Low prevalence of Pneumocystis pneumonia in hospitalized patients with systemic lupus erythematosus: review of a clinical data warehouse.

Objective In the era of powerful immunosuppression, opportunistic infections are an increasing concern in systemic lupus erythematosus. One of the best-studied opportunistic infections is Pneumocystis pneumonia; however, the prevalence of Pneumocystis pneumonia in systemic lupus erythematosus is not clearly defined. This study evaluates the prevalence of Pneumocystis pneumonia in hospitalized systemic lupus erythematosus patients, with a focus on validating the Pneumocystis pneumonia and systemic lupus erythematosus diagnoses with clinical information. Methods This retrospective cohort study evaluates the prevalence of Pneumocystis pneumonia in all systemic lupus erythematosus patients treated at Columbia University Medical Center-New York Presbyterian Hospital between January 2000 and September 2014, using electronic medical record data. Patients with human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) and patients with renal transplants (including both early and late post-transplant patients) represented immunocompromised control groups. Patients with systemic lupus erythematosus, Pneumocystis pneumonia, HIV/AIDS, or renal transplant were identified using diagnostic codes from the International Classification of Diseases, Ninth Revision (ICD-9). Results Out of 2013 hospitalized systemic lupus erythematosus patients, nine had presumed Pneumocystis pneumonia, yielding a low prevalence of Pneumocystis pneumonia in systemic lupus erythematosus of 0.45%. Three of the nine Pneumocystis pneumonia cases were patients with concomitant systemic lupus erythematosus and HIV/AIDS. Only one of these nine cases was histologically confirmed as Pneumocystis pneumonia, in a patient with concomitant systemic lupus erythematosus and HIV/AIDS and a CD4 count of 13 cells/mm3. The prevalence of Pneumocystis pneumonia in renal transplant patients and HIV/AIDS patients was 0.61% and 5.98%, respectively. Conclusion Given the reported high rate of adverse effects to trimethoprim-sulfamethoxazole in systemic lupus erythematosus and the low prevalence of Pneumocystis pneumonia in hospitalized systemic lupus erythematosus patients, our data do not substantiate the need for Pneumocystis pneumonia prophylaxis in systemic lupus erythematosus patients, except in those with concurrent HIV/AIDS.

Allosteric activation of VCP, a AAA unfoldase, by small molecule mimicry.

The loss of function of AAA (ATPases associated with diverse cellular activities) mechanoenzymes has been linked to diseases, and small molecules that activate these proteins can be powerful tools to probe mechanisms and test therapeutic hypotheses. Unlike chemical inhibitors that can bind a single conformational state to block enzyme activity, activator binding must be permissive to different conformational states needed for enzyme function. However, we do not know how AAA proteins can be activated by small molecules. Here, we focus on valosin-containing protein (VCP)/p97, a AAA unfoldase whose loss of function has been linked to protein aggregation-based disorders, to identify druggable sites for chemical activators. We identified VCP Activator 1 (VA1), a compound that dose-dependently stimulates VCP ATPase activity up to ∼3-fold. Our cryo-EM studies resulted in structures (∼2.9-3.5 Å-resolution) of VCP in apo and ADP-bound states, and revealed VA1 binding an allosteric pocket near the C-terminus in both states. Engineered mutations in the VA1 binding site confer resistance to VA1, and furthermore, modulate VCP activity to a similar level as VA1-mediated activation. The VA1 binding site can alternatively be occupied by a phenylalanine residue in the VCP C-terminal tail, a motif that is post-translationally modified and interacts with cofactors. Together, our findings uncover a druggable allosteric site and a mechanism of enzyme regulation that can be tuned through small molecule mimicry. Significance: The loss of function of valosin-containing protein (VCP/p97), a mechanoenzyme from the AAA superfamily that hydrolyzes ATP and uses the released energy to extract or unfold substrate proteins, is linked to protein aggregation-based disorders. However, druggable allosteric sites to activate VCP, or any AAA mechanoenzyme, have not been identified. Here, we report cryo-EM structures of VCP in two states in complex with VA1, a compound we identified that dose-dependently stimulates VCP's ATP hydrolysis activity. The VA1 binding site can also be occupied by a phenylalanine residue in the VCP C-terminal tail, suggesting that VA1 acts through mimicry of this interaction. Our study reveals a druggable allosteric site and a mechanism of enzyme regulation.

High-content microscopy reveals a morphological signature of bortezomib resistance.

Drug resistance is a challenge in anticancer therapy, particularly with targeted therapeutics and cytotoxic compounds. In many cases, cancers can be resistant to the drug prior to exposure, i.e., possess intrinsic drug resistance. However, we lack target-independent methods to anticipate resistance in cancer cell lines or characterize intrinsic drug resistance without a priori knowledge of its cause. We hypothesized that cell morphology could provide an unbiased readout of drug sensitivity prior to treatment. We therefore isolated clonal cell lines that were either sensitive or resistant to bortezomib, a well-characterized proteasome inhibitor and anticancer drug to which many cancer cells possess intrinsic resistance. We then measured high-dimensional single-cell morphology profiles using Cell Painting, a high-content microscopy assay. Our imaging- and computation-based profiling pipeline identified morphological features typically different between resistant and sensitive clones. These features were compiled to generate a morphological signature of bortezomib resistance, which correctly predicted the bortezomib treatment response in seven of ten cell lines not included in the training dataset. This signature of resistance was specific to bortezomib over other drugs targeting the ubiquitin-proteasome system. Our results provide evidence that intrinsic morphological features of drug resistance exist and establish a framework for their identification.

Eg5 is static in bipolar spindles relative to tubulin: evidence for a static spindle matrix.

We used fluorescent speckle microscopy to probe the dynamics of the mitotic kinesin Eg5 in Xenopus extract spindles, and compared them to microtubule dynamics. We found significant populations of Eg5 that were static over several seconds while microtubules flux towards spindle poles. Eg5 dynamics are frozen by adenylimidodiphosphate. Bulk turnover experiments showed that Eg5 can exchange between the spindle and the extract with a half life of <55 s. Eg5 distribution in spindles was not perturbed by inhibition of its motor activity with monastrol, but was perturbed by inhibition of dynactin with p50 dynamitin. We interpret these data as revealing the existence of a static spindle matrix that promotes Eg5 targeting to spindles, and transient immobilization of Eg5 within spindles. We discuss alternative interpretations of the Eg5 dynamics we observe, ideas for the biochemical nature of a spindle matrix, and implications for Eg5 function.

Probing spindle assembly mechanisms with monastrol, a small molecule inhibitor of the mitotic kinesin, Eg5.

Monastrol, a cell-permeable small molecule inhibitor of the mitotic kinesin, Eg5, arrests cells in mitosis with monoastral spindles. Here, we use monastrol to probe mitotic mechanisms. We find that monastrol does not inhibit progression through S and G2 phases of the cell cycle or centrosome duplication. The mitotic arrest due to monastrol is also rapidly reversible. Chromosomes in monastrol-treated cells frequently have both sister kinetochores attached to microtubules extending to the center of the monoaster (syntelic orientation). Mitotic arrest-deficient protein 2 (Mad2) localizes to a subset of kinetochores, suggesting the activation of the spindle assembly checkpoint in these cells. Mad2 localizes to some kinetochores that have attached microtubules in monastrol-treated cells, indicating that kinetochore microtubule attachment alone may not satisfy the spindle assembly checkpoint. Monastrol also inhibits bipolar spindle formation in Xenopus egg extracts. However, it does not prevent the targeting of Eg5 to the monoastral spindles that form. Imaging bipolar spindles disassembling in the presence of monastrol allowed direct observations of outward directed forces in the spindle, orthogonal to the pole-to-pole axis. Monastrol is thus a useful tool to study mitotic processes, detection and correction of chromosome malorientation, and contributions of Eg5 to spindle assembly and maintenance.

Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen.

Small molecules that perturb specific protein functions are valuable tools for dissecting complex processes in mammalian cells. A combination of two phenotype-based screens, one based on a specific posttranslational modification, the other visualizing microtubules and chromatin, was used to identify compounds that affect mitosis. One compound, here named monastrol, arrested mammalian cells in mitosis with monopolar spindles. In vitro, monastrol specifically inhibited the motility of the mitotic kinesin Eg5, a motor protein required for spindle bipolarity. All previously known small molecules that specifically affect the mitotic machinery target tubulin. Monastrol will therefore be a particularly useful tool for studying mitotic mechanisms.

CRIPT, a novel postsynaptic protein that binds to the third PDZ domain of PSD-95/SAP90.

The synaptic protein PSD-95/SAP90 binds to and clusters a variety of membrane proteins via its two N-terminal PDZ domains. We report a novel protein, CRIPT, which is highly conserved from mammals to plants and binds selectively to the third PDZ domain (PDZ3) of PSD-95 via its C terminus. While conforming to the consensus PDZ-binding C-terminal sequence (X-S/T-X-V-COOH), residues at the -1 position and upstream of the last four amino acids of CRIPT determine its specificity for PDZ3. In heterologous cells, CRIPT causes a redistribution of PSD-95 to microtubules. In brain, CRIPT colocalizes with PSD-95 in the postsynaptic density and can be coimmunoprecipitated with PSD-95 and tubulin. These findings suggest that CRIPT may regulate PSD-95 interaction with a tubulin-based cytoskeleton in excitatory synapses.

Roles of polymerization dynamics, opposed motors, and a tensile element in governing the length of Xenopus extract meiotic spindles.

Metaphase spindles assemble to a steady state in length by mechanisms that involve microtubule dynamics and motor proteins, but they are incompletely understood. We found that Xenopus extract spindles recapitulate the length of egg meiosis II spindles, by using mechanisms intrinsic to the spindle. To probe these mechanisms, we perturbed microtubule polymerization dynamics and opposed motor proteins and measured effects on spindle morphology and dynamics. Microtubules were stabilized by hexylene glycol and inhibition of the catastrophe factor mitotic centromere-associated kinesin (MCAK) (a kinesin 13, previously called XKCM) and destabilized by depolymerizing drugs. The opposed motors Eg5 and dynein were inhibited separately and together. Our results are consistent with important roles for polymerization dynamics in regulating spindle length, and for opposed motors in regulating the relative stability of bipolar versus monopolar organization. The response to microtubule destabilization suggests that an unidentified tensile element acts in parallel with these conventional factors, generating spindle shortening force.

Bipolarization and poleward flux correlate during Xenopus extract spindle assembly.

We investigated the mechanism by which meiotic spindles become bipolar and the correlation between bipolarity and poleward flux, using Xenopus egg extracts. By speckle microscopy and computational alignment, we find that monopolar sperm asters do not show evidence for flux, partially contradicting previous work. We account for the discrepancy by describing spontaneous bipolarization of sperm asters that was missed previously. During spontaneous bipolarization, onset of flux correlated with onset of bipolarity, implying that antiparallel microtubule organization may be required for flux. Using a probe for TPX2 in addition to tubulin, we describe two pathways that lead to spontaneous bipolarization, new pole assembly near chromatin, and pole splitting. By inhibiting the Ran pathway with excess importin-alpha, we establish a role for chromatin-derived, antiparallel overlap bundles in generating the sliding force for flux, and we examine these bundles by electron microscopy. Our results highlight the importance of two processes, chromatin-initiated microtubule nucleation, and sliding forces generated between antiparallel microtubules, in self-organization of spindle bipolarity and poleward flux.

Molecular basis for the binding of SH3 ligands with non-peptide elements identified by combinatorial synthesis.

BACKGROUND: Protein-structure-based combinatorial chemistry has recently been used to discover several ligands containing non-peptide binding elements to the Src SH3 domain. The encoded library used has the form Cap-M1-M2-M3-PLPPLP, in which the Cap and Mi's are composed of a diverse set of organic monomers. The PLPPLP portion provided a structural bias directing the non-peptide fragment Cap-M1-M2-M3 to the SH3 specificity pocket. Fifteen ligands were selected from > 1.1 million distinct compounds. The structural basis for selection was unknown. RESULTS: The solution structures of the Src SH3 domain complexed with two ligands containing non-peptide elements selected from the library were determined by multidimensional NMR spectroscopy. The non-peptide moieties of the ligands interact with the specificity pocket of Src SH3 domain differently from peptides complexed with SH3 domains. Structural information about the ligands was used to design various homologs, whose affinities for the SH3 domain were measured. The results provide a structural basis for understanding the selection of a few optimal ligands from a large library. CONCLUSIONS: The cycle of protein-structure-based combinatorial chemistry followed by structure determination of the few highest affinity ligands provides a powerful new tool for the field of molecular recognition.

Mechanistic studies of a signaling pathway activated by the organic dimerizer FK1012.

BACKGROUND: The T-cell receptor (TCR) signaling pathway is initiated by regulated association of TCR chains, including the zeta chain. A recently reported method for inducing the dimerization or oligomerization of targeted proteins in cells used the TCR pathway as a test system. In cells transfected with cDNA encoding MZF3E, a chimeric receptor comprising the intracellular domain of the zeta chain and three copies of FK506-binding protein (FKBP), low concentrations of a synthetic dimer of the natural product FK506 (FK1012) activated the expression of reporter genes. We set out to examine the signaling pathway initiated by FK1012. RESULTS: We characterized the effect of FK1012 on MZF3E and a second chimeric receptor, MZF1E, which contains the zeta chain and one copy of FKBP. Only MZF3E gave FK1012-activated signaling, as shown by an increase in the kinase activity associating with MZF3E, and the appearance of specific phosphotyrosine-containing proteins. Signaling required localization of MZF3E to the inner plasma membrane, and activation of gene transcription in response to FK1012 was dependent on the protein phosphatase calcineurin and the transcriptional activator NF-AT. Some signaling events in the pathway had different kinetics when activated by MZF3E instead of the TCR, however. An unexpected requirement for the prolonged activation of calcineurin was observed. CONCLUSIONS: Synthetic dimerizers can be used to gain control over cellular processes that require the association of specific intracellular proteins. The TCR signaling pathway was selected as an initial test system; we show here that one can indeed activate this signaling pathway by inducing the oligomerization of the cytoplasmic tail of the zeta chain with the cell-permeable reagent FK1012.

Allele-specific activators and inhibitors for kinesin.

Members of the kinesin superfamily are force-generating ATPases that drive movement and influence cytoskeleton organization in cells. Often, more than one kinesin is implicated in a cellular process, and many kinesins are proposed to have overlapping functions. By using conventional kinesin as a model system, we have developed an approach to activate or inhibit a specific kinesin allele in the presence of other similar motor proteins. Modified ATP analogs are described that do not activate either conventional kinesin or another superfamily member, Eg5. However, a kinesin allele with Arg-14 in its nucleotide binding pocket mutated to alanine can use a subset of these nucleotide analogs to drive microtubule gliding. Cyclopentyl-ATP is one such analog. Cyclopentyl-adenylylimidodiphosphate, a nonhydrolyzable form of this analog, inhibits the mutant allele in microtubule-gliding assays, but not wild-type kinesin or Eg5. We anticipate that the incorporation of kinesin mutants and allele-specific activators and inhibitors in in vitro assays should clarify the role of individual motor proteins in complex cellular processes.

Selection of gp41-mediated HIV-1 cell entry inhibitors from biased combinatorial libraries of non-natural binding elements.

The trimeric, alpha-helical coiled-coil core of the HIV-1 gp41 ectodomain is thought to be part of a transient, receptor-triggered intermediate in the refolding of the envelope glycoprotein into a fusion-active conformation. In an effort to discover small organic inhibitors that block gp41 activation, we have generated a biased combinatorial chemical library of non-natural binding elements targeted to the gp41 core. From this library of 61,275 potential ligands, we have identified elements that, when covalently attached to a peptide derived from the gp41 outer-layer alpha-helix, contribute to the formation of a stable complex with the inner core and to inhibition of gp41-mediated cell fusion.

The structure of an HIV-1 specific cell entry inhibitor in complex with the HIV-1 gp41 trimeric core.

The three-dimensional structure of the complex between an HIV-1 cell-entry inhibitor selected from screening a combinatorial library of non-natural building blocks and the central, trimeric, coiled-coil core of HIV-1 gp41 has been determined by X-ray crystallography. The biased combinatorial library was designed to identify ligands binding in nonpolar pockets on the surface of the coiled-coil core of gp41. The crystal structure shows that the non-peptide moiety of the inhibitor binds to the targeted cavity in two different binding modes. This result suggests a strategy for increasing inhibitor potency by use of a second-generation combinatorial library designed to give simultaneous occupancy of both binding sites.