MALT1 inhibition suppresses antigen-specific T cell responses.

The aim of this study was to assess the potential use of a selective small molecule MALT1 inhibitor in solid tumor treatment as an immunotherapy targeting regulatory T-cells (Tregs). In vitro, MALT1 inhibition suppressed the proteolytic cleavage of the MALT1-substrate HOIL1 and blocked IL-2 secretion in Jurkat cells. It selectively suppressed the proliferation of PBMC-derived Tregs, with no effect on conventional CD4+T-cells. In vivo, however, no evident anti-tumor effect was achieved by MALT1 inhibition monotherapy or in combination with anti-CTLA4 in the MB49 cancer model. Despite decreased Treg-frequencies in lymph nodes of tumor-bearing animals, intratumoral Treg depletion was not observed. We also showed that MALT1-inhibition caused a reduction of antigen-specific CD8+T-cells in an adoptive T-cell transfer model. Thus, selective targeting of Tregs would be required to improve the immunotherapeutic effect of MALT1-inhibition. Also, various dosing schedules and combination therapy strategies should be carefully designed and evaluated further.

PVT1 interacts with polycomb repressive complex 2 to suppress genomic regions with pro-apoptotic and tumour suppressor functions in multiple myeloma.

Multiple myeloma is a heterogeneous hematological disease that originates from the bone marrow and is characterized by the monoclonal expansion of malignant plasma cells. Despite novel therapies, multiple myeloma remains clinically challenging. A common feature among patients with poor prognosis is the increased activity of the epigenetic silencer EZH2, which is the catalytic subunit of the PRC2. Interestingly, the recruitment of PRC2 lacks sequence specificity and, to date, the molecular mechanisms that define which genomic locations are destined for PRC2-mediated silencing remain unknown. The presence of a long non-coding RNA (lncRNA)-binding pocket on EZH2 suggests that lncRNA could potentially mediate PRC2 recruitment to specific genomic regions. Here, we coupled RNA immunoprecipitation sequencing, RNA-sequencing and chromatin immunoprecipitation-sequencing analysis of human multiple myeloma primary cells and cell lines to identify potential lncRNA partners to EZH2. We found that the lncRNA plasmacytoma variant translocation 1 (PVT1) directly interacts with EZH2 and is overexpressed in patients with a poor prognosis. Moreover, genes predicted to be targets of PVT1 exhibited H3K27me3 enrichment and were associated with pro-apoptotic and tumor suppressor functions. In fact, PVT1 inhibition independently promotes the expression of the PRC2 target genes ZBTB7C, RNF144A and CCDC136. Altogether, our work suggests that PVT1 is an interacting partner in PRC2-mediated silencing of tumor suppressor and pro-apoptotic genes in multiple myeloma, making it a highly interesting potential therapeutic target.

SARS-CoV-2 Neutralizing Antibody Responses towards Full-Length Spike Protein and the Receptor-Binding Domain.

Tools to monitor SARS-CoV-2 transmission and immune responses are needed. We present a neutralization ELISA to determine the levels of Ab-mediated virus neutralization and a preclinical model of focused immunization strategy. The ELISA is strongly correlated with the elaborate plaque reduction neutralization test (ρ = 0.9231, p < 0.0001). The neutralization potency of convalescent sera strongly correlates to IgG titers against SARS-CoV-2 receptor-binding domain (RBD) and spike (ρ = 0.8291 and 0.8297, respectively; p < 0.0001) and to a lesser extent with the IgG titers against protein N (ρ = 0.6471, p < 0.0001). The preclinical vaccine NMRI mice models using RBD and full-length spike Ag as immunogens show a profound Ab neutralization capacity (IC50 = 1.9 × 104 to 2.6 × 104 and 3.9 × 103 to 5.2 × 103, respectively). Using a panel of novel high-affinity murine mAbs, we also show that a majority of the RBD-raised mAbs have inhibitory properties, whereas only a few of the spike-raised mAbs do. The ELISA-based viral neutralization test offers a time- and cost-effective alternative to the plaque reduction neutralization test. The immunization results indicate that vaccine strategies focused only on the RBD region may have advantages compared with the full spike.

Symptomatic and structural benefit of cathepsin K inhibition by MIV-711 in a subgroup with unilateral pain: post-hoc analysis of a randomised phase 2a clinical trial.

OBJECTIVES: Osteoarthritis (OA) development programmes face challenges due to discordance between structural changes and symptoms. A novel cathepsin-K inhibitor, MIV-711, recently reported structural benefits, but did not demonstrate a significant difference from placebo in symptoms. Previous work suggests that pain from non-target joints may confound OA pain outcomes. We therefore conducted an exploratory analysis in participants with predominantly unilateral knee pain from the MIV-711-201 trial. METHOSD: Participants scoring below median contralateral knee NRS pain at baseline from the MIV-711-201 phase 2a clinical trial (n=119) were analysed by treatment group for differences in change from baseline in WOMAC pain, quantitative magnetic resonance imaging bone area and cartilage thickness with a repeated-measures mixed model adjusting for relevant co-variates. RESULTS: In the subgroup with unilateral knee pain, treatment with MIV-711 100 mg led to greater reduction in WOMAC pain compared to placebo (-5.0, 95% CI: -8.69 to -1.3, p=0.008), while 200 mg did not (-2.5, 95% CI: -6.5 to 1.6, p=0.23). MIV-711 treatment was associated with a reduced change in bone area compared to placebo (200 mg; -19.6 mm2 , 95% CI: -36.2 to -3.0, p=0.02, and 100 mg; -12.5 mm2 , 95% CI: -27.8 to 2.8, p=0.11,). No observed differences between treatment groups in cartilage thickness were found in this subgroup. CONCLUSIONS: In a subgroup with predominantly unilateral knee pain, significant reduction in OA pain by MIV-711 100 mg treatment was found, with concurrent beneficial structural effects, highlighting the importance of appropriate pain inclusion criteria in OA trials.

Cell free protein synthesis versus yeast expression - A comparison using insulin as a model protein.

Expression of recombinant proteins traditionally require a cellular system to transcribe and translate foreign DNA to a desired protein. The process requires special knowledge of the specific cellular metabolism in use and is often time consuming and labour intensive. A cell free expression system provides an opportunity to express recombinant proteins without consideration of the living cell. Instead, a cell free system relies on either a cellular lysate or recombinant proteins to carry out protein synthesis, increasing overall production speed and ease of handling. The one-pot cell free setup is commonly known as an in vitro transcription/translation reaction (IVTT). Here we focused on a PURE (Protein synthesis Using Recombinant Elements) IVTT system based on recombinant proteins from Escherichia coli. We evaluated the cell free system's ability to express functional insulin analogues compared to Saccharomyces cerevisiae, a well-established system for large scale production of recombinant human insulin and insulin analogues. Significantly, it was found that correct insulin expression and folding was governed by the inherent properties of the primary amino acids sequence of insulin, whereas the eukaryotic features of the expression system apparently play a minor role. The IVTT system successfully produced insulin analogues identical in structure and with similar insulin receptor affinity to those produced by yeast. In conclusion we demonstrate that the PURE IVTT system is highly suited for expressing soluble molecules with higher order features and multiple disulphide bridges.

X-ray structure of human aquaporin 2 and its implications for nephrogenic diabetes insipidus and trafficking.

Human aquaporin 2 (AQP2) is a water channel found in the kidney collecting duct, where it plays a key role in concentrating urine. Water reabsorption is regulated by AQP2 trafficking between intracellular storage vesicles and the apical membrane. This process is tightly controlled by the pituitary hormone arginine vasopressin and defective trafficking results in nephrogenic diabetes insipidus (NDI). Here we present the X-ray structure of human AQP2 at 2.75 Å resolution. The C terminus of AQP2 displays multiple conformations with the C-terminal α-helix of one protomer interacting with the cytoplasmic surface of a symmetry-related AQP2 molecule, suggesting potential protein-protein interactions involved in cellular sorting of AQP2. Two Cd(2+)-ion binding sites are observed within the AQP2 tetramer, inducing a rearrangement of loop D, which facilitates this interaction. The locations of several NDI-causing mutations can be observed in the AQP2 structure, primarily situated within transmembrane domains and the majority of which cause misfolding and ER retention. These observations provide a framework for understanding why mutations in AQP2 cause NDI as well as structural insights into AQP2 interactions that may govern its trafficking.

Establishing a synthetic pathway for high-level production of 3-hydroxypropionic acid in Saccharomyces cerevisiae via ß-alanine.

Microbial fermentation of renewable feedstocks into plastic monomers can decrease our fossil dependence and reduce global CO2 emissions. 3-Hydroxypropionic acid (3HP) is a potential chemical building block for sustainable production of superabsorbent polymers and acrylic plastics. With the objective of developing Saccharomyces cerevisiae as an efficient cell factory for high-level production of 3HP, we identified the ß-alanine biosynthetic route as the most economically attractive according to the metabolic modeling. We engineered and optimized a synthetic pathway for de novo biosynthesis of ß-alanine and its subsequent conversion into 3HP using a novel ß-alanine-pyruvate aminotransferase discovered in Bacillus cereus. The final strain produced 3HP at a titer of 13.7±0.3gL(-1) with a 0.14±0.0C-molC-mol(-1) yield on glucose in 80h in controlled fed-batch fermentation in mineral medium at pH 5, and this work therefore lays the basis for developing a process for biological 3HP production.

Recombinant production of the human aquaporins in the yeast Pichia pastoris (Invited Review).

Aquaporins are water facilitating proteins embedded in the cellular membranes. Such channels have been identified in almost every living organism - including humans. These proteins are vital molecules and their malfunction can lead to several severe disorders and diseases. Hence, an increased understanding of their structure, function and regulation is of the utmost importance for developing current and future drugs. Heading towards this goal, the first problem to overcome is to acquire the proteins in sufficient amounts to enable functional and structural characterization. Using a suitable host organism, large amounts of target molecules can possibly be produced, but for membrane proteins limitations are frequently encountered. In the work described here, we have produced the 13 human aquaporins (hAQPs) in one of the most successful hosts for recombinant overproduction of eukaryotic proteins; the yeast Pichia pastoris, in order to explore the underlying bottleneck to a successful membrane protein production experiment. Here we present exceptional yield of hAQP1, whereas some other hAQPs were below the threshold needed for scaled up production. In the overproduction process, we have established methods for efficient production screening as well as for accurate determination of the initial production yield. Furthermore, we have optimized the yield of low producing targets, enabling studies of proteins previously out of reach, exemplified with hAQP4 as well as the homologue PfAQP. Taken together, our results. present insight into factors directing high production of eukaryotic membrane proteins together with suggestions on ways to optimize the recombinant production in the yeast P. pastoris.

CO2 permeability of cell membranes is regulated by membrane cholesterol and protein gas channels.

Recent observations that some membrane proteins act as gas channels seem surprising in view of the classical concept that membranes generally are highly permeable to gases. Here, we study the gas permeability of membranes for the case of CO(2), using a previously established mass spectrometric technique. We first show that biological membranes lacking protein gas channels but containing normal amounts of cholesterol (30-50 mol% of total lipid), e.g., MDCK and tsA201 cells, in fact possess an unexpectedly low CO(2) permeability (P(CO2)) of ∼0.01 cm/s, which is 2 orders of magnitude lower than the P(CO2) of pure planar phospholipid bilayers (∼1 cm/s). Phospholipid vesicles enriched with similar amounts of cholesterol also exhibit P(CO2) ≈ 0.01 cm/s, identifying cholesterol as the major determinant of membrane P(CO2). This is confirmed by the demonstration that MDCK cells depleted of or enriched with membrane cholesterol show dramatic increases or decreases in P(CO2), respectively. We demonstrate, furthermore, that reconstitution of human AQP-1 into cholesterol-containing vesicles, as well as expression of human AQP-1 in MDCK cells, leads to drastic increases in P(CO2), indicating that gas channels are of high functional significance for gas transfer across membranes of low intrinsic gas permeability.

A regulatory polymorphism in PDCD1 is associated with susceptibility to systemic lupus erythematosus in humans.

Systemic lupus erythematosus (SLE, OMIM 152700) is a complex autoimmune disease that affects 0.05% of the Western population, predominantly women. A number of susceptibility loci for SLE have been suggested in different populations, but the nature of the susceptibility genes and mutations is yet to be identified. We previously reported a susceptibility locus (SLEB2) for Nordic multi-case families. Within this locus, the programmed cell death 1 gene (PDCD1, also called PD-1) was considered the strongest candidate for association with the disease. Here, we analyzed 2,510 individuals, including members of five independent sets of families as well as unrelated individuals affected with SLE, for single-nucleotide polymorphisms (SNPs) that we identified in PDCD1. We show that one intronic SNP in PDCD1 is associated with development of SLE in Europeans (found in 12% of affected individuals versus 5% of controls; P = 0.00001, r.r. (relative risk) = 2.6) and Mexicans (found in 7% of affected individuals versus 2% of controls; P = 0.0009, r.r. = 3.5). The associated allele of this SNP alters a binding site for the runt-related transcription factor 1 (RUNX1, also called AML1) located in an intronic enhancer, suggesting a mechanism through which it can contribute to the development of SLE in humans.

Glycosylation increases the thermostability of human aquaporin 10 protein.

Human aquaporin10 (hAQP10) is a transmembrane facilitator of both water and glycerol transport in the small intestine. This aquaglyceroporin is located in the apical membrane of enterocytes and is believed to contribute to the passage of water and glycerol through these intestinal absorptive cells. Here we overproduced hAQP10 in the yeast Pichia pastoris and observed that the protein is glycosylated at Asn-133 in the extracellular loop C. This finding confirms one of three predicted glycosylation sites for hAQP10, and its glycosylation is unique for the human aquaporins overproduced in this host. Nonglycosylated protein was isolated using both glycan affinity chromatography and through mutating asparagine 133 to a glutamine. All three forms of hAQP10 where found to facilitate the transport of water, glycerol, erythritol, and xylitol, and glycosylation had little effect on functionality. In contrast, glycosylated hAQP10 showed increased thermostability of 3-6 °C compared with the nonglycosylated protein, suggesting a stabilizing effect of the N-linked glycan. Because only one third of hAQP10 was glycosylated yet the thermostability titration was mono-modal, we suggest that the presence of at least one glycosylated protein within each tetramer is sufficient to convey an enhanced structural stability to the remaining hAQP10 protomers of the tetramer.

Stat1 activation attenuates IL-6 induced Stat3 activity but does not alter apoptosis sensitivity in multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is at present an incurable malignancy, characterized by apoptosis-resistant tumor cells. Interferon (IFN) treatment sensitizes MM cells to Fas-induced apoptosis and is associated with an increased activation of Signal transducer and activator of transcription (Stat)1. The role of Stat1 in MM has not been elucidated, but Stat1 has in several studies been ascribed a pro-apoptotic role. Conversely, IL-6 induction of Stat3 is known to confer resistance to apoptosis in MM. METHODS: To delineate the role of Stat1 in IFN mediated sensitization to apoptosis, sub-lines of the U-266-1970 MM cell line with a stable expression of the active mutant Stat1C were utilized. The influence of Stat1C constitutive transcriptional activation on endogenous Stat3 expression and activation, and the expression of apoptosis-related genes were analyzed. To determine whether Stat1 alone would be an important determinant in sensitizing MM cells to apoptosis, the U-266-1970-Stat1C cell line and control cells were exposed to high throughput compound screening (HTS). RESULTS: To explore the role of Stat1 in IFN mediated apoptosis sensitization of MM, we established sublines of the MM cell line U-266-1970 constitutively expressing the active mutant Stat1C. We found that constitutive nuclear localization and transcriptional activity of Stat1 was associated with an attenuation of IL-6-induced Stat3 activation and up-regulation of mRNA for the pro-apoptotic Bcl-2 protein family genes Harakiri, the short form of Mcl-1 and Noxa. However, Stat1 activation alone was not sufficient to sensitize cells to Fas-induced apoptosis. In a screening of > 3000 compounds including bortezomib, dexamethasone, etoposide, suberoylanilide hydroxamic acid (SAHA), geldanamycin (17-AAG), doxorubicin and thalidomide, we found that the drug response and IC50 in cells constitutively expressing active Stat1 was mainly unaltered. CONCLUSION: We conclude that Stat1 alters IL-6 induced Stat3 activity and the expression of pro-apoptotic genes. However, this shift alone is not sufficient to alter apoptosis sensitivity in MM cells, suggesting that Stat1 independent pathways are operative in IFN mediated apoptosis sensitization.

Improving recombinant eukaryotic membrane protein yields in Pichia pastoris: the importance of codon optimization and clone selection.

In the last 15 years, 80% of all recombinant proteins reported in the literature were produced in the bacterium, Escherichia coli, or the yeast, Pichia pastoris. Nonetheless, developing effective general strategies for producing recombinant eukaryotic membrane proteins in these organisms remains a particular challenge. Using a validated screening procedure together with accurate yield quantitation, we therefore wished to establish the critical steps contributing to high yields of recombinant eukaryotic membrane protein in P. pastoris. Whilst the use of fusion partners to generate chimeric constructs and directed mutagenesis have previously been shown to be effective in bacterial hosts, we conclude that this approach is not transferable to yeast. Rather, codon optimization and the preparation and selection of high-yielding P. pastoris clones are effective strategies for maximizing yields of human aquaporins.

Acoustical and perceptual influence of duplex stringing in grand pianos.

This study investigates the acoustical and perceptual influence of the string parts outside the speaking length in grand pianos (front and rear duplex strings). Acoustical measurements on a grand piano in concert condition were conducted, measuring the fundamental frequencies of all main and duplex strings in the four octaves D4-C8. Considerable deviations from the nominal harmonic relations between the rear duplex and main string frequencies, as described by the manufacturer in a patent, were observed. Generally the rear duplex strings were tuned higher than the nominal harmonic relations with average and median deviations approaching +50 cent. Single keys reached +190 and -100 cent. The spread in deviation from harmonic relations within trichords was also substantial with average and median values around 25 cent, occasionally reaching 60 cent. Contributions from both front and rear duplex strings were observed in the bridge motion and sound. The audibility of the duplex strings was studied in an ABX listening test. Complete dampening of the front duplex was clearly perceptible both for an experiment group consisting of musicians and a control group with naive subjects. The contribution from the rear duplex could also be perceived, but less pronounced.

The Endothelin Receptor Antagonist Macitentan Inhibits Human Cytomegalovirus Infection.

Human cytomegalovirus (HCMV) infection is an important cause of morbidity and mortality in immunocompromised patients and a major etiological factor for congenital birth defects in newborns. Ganciclovir and its pro-drug valganciclovir are the preferred drugs in use today for prophylaxis and treatment of viremic patients. Due to long treatment times, patients are at risk for developing viral resistance to ganciclovir and to other drugs with a similar mechanism of action. We earlier found that the endothelin receptor B (ETBR) is upregulated during HCMV infection and that it plays an important role in the life cycle of this virus. Here, we tested the hypothesis that ETBR blockade could be used in the treatment of HCMV infection. As HCMV infection is specific to humans, we tested our hypothesis in human cell types that are relevant for HCMV pathogenesis; i.e., endothelial cells, epithelial cells and fibroblasts. We infected these cells with HCMV and treated them with the ETBR specific antagonist BQ788 or ETR antagonists that are approved by the FDA for treatment of pulmonary hypertension; macitentan, its metabolite ACT-132577, bosentan and ambrisentan, and as an anti-viral control, we used ganciclovir or letermovir. At concentrations expected to be relevant in vivo, macitentan, ACT-132577 and BQ788 effectively inhibited productive infection of HCMV. Of importance, macitentan also inhibited productive infection of a ganciclovir-resistant HCMV isolate. Our results suggest that binding or signaling through ETBR is crucial for viral replication, and that selected ETBR blockers inhibit HCMV infection.

A distinct metabolic response characterizes sensitivity to EZH2 inhibition in multiple myeloma.

Multiple myeloma (MM) is a heterogeneous haematological disease that remains clinically challenging. Increased activity of the epigenetic silencer EZH2 is a common feature in patients with poor prognosis. Previous findings have demonstrated that metabolic profiles can be sensitive markers for response to treatment in cancer. While EZH2 inhibition (EZH2i) has proven efficient in inducing cell death in a number of human MM cell lines, we hereby identified a subset of cell lines that despite a global loss of H3K27me3, remains viable after EZH2i. By coupling liquid chromatography-mass spectrometry with gene and miRNA expression profiling, we found that sensitivity to EZH2i correlated with distinct metabolic signatures resulting from a dysregulation of genes involved in methionine cycling. Specifically, EZH2i resulted in a miRNA-mediated downregulation of methionine cycling-associated genes in responsive cells. This induced metabolite accumulation and DNA damage, leading to G2 arrest and apoptosis. Altogether, we unveiled that sensitivity to EZH2i in human MM cell lines is associated with a specific metabolic and gene expression profile post-treatment.

Unraveling Human AQP5-PIP Molecular Interaction and Effect on AQP5 Salivary Glands Localization in SS Patients.

Saliva secretion requires effective translocation of aquaporin 5 (AQP5) water channel to the salivary glands (SGs) acinar apical membrane. Patients with Sjögren's syndrome (SS) display abnormal AQP5 localization within acinar cells from SGs that correlate with sicca manifestation and glands hypofunction. Several proteins such as Prolactin-inducible protein (PIP) may regulate AQP5 trafficking as observed in lacrimal glands from mice. However, the role of the AQP5-PIP complex remains poorly understood. In the present study, we show that PIP interacts with AQP5 in vitro and in mice as well as in human SGs and that PIP misexpression correlates with an altered AQP5 distribution at the acinar apical membrane in PIP knockout mice and SS hMSG. Furthermore, our data show that the protein-protein interaction involves the AQP5 C-terminus and the N-terminal of PIP (one molecule of PIP per AQP5 tetramer). In conclusion, our findings highlight for the first time the role of PIP as a protein controlling AQP5 localization in human salivary glands but extend beyond due to the PIP-AQP5 interaction described in lung and breast cancers.

TGF-beta enforces senescence in Myc-transformed hematopoietic tumor cells through induction of Mad1 and repression of Myc activity.

Inhibition of tumor growth factor (TGF)-beta-mediated cell cycle exit is considered an important tumorigenic function of Myc oncoproteins. Here we found that TGF-beta1 enforced G(1) cell cycle arrest and cellular senescence in human U-937 myeloid tumor cells ectopically expressing v-Myc, which contains a stabilizing mutation frequently found in lymphomas. This correlated with induced expression of the Myc antagonist Mad1, resulting in replacement of Myc for Mad1 at target promoters, reduced histone acetylation and strong repression of Myc-driven transcription. The latter was partially reversed by histone deacetylase (HDAC) inhibitors, consistent with involvement of Mad1. Importantly, knockdown of MAD1 expression prevented TGF-beta1-induced senescence, underscoring that Mad1 is a crucial component of this process. Enforced Mad1 expression sensitized U-937-myc cells to TGF-beta and restored phorbol ester-induced cell cycle exit, but could not alone induce G(1) arrest, suggesting that Mad1 is required but not sufficient for cellular senescence. Our results thus demonstrate that TGF-beta can override Myc activity despite a stabilizing cancer mutation and induce senescence in myeloid tumor cells, at least in part by induction of Mad1. TGF-beta-induced senescence, or signals mimicking this pathway, could therefore potentially be explored as a therapeutic principle for treating hematopoietic and other tumors with deregulated MYC expression.

Insight into factors directing high production of eukaryotic membrane proteins; production of 13 human AQPs in Pichia pastoris.

Membrane proteins are key players in all living cells. To achieve a better understanding of membrane protein function, significant amounts of purified protein are required for functional and structural analyses. Overproduction of eukaryotic membrane proteins, in particular, is thus an essential yet non-trivial task. Hence, improved understanding of factors which direct a high production of eukaryotic membrane proteins is desirable. In this study we have compared the overproduction of all human aquaporins in the eukaryotic host Pichia pastoris. We report quantitated production levels of each homologue and the extent of their membrane localization. Our results show that the protein production levels vary substantially, even between highly homologous aquaporins. A correlation between the extents of membrane insertion with protein function also emerged, with a higher extent of membrane insertion for pure water transporters compared to aquaporin family members with other substrate specificity. Nevertheless, the nucleic acid sequence of the second codon appears to play an important role in overproduction. Constructs containing guanine at the first position of this codon (being part of the mammalian Kozak sequence) are generally produced at a higher level, which is confirmed for hAQP8. In addition, mimicking the yeast consensus sequence (ATGTCT) apparently has a negative influence on the production level, as shown for hAQP1. Moreover, by mutational analysis we show that the yield of hAQP4 can be heavily improved by directing the protein folding pathway as well as stabilizing the aquaporin tetramer.

A lipidic-sponge phase screen for membrane protein crystallization.

A major current deficit in structural biology is the lack of high-resolution structures of eukaryotic membrane proteins, many of which are key drug targets for the treatment of disease. Numerous eukaryotic membrane proteins require specific lipids for their stability and activity, and efforts to crystallize and solve the structures of membrane proteins that do not address the issue of lipids frequently end in failure rather than success. To help address this problem, we have developed a sparse matrix crystallization screen consisting of 48 lipidic-sponge phase conditions. Sponge phases form liquid lipid bilayer environments which are suitable for conventional hanging- and sitting-drop crystallization experiments. Using the sponge phase screen, we obtained crystals of several different membrane proteins from bacterial and eukaryotic sources. We also demonstrate how the screen may be manipulated by incorporating specific lipids such as cholesterol; this modification led to crystals being recovered from a bacterial photosynthetic core complex.