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The decision to enter mitosis requires not only the control of DNA replication but also additional and crucial preparatory steps such as, for example, partial disassembly of the Golgi complex during G2. The Golgi complex is fundamental for the processing and sorting of proteins and lipids in the secretory pathway. It is organized as stacks of cisternae laterally connected by tubules to form a continuous Golgi ribbon. During G2, the Golgi ribbon is unlinked into isolated stacks in preparation for cell division. This structural reorganization is necessary for entry into mitosis, indicating that a “Golgi mitotic checkpoint” controls the correct segregation of this organelle. To understand the physiological significance of the pre-mitotic Golgi unlinking, we devised a strategy to accumulate cells in G2 with an intact Golgi ribbon and then induce entry into mitosis. Here, we show that forcing the entry of cells into mitosis with an intact Golgi ribbon causes remarkable cell division defects, including spindle multipolarity and binucleation, favoring cell transformation. We also find that the cells entering mitosis with an intact Golgi ribbon show reduced levels at the centrosome of the kinase Aurora-A, a pivotal regulator of spindle formation. Overexpression of Aurora-A rescues spindle formation, indicating that the Golgi-dependent Aurora-A recruitment has a crucial role in spindle formation. Thus, our results show that alterations of the pre-mitotic Golgi segregation have profound consequences on the fidelity of the mitotic process, representing potential risk factors for cell transformation and cancer development.

The Golgi complex of mammalian cells is organized in a ribbon-like structure often closely associated with the centrosome during interphase. Conversely, the Golgi complex assumes a fragmented and dispersed configuration away from the centrosome during mitosis. The structure of the Golgi complex and the relative position to the centrosome are dynamically regulated by microtubules. Many pieces of evidence reveal that this microtubule-mediated dynamic association between the Golgi complex and centrosome is of functional significance in cell polarization and division. Here, we summarize findings indicating how the Golgi complex and the centrosome cooperate in organizing the microtubule network for the directional protein transport and centrosome positioning required for cell polarization and regulating fundamental cell division processes.

Blocking the interaction between the apoptosis-inducing factor (AIF) and cyclophilin A (CypA) by the AIF fragment AIF(370-394) is protective against glutamate-induced neuronal cell death and brain injury in mice. Starting from AIF(370-394), we report the generation of the disulfide-bridged and shorter variant AIF(381-389) and its structural characterization by nuclear magnetic resonance (NMR) in the free and CypA-bound state. AIF(381-389) in both the free and bound states assumes a β-hairpin conformation similar to that of the fragment in the AIF protein and shows a highly reduced conformational flexibility. This peptide displays a similar in vitro affinity for CypA, an improved antiapoptotic activity in cells and an enhanced proteolytic stability compared to the parent peptide. The NMR-based 3D model of the AIF(381-389)/CypA complex provides a better understanding of the binding hot spots on both the peptide and the protein and can be exploited to design AIF/CypA inhibitors with improved pharmacokinetic and pharmacodynamics features.

Background The neuronal apoptotic process requires the nuclear translocation of Apoptosis Inducing Factor (AIF) in complex with Cyclophilin A (CypA) with consequent chromatin condensation and DNA degradation events. Targeting CypA by delivering an AIF-blocking peptide (AIF(370–394)) provides a significant neuroprotection, demonstrating the biological relevance of the AIF/CypA complex. To date pharmaceutical compounds targeting this complex are missing. Methods We designed and synthesized a set of mono and bicyclic AIF(370–394) analogs containing both disulfide and 1,2,3-triazole bridges, in the attempt to both stabilize the peptide conformation and improve its binding affinity to CypA. Peptide structures in solution and in complex with CypA have been studied by circular dichroism (CD), Nuclear Magnetic Resonance (NMR) and molecular modeling. The ability of stapled peptides to interact with CypA was evaluated by using Epic Corning label free technique and Isothermal Titration Calorimetry experiments. Results We identified a stapled peptide analogue of AIF(370–394) with a ten-fold improved affinity for CypA. Molecular modeling studies reveal that the new peptide acquires β-turn/β-fold structures and shares with the parent molecule the same binding region on CypA. Conclusions Data obtained provide invaluable assistance in designing new ligand of CypA for therapeutic approaches in neurodegenerative diseases. General significance Due to the crucial role of AIF/CypA complex formation in neurodegeneration, identification of selective inhibitors is of high importance for targeted therapies. We describe new bicyclic peptide inhibitors with improved affinity for CypA, investigating the kinetic, thermodynamic and structural effects of conformational constraints on the protein−ligand interaction, and their utility for drug design.

The complex formation between the proteins apoptosis-inducing factor (AIF) and cyclophilin A (CypA) following oxidative stress in neuronal cells has been suggested as a main target for reverting ischemia-stroke damage. Recently, a peptide encompassing AIF residues 370–394 has been developed to target the AIF-binding site on CypA, to prevent the association between the two proteins and suppress glutamate-induced cell death in neuronal cells. Using a combined approach based on NMR spectroscopy, synthesis and in vitro testing of all Ala-scan mutants of the peptide and molecular docking/molecular dynamics, we have generated a detailed model of the AIF (370–394)/CypA complex. The model suggests us that the central region of the peptide spanning residues V374–K384 mostly interacts with the protein and that for efficient complex inhibition and preservation of CypA activity, it is bent around amino acids F46–G75 of the protein. The model is consistent with experimental data also from previous works and supports the concept that the peptide does not interfere with other CypA activities unrelated to AIF activation; therefore, it may serve as an ideal template for generating future non-peptidic antagonists.

The Golgi complex (GC) has an essential role in the processing and sorting of proteins and lipids. The GC of mammalian cells is composed of stacks of cisternae connected by membranous tubules to create a continuous network, the Golgi ribbon, whose maintenance requires several core and accessory proteins. Despite this complex structural organization, the Golgi apparatus is highly dynamic, and this property becomes particularly evident during mitosis, when the ribbon undergoes a multistep disassembly process that allows its correct partitioning and inheritance by the daughter cells. Importantly, alterations of the Golgi structure are associated with a variety of physiological and pathological conditions. Here, we review the core mechanisms and signaling pathways involved in both the maintenance and disassembly of the Golgi ribbon, and we also report on the signaling pathways that connect the disassembly of the Golgi ribbon to mitotic entry and progression.

PREP1/p160 is a protein complex with relevant physiopathological roles in vivo. p160 regulates PREP1 transcriptional activity by preventing the formation of other PREP1-containing complexes, whereas PREP1 regulates p160 activity by increasing its stability. This induces the repression of the insulin-regulated glucose transporter GLUT4 dampening insulin sensitivity. In spite of the considerable amount of functional studies performed on the PREP1/p160 complex in vivo, a biochemical and structural characterization of this complex has not been so far undertaken, given the poor stability of the recombinant full-length proteins. Here, we report the design and preparation of PREP1 and p160 domains together with preliminary structural and binding studies. PREP1, residues 45-155, and p160, residues 20-160, have been expressed and purified as folded, monomeric domains. The two domains show both all-alpha secondary structures, as demonstrated by CD studies and are endowed with unusually high thermal stabilities. We have also estimated for the first time the PREP1-p160 interaction strength finding that the two recombinant domains interact with a KD ranging between about 0.3 and 1 μM. Altogether, data suggest that the selected PREP1 and p160 domains are structurally independent and that their structure is underlined by high stability and a prevailing alpha-helical organization.

In this work, a sensitive and convenient protease-based fluorimetric high-throughput screening (HTS) assay for determining peptidyl-prolyl cis-trans isomerase activity was developed. The assay was based on a new intramolecularly quenched substrate, whose fluorescence and structural properties were examined together with kinetic constants and the effects of solvents on its isomerization process. Pilot screens performed using the Library of Pharmacologically Active Compounds (LOPAC) and cyclophilin A (CypA), as isomerase model enzyme, indicated that the assay was robust for HTS, and that comparable results were obtained with a CypA inhibitor tested both manually and automatically. Moreover, a new compound that inhibits CypA activity with an IC50 in the low micromolar range was identified. Molecular docking studies revealed that the molecule shows a notable shape complementarity with the catalytic pocket confirming the experimental observations. Due to its simplicity and precision in the determination of extent of inhibition and reaction rates required for kinetic analysis, this assay offers many advantages over other commonly used assays.

In this work, a sensitive and convenient protease-based fluorimetric high-throughput screening (HTS) assay for determining peptidyl-prolyl cis-trans isomerase activity was developed. The assay was based on a new intramolecularly quenched substrate, whose fluorescence and structural properties were examined together with kinetic constants and the effects of solvents on its isomerization process. Pilot screens performed using the Library of Pharmacologically Active Compounds (LOPAC) and cyclophilin A (CypA), as isomerase model enzyme, indicated that the assay was robust for HTS, and that comparable results were obtained with a CypA inhibitor tested both manually and automatically. Moreover, a new compound that inhibits CypA activity with an IC50 in the low micromolar range was identified. Molecular docking studies revealed that the molecule shows a notable shape complementarity with the catalytic pocket confirming the experimental observations. Due to its simplicity and precision in the determination of extent of inhibition and reaction rates required for kinetic analysis, this assay offers many advantages over other commonly used assays.