Oxidized LDL receptors: a recent update.

PURPOSE OF REVIEW: LDL in its oxidized form, or 'oxLDL', is now generally acknowledged to be highly proatherogenic and to play a significant role in atherosclerotic plaque formation. Therefore, there has been increasing interest in understanding the significance of oxLDL and its receptors in different phases of atherosclerosis, leading to the accumulation of additional data at the cellular, structural, and physiological levels. This review focuses on the most recent discoveries about these receptors and how they influence lipid absorption, metabolism, and inflammation in various cell types. RECENT FINDINGS: Two crystal structures of lectin-like oxLDL receptor-1 (LOX-1), one with a small molecule inhibitor and the other with a monoclonal antibody have been published. We recently demonstrated that the 'surface site' of LOX1, adjacent to the positively charged 'basic spine region' that facilitates oxLDL binding, is a targetable site for drug development. Further, recent human studies showed that soluble LOX-1 holds potential as a biomarker for cardiovascular disease diagnosis, prognosis, and assessing the efficacy of therapy. SUMMARY: Receptor-mediated oxLDL uptake results in cellular dysfunction of various cell types involved in atherogenesis and plaque development. The current advancements clearly demonstrate that targeting oxLDL-LOX-1 axis may lead to development of future therapeutics for the treatment of atherosclerotic cardiovascular and cerebrovascular diseases.

Exploring the druggability of oxidized low-density lipoprotein (ox-LDL) receptor, LOX-1, a proatherogenic drug target involved in atherosclerosis.

Lectin-like oxidized low-density lipoprotein (ox-LDL) receptor 1 (LOX-1) is a vital scavenger receptor involved in ox-LDL binding, internalization, and subsequent proatherogenic signaling leading to cellular dysfunction and atherosclerotic plaque formation. Existing data suggest that modulation of ox-LDL - LOX-1 interaction can prevent or slow down atherosclerosis. Therefore, we utilized computational methods such as multi-solvent simulation and characterized two top-ranked druggable sites. Using systematic molecular docking followed by atomistic molecular dynamics simulation, we have identified and shortlisted small molecules from the NCI library that target two key binding sites. We demonstrate, using surface plasmon resonance (SPR), that four of the shortlisted molecules bind one-on-one to the purified C-terminal domain (CTLD) of LOX-1 receptor with high affinity (KD), ranging from 4.9 nM to 20.1 µM. Further, we performed WaterMap analysis to understand the role of individual water molecules in small molecule binding and the LOX-1-ligand complex stability. Our data clearly show that LOX-1 is druggable with small molecules. Our study provides strategies to identify novel inhibitors to attenuate ox-LDL - LOX-1 interaction.

Drug resistance in Mycobacterium tuberculosis and targeting the l,d-transpeptidase enzyme.

Tuberculosis (TB) is a disease that has afflicted mankind for thousands of years, but in the last seven decades, much progress has been made in anti-TB therapy. Early drugs, such as para-aminosalicylic acid, streptomycin, isoniazid, and rifamycins were very effective in combatting the disease, giving rise to the hope that TB would be eradicated from the face of the earth by 2010. Despite that optimism, TB continues to kill more than a million people annually worldwide. A major reason for our inability to contain TB is the emergence drug resistance in Mycobacterium tuberculosis. This commentary is based on our recent publication on the structure of l,d-transpeptidase enzyme, relevant to drug resistance. As a background, we briefly outline the history and development of anti-TB therapy. Based on the crystal structure, we suggest a potential direction for designing more potent drugs against TB.

Binding of alpha-thrombin to surface-anchored platelet glycoprotein Ib(alpha) sulfotyrosines through a two-site mechanism involving exosite I.

The involvement of exosite I in α-thrombin (FIIa) binding to platelet glycoprotein Ibα (GPIbα), which could influence interactions with other substrates, remains undefined. To address the problem, we generated the GPIbα amino terminal domain (GPIbα-N) fully sulfated on three tyrosine residues and solved the structure of its complex with FIIa. We found that sulfotyrosine (Tys) 278 enhances the interaction mainly by establishing contacts with exosite I. We then evaluated how substituting tyrosine with phenylalanine, which cannot be sulfated, affects FIIa binding to soluble or surface-immobilized GPIbα-N. Mutating Tyr(276), which mostly contacts exosite II residues, markedly reduced FIIa interaction with both soluble and immobilized GPIbα-N; mutating Tyr(278) or Tyr(279), which mostly contact exosite I residues, reduced FIIa complexing in solution by 0-20% but affinity for immobilized GPIbα-N 2 to 6-fold, respectively. Moreover, three exosite I ligands--aptamer HD1, hirugen, and lepirudin--did not interfere with soluble FIIa complexing to GPIbα-N, excluding that their binding caused allosteric effects influencing the interaction; nonetheless, all impaired FIIa binding to immobilized GPIbα-N and platelet GPIb nearly as much as aptamer HD22 and heparin, both exosite II ligands. Bound HD1 and hirugen alter Trp(148) orientation in a loop near exosite I preventing contacts with the sulfate oxygen atoms of Tys(279). These results support a mechanism in which binding occurs when the two exosites of one FIIa molecule independently interact with two immobilized GPIbα molecules. Through exosite engagement, GPIbα may influence FIIa-dependent processes relevant to hemostasis and thrombosis.

Molecular dynamics guided design of tocoflexol: a new radioprotectant tocotrienol with enhanced bioavailability.

There is a pressing need to develop safe and effective radioprotector/radiomitigator agents for use in accidental or terrorist-initiated radiological emergencies. Naturally occurring vitamin E family constituents, termed tocols, that include the tocotrienols, are known to have radiation-protection properties. These agents, which work through multiple mechanisms, are promising radioprotectant agents having minimal toxicity. Although α-tocopherol (AT) is the most commonly studied form of vitamin E, the tocotrienols are more potent than AT in providing radioprotection and radiomitigation. Unfortunately, despite their very significant radioprotectant activity, tocotrienols have very short plasma half-lives and require dosing at very high levels to achieve necessary therapeutic benefits. Thus, it would be highly desirable to develop new vitamin E analogues with improved pharmacokinetic properties, specifically increased elimination half-life and increased area under the plasma level versus time curve. The short elimination half-life of the tocotrienols is related to their low affinity for the α-tocopherol transfer protein (ATTP), the protein responsible for maintaining the plasma level of the tocols. Tocotrienols have less affinity for ATTP than does AT, and thus have a longer residence time in the liver, putting them at higher risk for metabolism and biliary excretion. We hypothesized that the low-binding affinity of tocotrienols to ATTP is due to the relatively more rigid tail structure of the tocotrienols in comparison with that of the tocopherols. Therefore, compounds with a more flexible tail would have better binding to ATTP and consequently would have longer elimination half-life and, consequently, an increased exposure to drug, as measured by area under the plasma drug level versus time curve (AUC). This represents an enhanced residence of drug in the systemic circulation. Based on this hypothesis, we developed a new class of vitamin E analogues, the tocoflexols, which maintain the superior bioactivity of the tocotrienols with the potential to achieve the longer half-life and larger AUC of the tocopherols.

Molecular and functional mapping of Plekhm1-Rab7 interaction in osteoclasts.

Mutations in PLEKHM1 cause osteopetrosis in humans and rats. The germline and osteoclast conditional deletions of Plekhm1 gene in mice lead to defective osteoclast bone resorption and increased trabecular bone mass without overt abnormalities in other organs. As an adaptor protein, pleckstrin homology and RUN domain containing M1 (PLEKHM1) interacts with the key lysosome regulator small GTPase RAB7 via its C-terminal RUBICON homologous (RH) domain. In this study, we have conducted a structural-functional study of the PLEKHM1 RH domain and RAB7 interaction in osteoclasts in vitro. The single mutations of the key residues in the Plekhm1 RH predicted from the crystal structure of the RUBICON RH domain and RAB7 interface failed to disrupt the Plekhm1-Rab7 binding, lysosome trafficking, and bone resorption. The compound alanine mutations at Y949-R954 and L1011-I1018 regions decreased Plekhm1 protein stability and Rab7-binding, respectively, thereby attenuated lysosome trafficking and bone resorption in osteoclasts. In contrast, the compound alanine mutations at R1060-Q1068 region were dispensable for Rab7-binding and Plekhm1 function in osteoclasts. These results indicate that the regions spanning Y949-R954 and L1011-I1018 of Plekhm1 RH domain are functionally important for Plekhm1 in osteoclasts and offer the therapeutic targets for blocking bone resorption in treatment of osteoporosis and other metabolic bone diseases.

(E)-13-(4-Amino-phen-yl)parthenolide.

The title compound, C21H25NO3 [systematic name: (3aS,9aR,10aR,10bS,E)-3-[(E)-4-(4-amino-benzyl-idene)-6,9a-dimethyl-3a,4,5,8,9,9a,10a,10b-octa-hydro-oxireno[2',3':9,10]cyclo-deca-[1,2-b]furan-2(3H)-one] was obtained from the reaction of parthenolide [synonym: 4,5-ep-oxy-germacra-1(10),11(13)-dieno-12,6-lactone] with 4-iodo-aniline under Heck reaction conditions. It was identified as the E-isomer (conformation about the exocyclic methyl-idene C=C bond; the conformation about the C=C bond in the ten-membered ring is also E). The mol-ecule is built up from fused ten-, five- (lactone) and three-membered (epoxide) rings with a 4-amino-phenyl group as a substituent. The ten-membered ring displays an approximate chair-chair conformation, while the lactone ring has an envelope conformation with the C atom bonded to the ring O atom as the flap. The dihedral angle between the benzene ring of the 4-amino-phenyl moiety and the lactone ring mean plane is 23.50 (8)°. In the crystal, mol-ecules are linked via N-H⋯O hydrogen bonds, between the amine group and the lactone and epoxide ring O atoms, forming chains propagating along the b-axis direction. Adjacent chains are linked via C-H⋯O inter-actions, forming an undulating two-dimensional network lying parallel to the plane (001). The absolute structure of the mol-ecule in the crystal was confirmed by resonance scattering [Flack parameter = 0.03 (3)].

In silico analysis of TUBA4A mutations in Amyotrophic Lateral Sclerosis to define mechanisms of microtubule disintegration.

Amyotrophic lateral sclerosis (ALS) is an inexorably progressive and degenerative disorder of motor neurons with no currently-known cure. Studies to determine the mechanism of neurotoxicity and the impact of ALS-linked mutations (SOD1, FUS, TARDP, C9ORF72, PFN1, TUBA4A and others) have greatly expanded our knowledge of ALS disease mechanisms and have helped to identify potential targets for ALS therapy. Cellular pathologies (e.g., aggregation of mutant forms of SOD1, TDP43, FUS, Ubiqulin2, PFN1, and C9ORF72), mitochondrial dysfunction, neuroinflammation, and oxidative damage are major pathways implicated in ALS. Nevertheless, the selective vulnerability of motor neurons remains unexplained. The importance of tubulins for long-axon infrastructure, and the special morphology and function of motor neurons, underscore the central role of the cytoskeleton. The recent linkage of mutations to the tubulin α chain, TUBA4A, to familial and sporadic cases of ALS provides a new investigative opportunity to shed light on both mechanisms of ALS and the vulnerability of motor neurons. In the current study we investigate TUBA4A, a structural microtubule protein with mutations causal to familial ALS, using molecular-dynamic (MD) modeling of protein structure to predict the effects of each mutation and its overall impact on GTP binding, chain stability, tubulin assembly, and aggregation propensity. These studies predict that each of the reported mutations will cause notable structural changes to the TUBA4A (α chain) tertiary protein structure, adversely affecting its physical properties and functions. Molecular docking and MD simulations indicate certain α chain mutations (e.g. K430N, R215C, and W407X) may cause structural deviations that impair GTP binding, and plausibly prevent or destabilize tubulin polymerization. Furthermore, several mutations (including R320C and K430N) confer a significant increase in predicted aggregation propensity of TUBA4A mutants relative to wild-type. Taken together, these in silico modeling studies predict structural perturbations and disruption of GTP binding, culminating in failure to form a stable tubulin heterocomplex, which may furnish an important pathogenic mechanism to trigger motor neuron degeneration in ALS.

ATP forms a stable complex with the essential histidine kinase WalK (YycG) domain.

In Bacillus subtilis, the WalRK (YycFG) two-component system coordinates murein synthesis with cell division. It regulates the expression of autolysins that function in cell-wall remodeling and of proteins that modulate autolysin activity. The transcription factor WalR is activated upon phosphorylation by the histidine kinase WalK, a multi-domain homodimer. It autophosphorylates one of its histidine residues by transferring the γ-phosphate from ATP bound to its ATP-binding domain. Here, the high-resolution crystal structure of the ATP-binding domain of WalK in complex with ATP is presented at 1.61 Šresolution. The bound ATP remains intact in the crystal lattice. It appears that the strong binding interactions and the nature of the binding pocket contribute to its stability. The triphosphate moiety of ATP wraps around an Mg(2+) ion, providing three O atoms for coordination in a near-ideal octahedral geometry. The ATP molecule also makes strong interactions with the protein. In addition, there is a short contact between the exocyclic O3' of the sugar ring and O2B of the ß-phosphate, implying an internal hydrogen bond. The stability of the WalK-ATP complex in the crystal lattice suggests that such a complex may exist in vivo poised for initiation of signal transmission. This feature may therefore be part of the sensing mechanism by which the WalRK two-component system is so rapidly activated when cells encounter conditions conducive for growth.

The structural basis of effective LOX-1 inhibition.

Along with other scavenger receptors, splice variants of LOX-1 play an important role in modulating numerous subcellular mechanisms such as normal cell development, differentiation and growth in response to physiological stimuli. Thus, LOX-1 activity is a key regulator in determining the severity of many genetic, metabolic, cardiovascular, renal, and neurodegenerative diseases and/or cancer. Increased expression of LOX-1 precipitates pathological disorders during the aging process. Therefore, it becomes important to develop novel LOX-1 inhibitors based on its ligand binding polarity and/or affinity and disrupt the uptake of its ligand: oxidized low-density lipoproteins (ox-LDL). In this review, we shed light on the presently studied and developed novel LOX-1 inhibitors that may have potential for treatment of diseases characterized by LOX-1 activation.

Amino acid identity at one position within the alpha1 helix of both the histidine kinase and the response regulator of the WalRK and PhoPR two-component systems plays a crucial role in the specificity of phosphotransfer.

Two-component systems usually function as cognate pairs, thereby ensuring an appropriate response to the detected signal. The ability to exclusively phosphorylate a partner protein, often in the presence of many competing homologous substrates, demonstrates a high level of specificity that must derive from the interacting surfaces of the two-component system. Here, we identify positions within the histidine kinases and response regulators of the WalRK and PhoPR two-component systems of Bacillus subtilis that make a major contribution to the specificity of phosphotransfer. Changing the identity of the amino acid at position 11 within the alpha1 helix of WalK and at position 17 within the alpha1 helix of PhoP altered discrimination and allowed phosphotransfer to occur with the non-cognate partner. Changing amino acids at additional positions of the WalK kinase increased phosphotransfer, while changes at additional positions in PhoP only had an effect in the presence of the change at position 17. The importance of amino acid identity at these two positions is supported by the fact that the amino acid combinations of Ile and Ser in WalRK, and Leu and Gly in PhoPR, are very highly conserved among orthologues, while modelling indicates that these amino acid pairs are juxtaposed in the WalRK and PhoPR complexes.

Crystal structure of the transcriptional repressor PagR of Bacillus anthracis.

PagR is a transcriptional repressor in Bacillus anthracis that controls the chromosomal S-layer genes eag and sap, and downregulates the protective antigen pagA gene by direct binding to their promoter regions. The PagR protein sequence is similar to those of members of the ArsR repressor family involved in the repression of arsenate-resistance genes in numerous bacteria. The crystal structure of PagR was solved using multi-wavelength anomalous diffraction (MAD) techniques and was refined with 1.8 A resolution diffraction data. The PagR molecules form dimers, as observed in all SmtB/ArsR repressor family proteins. In the crystal lattice four PagR dimers pack together to form an inactive octamer. Model-building studies suggest that the dimer binds to a DNA duplex with a bend of around 4 degrees.

Structure and Inhibitor Specificity of L,D-Transpeptidase (LdtMt2) from Mycobacterium tuberculosis and Antibiotic Resistance: Calcium Binding Promotes Dimer Formation.

The final step of peptidoglycan (PG) synthesis in all bacteria is the formation of cross-linkage between PG-stems. The cross-linking between amino acids in different PG chains gives the peptidoglycan cell wall a 3-dimensional structure and adds strength and rigidity to it. There are two distinct types of cross-linkages in bacterial cell walls. D,D-transpeptidase (D,D-TPs) generate the classical 4➔3 cross-linkages and the L,D-transpeptidase (L,D-TPs) generate the 3➔3 non-classical peptide cross-linkages. The present study is aimed at understanding the nature of drug resistance associated with L,D-TP and gaining insights for designing novel antibiotics against multi-drug resistant bacteria. Penicillin and cephalosporin classes of ß-lactams cannot inhibit L,D-TP function; however, carbapenems inactivate its function. We analyzed the structure of L,D-TP of Mycobacterium tuberculosis in the apo form and in complex with meropenem and imipenem. The periplasmic region of L,D-TP folds into three domains. The catalytic residues are situated in the C-terminal domain. The acylation reaction occurs between carbapenem antibiotics and the catalytic Cys-354 forming a covalent complex. This adduct formation mimics the acylation of L,D-TP with the donor PG-stem. A novel aspect of this study is that in the crystal structures of the apo and the carbapenem complexes, the N-terminal domain has a muropeptide unit non-covalently bound to it. Another interesting observation is that the calcium complex crystallized as a dimer through head and tail interactions between the monomers.

ALS-causing mutations in profilin-1 alter its conformational dynamics: A computational approach to explain propensity for aggregation.

Profilin-1 (PFN1) is a 140-amino-acid protein with two distinct binding sites-one for actin and one for poly-L-proline (PLP). The best-described function of PFN1 is to catalyze actin elongation and polymerization. Thus far, eight DNA mutations in the PFN1 gene encoding the PFN1 protein are associated with human amyotrophic lateral sclerosis (ALS). We and others recently showed that two of these mutations (Gly118Val or G118V and Cys71Gly or C71G) cause ALS in rodents. In vitro studies suggested that Met114Thr and Thr109Met cause the protein to behave abnormally and cause neurotoxicity. The mechanism by which a single amino acid change in human PFN1 causes the degeneration of motor neurons is not known. In this study, we investigated the structural perturbations of PFN1 caused by each ALS-associated mutation. We used molecular dynamics simulations to assess how these mutations alter the secondary and tertiary structures of human PFN1. Herein, we present our in silico data and analysis on the effect of G118V and T109M mutations on PFN1 and its interactions with actin and PLP. The substitution of valine for glycine reduces the conformational flexibility of the loop region between the α-helix and ß-strand and enhances the hydrophobicity of the region. Our in silico analysis of T109M indicates that this mutation alters the shape of the PLP-binding site and reduces the flexibility of this site. Simulation studies of PFN1 in its wild type (WT) and mutant forms (both G118V and T109M mutants) revealed differential fluctuation patterns and the formation of salt bridges and hydrogen bonds between critical residues that may shed light on differences between WT and mutant PFN1. In particular, we hypothesize that the flexibility of the actin- and PLP-binding sites in WT PFN1 may allow the protein to adopt slightly different conformations in its free and bound forms. These findings provide new insights into how each of these mutations in PFN1 might increase its propensity for misfolding and aggregation, leading to its dysfunction.

Sporulation phosphorelay proteins and their complexes: crystallographic characterization.

Bacteria use two-component systems to adapt to changes in environmental conditions. In response to deteriorating conditions of growth, certain types of bacteria form spores instead of proceeding with cell division. The formation of spores is controlled by an expanded version of two-component systems called the phosphorelay. The phosphorelay comprises a primary kinase that receives the signal/stimulus and undergoes autophosphorylation, followed by two intermediate messengers that regulate the flow of the phosphoryl group to the ultimate response regulator/transcription factor. Sporulation is initiated when the level of phosphorylation of the transcription factor reaches a critical point. This chapter describes efforts to understand the mechanism of initiation of sporulation at the molecular level using X-ray crystallography as a tool. Structural analyses of individual members, as well as their complexes, provide insight into the mechanism of phosphoryl transfer and the origin of specificity in signal transduction.

Structural origins of hemostasis and adaptive immunity.

BACKGROUND: Adaptive immunity in jawless fishes is performed by a unique set of proteins termed variable lymphocyte receptors (VLRs). Here we compare the crystallographic structures of VLRs and the human primary hemostasis receptor, glycoprotein (GP) Ib. It has been estimated jawless fish vertebrates diverged from jawed vertebrates 500 million years ago. Identifying structural similarities provides insights into the origins of primary hemostasis and the unique adaptive immunity of jawless fishes. METHODS: Three-dimensional structures obtained from crystallographic data and primary sequences alignments are compared. The results focus on overall domain arrangement to include the structural roles of leucine-rich repeats (LRRs), disulfide bond, and disulfide loop arrangements. RESULTS: The crystal structures of human GPIb (GPIbαN) and jawless fish VLRs are made up of three common segments each. The N-terminal cap and the C-terminal cap are characterized by disulfide bonds conserved in both GPIbαN and VLRs. The body of each molecule consists of LRRs which varies depending on the number of LRRs present in each molecule. The stacking of the LRRs results in the formation of a concave surface which serves as a motif to build ligand-binding specificity with the flanking regions. CONCLUSION: A comparison of VLR and GPIb structures reveals a phylogenetic trail of cellular differentiation contributing to mammalian hemostasis and jawless fish adaptive immunity. The results provide a structural basis to explain some of the interrelationships between hemostasis and immunity in vertebrates and potentially identifies a common ancestral motif linking hemostasis and immunity.

LOX-1 in Atherosclerosis and Myocardial Ischemia: Biology, Genetics, and Modulation.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), one of the scavenger receptors for oxidized low-density lipoprotein cholesterol (ox-LDL), plays a crucial role in the uptake of ox-LDL by cells in the arterial wall. Mounting evidence suggests a role for LOX-1 in various steps of the atherosclerotic process, from initiation to plaque destabilization. Studies of the genetic structure of LOX-1 have also uncovered various genetic polymorphisms that could modulate the risk of atherosclerotic cardiovascular events. As evidence supporting the vital role of LOX-1 in atherogenesis keeps accumulating, there is growing interest in LOX-1 as a potential therapeutic target. This review discusses the discovery and genetics of LOX-1; describes existing evidence supporting the role of LOX-1 in atherogenesis and its major complication, myocardial ischemia; and summarizes LOX-1 modulation by some naturally occurring compounds and efforts toward development of small molecules and biologics that could be of therapeutic use.

The crystal structure of YycH involved in the regulation of the essential YycFG two-component system in Bacillus subtilis reveals a novel tertiary structure.

The Bacillus subtilis YycFG two-component signal transduction system is essential for cell viability, and the YycH protein is part of the regulatory circuit that controls its activity. The crystal structure of YycH was solved by two-wavelength selenium anomalous dispersion data, and was refined using 2.3 A data to an R-factor of 25.2%. The molecule is made up of three domains, and has a novel three-dimensional structure. The N-terminal domain features a calcium binding site and the central domain contains two conserved loop regions.

DNA complexed structure of the key transcription factor initiating development in sporulating bacteria.

Sporulation in Bacillus species, the ultimate bacterial adaptive response, requires the precisely coordinated expression of a complex genetic pathway, and is initiated through the accumulation of the phosphorylated form of Spo0A, a pleiotropic response regulator transcription factor. Spo0A controls the transcription of several hundred genes in all spore-forming Bacilli including genes for sporulation and toxin regulation in pathogens such as Bacillus anthracis. The crystal structure of the effector domain of Spo0A from Bacillus subtilis in complex with its DNA target was determined. In the crystal lattice, two molecules form a tandem dimer upon binding to adjacent sites on DNA. The protein:protein and protein:DNA interfaces revealed in the crystal provide a basis for interpreting the transcription activation process and for the design of drugs to counter infections by these bacteria.

LIS1 Regulates Osteoclastogenesis through Modulation of M-SCF and RANKL Signaling Pathways and CDC42.

We have previously reported that depletion of LIS1, a key regulator of microtubules and cytoplasmic dynein motor complex, in osteoclast precursor cells by shRNAs attenuates osteoclastogenesis in vitro. However, the underlying mechanisms remain unclear. In this study, we show that conditional deletion of LIS1 in osteoclast progenitors in mice led to increased bone mass and decreased osteoclast number on trabecular bone. In vitro mechanistic studies revealed that loss of LIS1 had little effects on cell cycle progression but accelerated apoptosis of osteoclast precursor cells. Furthermore, deletion of LIS1 prevented prolonged activation of ERK by M-CSF and aberrantly enhanced prolonged JNK activation stimulated by RANKL. Finally, lack of LIS1 abrogated M-CSF and RANKL induced CDC42 activation and retroviral transduction of a constitutively active form of CDC42 partially rescued osteoclastogenesis in LIS1-deficient macrophages. Therefore, these data identify a key role of LIS1 in regulation of cell survival of osteoclast progenitors by modulating M-CSF and RANKL induced signaling pathways and CDC42 activation.