Small Peptide Inhibitors Disrupt a High-Affinity Interaction between Cytoplasmic Dynein and a Viral Cargo Protein

Several viruses target the microtubular motor system in early stages of the viral life cycle. African swine fever virus (ASFV) protein p54 hijacks the microtubule-dependent transport by interaction with a dynein light chain (DYNLL1/DLC8). This was shown to be a high-affinity interaction, and the residues gradually disappearing were mapped on DLC8 to define a putative p54 binding surface by nuclear magnetic resonance (NMR) spectroscopy. The potential of short peptides targeting the binding domain to disrupt this high-affinity protein-protein interaction was assayed, and a short peptide sequence was shown to bind and compete with viral protein binding to dynein. Given the complexity and number of proteins involved in cellular transport, the prevention of this viral-DLC8 interaction might not be relevant for successful viral infection. Thus, we tested the capacity of these peptides to interfere with viral infection by disrupting dynein interaction with viral p54. Using this approach, we report on short peptides that inhibit viral growth.To enter the host cell, a virus must cross several barriers to reach the nucleus. Many viruses hijack the microtubular network to be transported along the cytoplasm (7, 18). Dynein is a microtubular motor protein, part of a large macromolecular complex called the microtubular motor complex. Dynein is involved in early stages of the viral life cycle of diverse infections, the first stage being the intracellular transport of the incoming virus along microtubules. Once transported throughout the cytosol, the virus rapidly gains the perinuclear area or the nucleus, where virus replication takes place. The disruption of microtubules or microtubular motor dynein function impairs the transport of a number of viruses; however, the intrinsic mechanism of this transport is unclear. Also, it has not been firmly established whether there is a common mechanism by which these viruses hijack a component of the microtubular motor complex for this purpose (7). A direct interaction between a given viral protein and cytoplasmic dynein for transport has been reported for HIV, herpes simplex virus, African swine fever virus (ASFV), and rabies virus (4, 14, 22, 25). In adenoviruses, a direct interaction of the viral capsid hexon subunit with cytoplasmic dynein has been described recently (5).One of these viruses, ASFV, which is a large DNA virus, enters the cell by dynamin- and clathrin-dependent endocytosis (12), and its infectivity is dependent on the acidification of the endosome. ASFV protein p54, a major protein of virion membranes, interacts with the light-chain dynein of 8 kDa (DLC8), which allows the transport of the virus to the perinuclear area (4), in a region called the microtubular organizing center (MTOC). In this zone, the virus starts replication in the viral factory, a secluded compartment where newly formed virions assemble (11, 13). By binding DLC8, the virus masters intracellular transport to ensure successful infection. However, due to the complexity of the system, the mechanism of this interaction is still elusive.A variety of names have been used for the subunits of the cytoplasmic dynein complex. A new classification for mammalian cytoplasmic dynein subunit genes based on their phylogenetic relationships has been reported in which the DLC8 gene was named DYNLL1 (26).Light dynein chains are responsible for direct cargo binding in the cell, but how do they select so many different cargos? It is not known whether the mode and site of binding is the same for viral proteins and physiological cargos. Within these multimeric complexes, there are a number of molecules that theoretically could interact with a given viral protein. However, to date viral proteins have been described to bind only light or intermediate dynein chains, such as DLC8 and TcTex1 (4, 5, 8). A candidate viral protein would bind one of the DLC binding domains, which in DLC8 are located between the two dimers of the DLC8 molecule (LysXThrThr). Here, we analyzed this interaction between a viral protein and DLC8 in an attempt to elucidate its requirements and relevance for viral infection.To determine whether this interaction is crucial for viral replication or whether it is just one of a number of alternatives for the virus-host interplay, we analyzed the capacity of a set of inhibitor peptides targeting a determined binding domain of the DLC8 molecule to interfere with viral infection by disrupting dynein interaction with viral p54.     

Identification and Characterization of Novel Classes of Macrophage Migration Inhibitory Factor (MIF) Inhibitors with Distinct Mechanisms of Action

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is considered an attractive therapeutic target in multiple inflammatory and autoimmune disorders. In addition to its known biologic activities, MIF can also function as a tautomerase. Several small molecules have been reported to be effective inhibitors of MIF tautomerase activity in vitro. Herein we employed a robust activity-based assay to identify different classes of novel inhibitors of the catalytic and biological activities of MIF. Several novel chemical classes of inhibitors of the catalytic activity of MIF with IC₅₀ values in the range of 0.2–15.5 μm were identified and validated. The interaction site and mechanism of action of these inhibitors were defined using structure-activity studies and a battery of biochemical and biophysical methods. MIF inhibitors emerging from these studies could be divided into three categories based on their mechanism of action: 1) molecules that covalently modify the catalytic site at the N-terminal proline residue, Pro¹; 2) a novel class of catalytic site inhibitors; and finally 3) molecules that disrupt the trimeric structure of MIF. Importantly, all inhibitors demonstrated total inhibition of MIF-mediated glucocorticoid overriding and AKT phosphorylation, whereas ebselen, a trimer-disrupting inhibitor, additionally acted as a potent hyperagonist in MIF-mediated chemotactic migration. The identification of biologically active compounds with known toxicity, pharmacokinetic properties, and biological activities in vivo should accelerate the development of clinically relevant MIF inhibitors. Furthermore, the diversity of chemical structures and mechanisms of action of our inhibitors makes them ideal mechanistic probes for elucidating the structure-function relationships of MIF and to further determine the role of the oligomerization state and catalytic activity of MIF in regulating the function(s) of MIF in health and disease.     

IDO upregulates regulatory T cells via tryptophan catabolite and suppresses encephalitogenic T cell responses in experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a Th1 and Th17 cell-mediated autoimmune disease of the CNS. IDO and tryptophan metabolites have inhibitory effects on Th1 cells in EAE. For Th17 cells, IDO-mediated tryptophan deprivation and small molecule halofuginone-induced amino acid starvation response were shown to activate general control nonrepressed 2 (GCN2) kinase that directly or indirectly inhibits Th17 cell differentiation. However, it remains unclear whether IDO and tryptophan metabolites impact the Th17 cell response by mechanisms other than the GCN2 kinase pathway. In this article, we show that IDO-deficient mice develop exacerbated EAE with enhanced encephalitogenic Th1 and Th17 cell responses and reduced regulatory T cell (Treg) responses. Administration of the downstream tryptophan metabolite 3-hydroxyanthranillic acid (3-HAA) enhanced the percentage of Tregs, inhibited Th1 and Th17 cells, and ameliorated EAE. We further demonstrate that Th17 cells are less sensitive to direct suppression by 3-HAA than are Th1 cells. 3-HAA treatment in vitro reduced IL-6 production by activated spleen cells and increased expression of TGF-β in dendritic cells (DCs), which correlated with enhanced levels of Tregs, suggesting that 3-HAA-induced Tregs contribute to inhibition of Th17 cells. By using a DC-T cell coculture, we found that 3-HAA-treated DCs expressed higher levels of TGF-β and had properties to induce generation of Tregs from anti-CD3/anti-CD28-stimulated naive CD4(+) T cells. Thus, our data support the hypothesis that IDO induces the generation of Tregs via tryptophan metabolites, such as 3-HAA, which enhances TGF-β expression from DCs and promotes Treg differentiation.     

Autochthony and HLA frequencies in the Netherlands: when surnames are useless markers

To study the genetic variability of the HLA loci A, B, DR, and DQ in the Netherlands, we analyzed more than 13,000 typings provided by the Dutch National Reference Laboratory for Histocompatibility. To investigate any possibly existing population structure, we subdivided the typings by the geographic location of residency of donors and by the historical belonging of their surnames to given provinces. Concerning possible geographic patterns, we found no significant differences between the four provinces examined (North Holland, South Holland, Utrecht, Zeeland). To assess whether such a negative result was related to recent immigration to the area (the richest of the country) that erased possible preexisting patterns of HLA diversity, we reprocessed the database according to the surnames of HLA donors. We obtained two groups: (1) those having a surname typical of the four provinces they inhabit and (2) those with surnames coming from elsewhere. Such an analysis was made possible because of the availability of a database concerning the geographic origin of most Dutch surnames. Even with this surname-based approach, no major differences were found. We conclude that either the western part of the Netherlands was genetically homogeneous before the official introduction of Dutch surnames two centuries ago by Napoleon or surnames have no power in dissecting HLA variability; that is, such variability is the result of recombination phenomena that surnames cannot mirror because they are transmitted virtually unchanged generation after generation. A comparable study by other investigators recommended the use of family names to identify rare HLA haplotypes in France, but now, concerning the Netherlands, we find opposite results. We suggest that a few typing centers may be sufficient to type bone marrow donors, because HLA genetic differences between the different provinces of the Netherlands are extremely low. To maximize the number of donors, such centers should be located in areas providing the easiest access to the largest population of possible donors, thus disregarding the search for a local variability that we did not find.     

Phase state and surface topography of palmitoyl-ceramide monolayers

In cell biology (and in many biophysical) studies there is a natural tendency to consider ceramide as a highly condensed, solid-type lipid conferring rigidity and close packing to biomembranes. In the present work we advanced the understanding of the phase behavior of palmitoyl-ceramide restricted to a planar interface using Langmuir monolayers under strictly controlled and known surface packing conditions. Surface pressure–molecular area isotherms were complemented with molecular area–temperature isobars and with observations of the surface topography by Brewster Angle Microscopy. The results described herein indicate that palmitoyl-ceramide can exhibit expanded, as well as condensed phase states. Formation of three phases was found, depending on the surface pressure and temperature: a solid (1.80 nm thick), a liquid-condensed (1.73 nm thick, likely tilted) and a liquid-expanded (1.54 nm thick) phase over the temperature range 5–62 °C. A large hysteretic behavior is observed for the S phase monolayer that may indicate high resistance to domain boundary deformation. A second (or higher) order S → LC phase transition is observed at about room temperature while a first order LC → LE transition occurs in a range of temperature encompassing the physiological one (observed above 30 °C at low surface pressure). This phase behavior broadens the view of ceramide as a type of lipid not-always-rigid but able to exhibit polymorphic properties.     

Robustness of positional specification by the Hedgehog morphogen gradient

Spatial gradients of Hedgehog signalling play a central role in many patterning events during animal development, regulating cell fate determination and tissue growth in a variety of tissues and developmental stages. Experimental evidence suggests that many of the proteins responsible for regulating Hedgehog signalling and transport are themselves targets of Hedgehog signalling, leading to multiple levels of feedback within the system. We use mathematical modelling to analyse how these overlapping feedbacks combine to regulate patterning and potentially enhance robustness in the Drosophila wing imaginal disc. Our results predict that the regulation of Hedgehog transport and stability by glypicans, as well as multiple overlapping feedbacks in the Hedgehog response network, can combine to enhance the robustness of positional specification against variability in Hedgehog levels. We also discuss potential trade-offs between robustness and additional features of the Hedgehog gradient, such as signalling range and size regulation.     

Intermolecular Autophosphorylation Regulates Myosin IIIa Activity and Localization in Parallel Actin Bundles

Myosin IIIa (Myo3A) transports cargo to the distal end of actin protrusions and contains a kinase domain that is thought to autoregulate its activity. Because Myo3A tends to cluster at the tips of actin protrusions, we investigated whether intermolecular phosphorylation could regulate Myo3A biochemical activity, cellular localization, and cellular function. Inactivation of Myo3A 2IQ kinase domain with the point mutation K50R did not alter maximal ATPase activity, whereas phosphorylation of Myo3A 2IQ resulted in reduced maximal ATPase activity and actin affinity. The rate and degree of Myo3A 2IQ autophosphorylation was unchanged by the presence of actin but was found to be dependent upon Myo3A 2IQ concentration within the range of 0.1 to 1.2 μm, indicating intermolecular autophosphorylation. In cultured cells, we observed that the filopodial tip localization of Myo3A lacking the kinase domain decreased when co-expressed with kinase-active, full-length Myo3A. The cellular consequence of reduced Myo3A tip localization was decreased filopodial density along the cell periphery, identifying a novel cellular function for Myo3A in mediating the formation and stability of actin-based protrusions. Our results suggest that Myo3A motor activity is regulated through a mechanism involving concentration-dependent autophosphorylation. We suggest that this regulatory mechanism plays an essential role in mediating the transport and actin bundle formation/stability functions of Myo3A.     

AMPK activation restores the stimulation of glucose uptake in an in vitro model of insulin-resistant cardiomyocytes via the activation of protein kinase B

Diabetic hearts are known to be more susceptible to ischemic disease. Biguanides, like metformin, are known antidiabetic drugs that lower blood glucose concentrations by decreasing hepatic glucose production and increasing glucose disposal in muscle. Part of these metabolic effects is thought to be mediated by the activation of AMP-activated protein kinase (AMPK). In this work, we studied the relationship between AMPK activation and glucose uptake stimulation by biguanides and oligomycin, another AMPK activator, in both insulin-sensitive and insulin-resistant cardiomyocytes. In insulin-sensitive cardiomyocytes, insulin, biguanides and oligomycin were able to stimulate glucose uptake with the same efficiency. Stimulation of glucose uptake by insulin or biguanides was correlated to protein kinase B (PKB) or AMPK activation, respectively, and were additive. In insulin-resistant cardiomyocytes, where insulin stimulation of glucose uptake was greatly reduced, biguanides or oligomycin, in the absence of insulin, induced a higher stimulation of glucose uptake than that obtained in insulin-sensitive cells. This stimulation was correlated with the activation of both AMPK and PKB and was sensitive to the phosphatidylinositol-3-kinase/PKB pathway inhibitors. Finally, an adenoviral-mediated expression of a constitutively active form of AMPK increased both PKB phosphorylation and glucose uptake in insulin-resistant cardiomyocytes. We concluded that AMPK activators, like biguanides and oligomycin, are able to restore glucose uptake stimulation, in the absence of insulin, in insulin-resistant cardiomyocytes via the additive activation of AMPK and PKB. Our results suggest that AMPK activation could restore normal glucose metabolism in diabetic hearts and could be a potential therapeutic approach to treat insulin resistance.