absence of STLC was associated with strong non-exchangeable binding to microtubules causing them to bundle. Biochemical assays showed that in contrast to the wild type Eg5-STLC complex, the ATP binding site of the Eg5(T107N) is accessible for nucleotide exchange only when the inhibitor is present. We predict that resistance can be overcome by inhibitors that bind to other than the Eg5 loop-L5 binding site having different chemical scaffolds, and that allostery-dependent resistance to Eg5 inhibitors may also occur in cells and may have positive implications in chemotherapy since once diagnosed may be beneficial following cessation of the chemotherapeutic regimen., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Indorato, DeBonis, Garcia-Saez and Skoufias.)

HIV-1 Rev mediates the nuclear export of intron-containing viral RNA transcripts and is essential for viral replication. Rev is imported into the nucleus by the host protein importin β (Impβ), but how Rev associates with Impβ is poorly understood. Here, we report biochemical, mutational, and biophysical studies of the Impβ/Rev complex. We show that Impβ binds two Rev monomers through independent binding sites, in contrast to the 1:1 binding stoichiometry observed for most Impβ cargos. Peptide scanning data and charge-reversal mutations identify the N-terminal tip of Rev helix α2 within Rev's arginine-rich motif (ARM) as a primary Impβ-binding epitope. Cross-linking mass spectrometry and compensatory mutagenesis data combined with molecular docking simulations suggest a structural model in which one Rev monomer binds to the C-terminal half of Impβ with Rev helix α2 roughly parallel to the HEAT-repeat superhelical axis, whereas the other monomer binds to the N-terminal half. These findings shed light on the molecular basis of Rev recognition by Impβ and highlight an atypical binding behavior that distinguishes Rev from canonical cellular Impβ cargos., (© 2022 Spittler et al.)

Taking advantage of the evolutionary conserved nature of ATAD2, we report here a series of parallel functional studies in human, mouse, and Schizosaccharomyces pombe to investigate ATAD2's conserved functions. In S. pombe , the deletion of ATAD2 ortholog, abo1 , leads to a dramatic decrease in cell growth, with the appearance of suppressor clones recovering normal growth. The identification of the corresponding suppressor mutations revealed a strong genetic interaction between Abo1 and the histone chaperone HIRA. In human cancer cell lines and in mouse embryonic stem cells, we observed that the KO of ATAD2 leads to an accumulation of HIRA. A ChIP-seq mapping of nucleosome-bound HIRA and FACT in Atad2 KO mouse ES cells demonstrated that both chaperones are trapped on nucleosomes at the transcription start sites of active genes, resulting in the abnormal presence of a chaperone-bound nucleosome on the TSS-associated nucleosome-free regions. Overall, these data highlight an important layer of regulation of chromatin dynamics ensuring the turnover of histone-bound chaperones., (© 2021 Wang et al.)

Eg5, the product of Kif11 gene, also known as kinesin spindle protein, is a motor protein involved in the proper establishment of a bipolar mitotic spindle. Eg5 is one of the 45 different kinesins coded in the human genome of the kinesin motor protein superfamily. Over the last three decades Eg5 has attracted great interest as a promising new mitotic target. The identification of monastrol as specific inhibitor of the ATPase activity of the motor domain of Eg5 inhibiting the Eg5 microtubule motility in vitro and in cellulo sparked an intense interest in academia and industry to pursue the identification of novel small molecules that target Eg5 in order to be used in cancer chemotherapy based on the anti-mitotic strategy. Several Eg5 inhibitors entered clinical trials. Currently the field is faced with the problem that most of the inhibitors tested exhibited only limited efficacy. However, one Eg5 inhibitor, Arry-520 (clinical name filanesib), has demonstrated clinical efficacy in patients with multiple myeloma and is scheduled to enter phase III clinical trials. At the same time, new trends in Eg5 inhibitor research are emerging, including an increased interest in novel inhibitor binding sites and a focus on drug synergy with established antitumor agents to improve chemotherapeutic efficacy. This review presents an updated view of the structure and function of Eg5-inhibitor complexes, traces the possible development of resistance to Eg5 inhibitors and their potential therapeutic applications, and surveys the current challenges and future directions of this active field in drug discovery., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Chromatin adopts a diversity of regular and irregular fiber structures in vitro and in vivo. However, how an array of nucleosomes folds into and switches between different fiber conformations is poorly understood. We report the 9.7 Å resolution crystal structure of a 6-nucleosome array bound to linker histone H1 determined under ionic conditions that favor incomplete chromatin condensation. The structure reveals a flat two-start helix with uniform nucleosomal stacking interfaces and a nucleosome packing density that is only half that of a twisted 30-nm fiber. Hydroxyl radical footprinting indicates that H1 binds the array in an on-dyad configuration resembling that observed for mononucleosomes. Biophysical, cryo-EM, and crosslinking data validate the crystal structure and reveal that a minor change in ionic environment shifts the conformational landscape to a more compact, twisted form. These findings provide insights into the structural plasticity of chromatin and suggest a possible assembly pathway for a 30-nm fiber., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Organelles have unique structures and molecular compositions for their functions and have been classified accordingly. However, many organelles are heterogeneous and in the process of maturation and differentiation. Because traditional methods have a limited number of parameters and spatial resolution, they struggle to capture the heterogeneous landscapes of organelles. Here, we present a method for multiparametric particle-based analysis of organelles. After disrupting cells, fluorescence microscopy images of organelle particles labeled with 6 to 8 different organelle markers were obtained, and their multidimensional data were represented in two-dimensional uniform manifold approximation and projection (UMAP) spaces. This method enabled visualization of landscapes of 7 major organelles as well as the transitional states of endocytic organelles directed to the recycling and degradation pathways. Furthermore, endoplasmic reticulum–mitochondria contact sites were detected in these maps. Our proposed method successfully detects a wide array of organelles simultaneously, enabling the analysis of heterogeneous organelle landscapes. Organelles have unique structures and molecular composition, but can form heterogeneous populations and change substantially during maturation and differentiation, and traditional methods have failed to reflect these variations. In this study, the authors introduce a multi-parametric, fluorescence spectroscopy method that allows simultaneous visualization of a wide array of organelles in different maturation states. [ABSTRACT FROM AUTHOR]

Centrosome amplification is a feature of cancer cells associated with chromosome instability and invasiveness. Enhancing chromosome instability and subsequent cancer cell death via centrosome unclustering and multipolar divisions is an aimed-for therapeutic approach. Here, we show that centrosome amplification potentiates responses to conventional chemotherapy in addition to its effect on multipolar divisions and chromosome instability. We perform single-cell live imaging of chemotherapy responses in epithelial ovarian cancer cell lines and observe increased cell death when centrosome amplification is induced. By correlating cell fate with mitotic behaviors, we show that enhanced cell death can occur independently of chromosome instability. We identify that cells with centrosome amplification are primed for apoptosis. We show they are dependent on the apoptotic inhibitor BCL-XL and that this is not a consequence of mitotic stresses associated with centrosome amplification. Given the multiple mechanisms that promote chemotherapy responses in cells with centrosome amplification, we assess such a relationship in an epithelial ovarian cancer patient cohort. We show that high centrosome numbers associate with improved treatment responses and longer overall survival. Our work identifies apoptotic priming as a clinically relevant consequence of centrosome amplification, expanding our understanding of this pleiotropic cancer cell feature. What are the effects of centrosome amplification in cancer? This study shows that extra centrosomes prime ovarian cancer cells for apoptosis, acting in synergy with chemotherapy to induce cell death. High centrosome numbers correlate with improved treatment responses and longer survival. [ABSTRACT FROM AUTHOR]

Determining the mechanism of action of drugs and their target specificity in cells remains a major challenge. Here we describe the use of cell lines expressing two point mutations in the allosteric inhibitor binding pocket of the mitotic kinesin Eg5 (D130A, in the loop L5 region and L214A in helix α3), which following transfection, were selected for their ability to proliferate normally in the presence of STLC, a well known Eg5 inhibitor. The cell lines were used to discriminate the mechanism of action of other chemically distinct small molecule inhibitors of Eg5 that differ in their mode of action. The STLC resistant cells were capable of continuous proliferation in the presence of ATP uncompetitive inhibitors, such as K858 and dimethylenastron, but were still sensitive to ATP competitive inhibitors that are thought to bind to a distinct site on Eg5 than the allosteric binding pocket. The STLC resistant cell lines can therefore be used as a filter to distinguish Eg5 loop L5 binding drugs from drugs binding to other pockets without prior structural information. Additionally, the cells can be used to analyze whether inhibitors of Eg5 are specific to this potential drug target or whether they have additional targets in dividing cells., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Structures of homologous proteins are usually conserved during evolution, as are critical active site residues. This is the case for actin and tubulin, the two most important cytoskeleton proteins in eukaryotes. Actins and their related proteins (Arps) constitute a large superfamily whereas the tubulin family has fewer members. Unaligned sequences of these two protein families were analysed by searching for short groups of family-specific amino acid residues, that we call motifs, and by counting the number of residues from one motif to the next. For each sequence, the set of motif-to-motif residue counts forms a subfamily-specific pattern (landmark pattern) allowing actin and tubulin superfamily members to be identified and sorted into subfamilies. The differences between patterns of individual subfamilies are due to inserts and deletions (indels). Inserts appear to have arisen at an early stage in eukaryote evolution as suggested by the small but consistent kingdom-dependent differences found within many Arp subfamilies and in gamma-tubulins. Inserts tend to be in surface loops where they can influence subfamily-specific function without disturbing the core structure of the protein. The relatively few indels found for tubulins have similar positions to established results, whereas we find many previously unreported indel positions and lengths for the metazoan Arps.

The crystal structures of the universally widespread metallo-beta-lactamase (MBL) Verona integron-encoded MBL (VIM)-2 from Pseudomonas aeruginosa have been solved in their native form as well as in an unexpected oxidised form. This carbapenem-hydrolysing enzyme belongs to the so-called B1 subfamily of MBLs and shares the folding of alpha beta/beta alpha sandwich, consisting of a core of beta-sheet surrounded by alpha-helices. Surprisingly, it showed a high tendency to be strongly oxidised at the catalytic cysteine located in the Cys site, Cys221, which, in the oxidised structure, becomes a cysteinesulfonic residue. Its native structure was obtained only in the presence of Tris(2-carboxyethyl)phosphine. This oxidation might be a consequence of a lower affinity for the second Zn located in the Cys site that would also explain the observed susceptibility of VIM-2 to chelating agents. This modification, if present in nature, might play a role in catalytic down-regulation. Comparison between native and oxidised VIM-2 and a predicted model of VIM-1 (which shows one residue different in the Cys site compared with VIM-2) is performed to explain the different activities and antibiotic specificities.

Drugs that target mitotic spindle proteins have been proven useful for tackling tumor growth. Eg5, a kinesin-5 family member, represents a potential target, since its inhibition leads to prolonged mitotic arrest through the activation of the mitotic checkpoint and apoptotic cell death. Monastrol, a specific dihydropyrimidine inhibitor of Eg5, shows stereo-specificity, since predominantly the (S)-, but not the (R)-, enantiomer has been shown to be the biologically active compound in vitro and in cell-based assays. Here, we solved the crystal structure (2.7A) of the complex between human Eg5 and a new keto derivative of monastrol (named mon-97), a potent antimitotic inhibitor. Surprisingly, we identified the (R)-enantiomer bound in the active site, and not, as for monastrol, the (S)-enantiomer. The absolute configuration of this more active (R)-enantiomer has been unambiguously determined via chemical correlation and x-ray analysis. Unexpectedly, both the R- and the S-forms inhibit Eg5 ATPase activity with IC(50) values of 110 and 520 nM (basal assays) and 150 nm and 650 nm (microtubule-stimulated assays), respectively. However, the difference was large enough for the protein to select the (R)- over the (S)-enantiomer. Taken together, these results show that in this new monastrol family, both (R)- and (S)-enantiomers can be active as Eg5 inhibitors. This considerably broadens the alternatives for rational drug design.

Eukaryotic NMEs/NDP kinases are a family of 10 multifunctional proteins that occur in different cellular compartments and interact with various cellular components (proteins, membranes, and DNA). In contrast to the well-studied Group I NMEs (NME1–4), little is known about the more divergent Group II NMEs (NME5–9). Three recent publications now shed new light on NME6. First, NME6 is a third mitochondrial NME, largely localized in the matrix space, associated with the mitochondrial inner membrane. Second, while its monomeric form is inactive, NME6 gains NDP kinase activity through interaction with mitochondrial RCC1L. This challenges the current notion that mammalian NMEs require the formation of hexamers to become active. The formation of complexes between NME6 and RCC1L, likely heterodimers, seemingly obviates the necessity for hexamer formation, stabilizing a NDP kinase-competent conformation. Third, NME6 is involved in mitochondrial gene maintenance and expression by providing (d)NTPs for replication and transcription (in particular the pyrimidine nucleotides) and by a less characterized mechanism that supports mitoribosome function. This review offers an overview of NME evolution and structure and highlights the new insight into NME6. The new findings position NME6 as the most comprehensively studied protein in NME Group II and may even suggest it as a new paradigm for related family members. [ABSTRACT FROM AUTHOR]

Skeletal dysplasias are group of rare genetic diseases resulting from mutations in genes encoding structural proteins of the cartilage extracellular matrix (ECM), signaling molecules, transcription factors, epigenetic modifiers, and several intracellular proteins. Cell division, organelle maintenance, and intracellular transport are all orchestrated by the cytoskeleton-associated proteins, and intracellular processes affected through microtubule-associated movement are important for the function of skeletal cells. Among microtubule-associated motor proteins, kinesins in particular have been shown to play a key role in cell cycle dynamics, including chromosome segregation, mitotic spindle formation, and ciliogenesis, in addition to cargo trafficking, receptor recycling, and endocytosis. Recent studies highlight the fundamental role of kinesins in embryonic development and morphogenesis and have shown that mutations in kinesin genes lead to several skeletal dysplasias. However, many questions concerning the specific functions of kinesins and their adaptor molecules as well as specific molecular mechanisms in which the kinesin proteins are involved during skeletal development remain unanswered. Here we present a review of the skeletal dysplasias resulting from defects in kinesins and discuss the involvement of kinesin proteins in the molecular mechanisms that are active during skeletal development. [ABSTRACT FROM AUTHOR]

Phosphohistidine (pHis) is a reversible protein post-translational modification (PTM) that is currently poorly understood. The P-N bond in pHis is heat and acid-sensitive, making it more challenging to study than the canonical phosphoamino acids pSer, pThr, and pTyr. As advancements in the development of tools to study pHis have been made, the roles of pHis in cells are slowly being revealed. To date, a handful of enzymes responsible for controlling this modification have been identified, including the histidine kinases NME1 and NME2, as well as the phosphohistidine phosphatases PHPT1, LHPP, and PGAM5. These tools have also identified the substrates of these enzymes, granting new insights into previously unknown regulatory mechanisms. Here, we discuss the cellular function of pHis and how it is regulated on known pHis-containing proteins, as well as cellular mechanisms that regulate the activity of the pHis kinases and phosphatases themselves. We further discuss the role of the pHis kinases and phosphatases as potential tumor promoters or suppressors. Finally, we give an overview of various tools and methods currently used to study pHis biology. Given their breadth of functions, unraveling the role of pHis in mammalian systems promises radical new insights into existing and unexplored areas of cell biology. [ABSTRACT FROM AUTHOR]

Nucleoside diphosphate (NDP) kinases 1 and 2 (NME1/2) are well-characterized enzymes known for their NDP kinase activity. Recently, these enzymes have been shown by independent studies to bind coenzyme A (CoA) or acyl-CoA. These findings suggest a hitherto unknown role for NME1/2 in the regulation of CoA/acyl-CoA-dependent metabolic pathways, in tight correlation with the cellular NTP/NDP ratio. Accordingly, the regulation of NME1/2 functions by CoA/acyl-CoA binding has been described, and additionally, NME1/2 have been shown to control the cellular pathways consuming acetyl-CoA, such as histone acetylation and fatty acid synthesis. NME1/2-controlled histone acetylation in turn mediates an important transcriptional response to metabolic changes, such as those induced following a high-fat diet (HFD). This review discusses the CoA/acyl-CoA-dependent NME1/2 activities and proposes that these enzymes be considered as the first identified carriers of CoA/short-chain acyl-CoAs. [ABSTRACT FROM AUTHOR]