Explore the vast range of titles available.

Leucine is a proteogenic amino acid that also regulates many aspects of mammalian physiology, in large part by activating the mTOR complex 1 (mTORC1) protein kinase, a master growth controller. Amino acids signal to mTORC1 through the Rag guanosine triphosphatases (GTPases). Several factors regulate the Rags, including GATOR1, a GTPase-activating protein; GATOR2, a positive regulator of unknown function; and Sestrin2, a GATOR2-interacting protein that inhibits mTORC1 signaling. We find that leucine, but not arginine, disrupts the Sestrin2-GATOR2 interaction by binding to Sestrin2 with a dissociation constant of 20 micromolar, which is the leucine concentration that half-maximally activates mTORC1. The leucine-binding capacity of Sestrin2 is required for leucine to activate mTORC1 in cells. These results indicate that Sestrin2 is a leucine sensor for the mTORC1 pathway.

The chemotherapeutic drug methotrexate inhibits the enzyme dihydrofolate reductase 1 , which generates tetrahydrofolate, an essential cofactor in nucleotide synthesis 2 . Depletion of tetrahydrofolate causes cell death by suppressing DNA and RNA production 3 . Although methotrexate is widely used as an anticancer agent and is the subject of over a thousand ongoing clinical trials 4 , its high toxicity often leads to the premature termination of its use, which reduces its potential efficacy 5 . To identify genes that modulate the response of cancer cells to methotrexate, we performed a CRISPR–Cas9-based screen 6 , 7 . This screen yielded FTCD , which encodes an enzyme—formimidoyltransferase cyclodeaminase—that is required for the catabolism of the amino acid histidine 8 , a process that has not previously been linked to methotrexate sensitivity. In cultured cancer cells, depletion of several genes in the histidine degradation pathway markedly decreased sensitivity to methotrexate. Mechanistically, histidine catabolism drains the cellular pool of tetrahydrofolate, which is particularly detrimental to methotrexate-treated cells. Moreover, expression of the rate-limiting enzyme in histidine catabolism is associated with methotrexate sensitivity in cancer cell lines and with survival rate in patients. In vivo dietary supplementation of histidine increased flux through the histidine degradation pathway and enhanced the sensitivity of leukaemia xenografts to methotrexate. The histidine degradation pathway markedly influences the sensitivity of cancer cells to methotrexate and may be exploited to improve methotrexate efficacy through a simple dietary intervention. Histidine metabolism influences the sensitivity of cancer cells to methotrexate, with mice bearing leukaemia xenografts showing increased response to the drug upon histidine supplementation.

A number of heterologous enzymes have been investigated for cancer treatment and other therapeutic applications; however, immunogenicity issues have limited their clinical utility. Here, a new approach has been created for heterologous enzyme deimmunization whereby combinatorial saturation mutagenesis is coupled with a screening strategy that capitalizes on the evolutionary biology concept of neutral drift, and combined with iterative computational prediction of T-cell epitopes to achieve extensive reengineering of a protein sequence for reduced MHC-II binding propensity without affecting catalytic and pharmacological properties. Escherichia coli L-asparaginase II (EcAll), the only nonhuman enzyme approved for repeated administration, is critical in treatment of childhood acute lymphoblastic leukemia (ALL), but elicits adverse antibody responses in a significant fraction of patients. The neutral drift screening of combinatorial saturation mutagenesis libraries at a total of 12 positions was used to isolate an EcAll variant containing eight amino acid substitutions within computationally predicted T-cell epitopes—of which four were nonconservative—while still exhibiting k cat /K M = 10⁶ M⁻¹ s⁻¹ for L-Asn hydrolysis. Further, immunization of HLA-transgenic mice expressing the ALL-associated DRB1*0401 allele with the engineered variant resulted in significantly reduced T-cell responses and a 10-fold reduction in anti-EcAll IgG titers relative to the existing therapeutic. This significant reduction in the immunogenicity of EcAll may be clinically relevant for ALL treatment and illustrates the potential of employing neutral drift screens to achieve large jumps in sequence space as may be required for the deimmunization of heterologous proteins.

Tumours are a low-oxygen environment, in this study glioblastoma cells are found to overexpress the serine hydroxymethyltransferase SHMT2; SHMT acts to reduce oxygen consumption, which confers the tumour cells with a survival advantage. Low oxygen gives tumour cells an edge Tumour cells thrive in a low-oxygen environment, and in this study David Sabatini and colleagues demonstrate a mechanism that operates in the ischaemic zone of glioblastoma cells to give tumour cells a survival advantage. Glioblastoma cells are shown to overexpress the serine hydroxymethyltransferase (SHMT2) and glycine decarboxylase (GLDC). SHMT2 favours poorly vascularized tumour cells by reducing oxygen consumption but at the same time it exposes a selective vulnerability. Glycine, the product of SHMT2 activity, if allowed to accumulate in excess within the cell can be converted into toxic molecules, hence it may be possible to target tumorigenic glioblastoma cells by inhibiting GLDC. Cancer cells adapt their metabolic processes to support rapid proliferation, but less is known about how cancer cells alter metabolism to promote cell survival in a poorly vascularized tumour microenvironment 1 , 2 , 3 . Here we identify a key role for serine and glycine metabolism in the survival of brain cancer cells within the ischaemic zones of gliomas. In human glioblastoma multiforme, mitochondrial serine hydroxymethyltransferase (SHMT2) and glycine decarboxylase (GLDC) are highly expressed in the pseudopalisading cells that surround necrotic foci. We find that SHMT2 activity limits that of pyruvate kinase (PKM2) and reduces oxygen consumption, eliciting a metabolic state that confers a profound survival advantage to cells in poorly vascularized tumour regions. GLDC inhibition impairs cells with high SHMT2 levels as the excess glycine not metabolized by GLDC can be converted to the toxic molecules aminoacetone and methylglyoxal. Thus, SHMT2 is required for cancer cells to adapt to the tumour environment, but also renders these cells sensitive to glycine cleavage system inhibition.

Herein, we describe a methodology for the construction of targeted libraries intended to modify the substrate specificity of proteases expressed on the cell surface of Escherichia coli . The native outer membrane protease, OmpT, is used as a model system. The protocol relies on gene assembly using oligonucleotides and is easily adaptable to any enzyme in which information is available on the putative active site residues. Increasingly complex libraries can be generated in a systematic manner and screened using flow cytometry (fluorescence-activated cell sorting, FACS) for variants displaying altered function. Furthermore, if the substrate-binding pockets have not been elucidated, a protocol for partial multi-site saturation library construction is presented that allows for sampling a large number of residues, while maintaining an appropriate level of protein function. The entire procedure, from start to finish, should take approximately 2–3 weeks.

Hexokinase (HK) catalyzes the synthesis of glucose-6-phosphate, marking the first committed step of glucose metabolism. Most cancer cells express two homologous isoforms (HK1 and HK2) that can each bind to the outer mitochondrial membrane (OMM). CRISPR screens across hundreds of cancer cell lines indicate that both are dispensable for cell growth in traditional culture media. By contrast, deletion impairs cell growth in Human Plasma-Like Medium (HPLM). Here, we find that HK2 is required to maintain sufficient cytosolic (OMM-detached) HK activity under conditions that enhance HK1 binding to the OMM. Notably, OMM-detached rather than OMM-docked HK promotes \"aerobic glycolysis\" (Warburg effect), an enigmatic phenotype displayed by most proliferating cells. We show that several proposed theories for this phenotype cannot explain the dependence and instead find that deletion severely impairs glycolytic ATP production with little impact on total ATP yield for cells in HPLM. Our results reveal a basis for conditional essentiality and suggest that demand for compartmentalized ATP synthesis underlies the Warburg effect.

Maturing human pluripotent stem cell‐derived cardiomyocytes (hPSC‐CMs) in vitro is critical for advancing drug discovery and cardiotoxicity screening applications of these cells. However, the metabolic compositions of basal media used for hPSC‐CM culture typically offer limited relevance to human cardiac physiology. Here, we examined how culture in human plasma‐like medium (HPLM) versus conventional basal media affects the behavior of hPSC‐CMs. Starting with Day 16 hPSC‐CMs, we cultured cells for 2 weeks in either HPLM or RPMI‐based media and then assessed maturation outcomes at Day 30. Compared to RPMI/B27 media containing either RPMI‐defined (11.1 mM) or physiologic glucose levels (5 mM), HPLM/B27 enhanced hPSC‐CM maturity as evinced by concerted transcriptomic, structural, and metabolic phenotypes. These effects included a higher extent of myosin heavy chain isoform switching (α‐MHC to β‐MHC), accelerated ventricular‐specific myosin light chain isoform switching (MLC2a to MLC2v), elongated sarcomeres, increased multinucleation, enhanced calcium transient kinetics, and coordinated activation of oxidative and glycolytic metabolism. Collectively, these findings demonstrate that medium composition has substantial effects on hPSC‐CM biology and also establish HPLM as a basal medium for driving hPSC‐CM maturation in vitro.

Chemical screening studies have identified drug sensitivities across hundreds of cancer cell lines but most putative therapeutics fail to translate. Discovery and development of drug candidates in models that more accurately reflect nutrient availability in human biofluids may help in addressing this major challenge. Here we performed high-throughput screens in conventional versus Human Plasma-Like Medium (HPLM). Sets of conditional anticancer compounds span phases of clinical development and include non-oncology drugs. Among these, we characterize a unique dual-mechanism of action for brivudine, an agent otherwise approved for antiviral treatment. Using an integrative approach, we find that brivudine affects two independent targets in folate metabolism. We also traced conditional phenotypes for several drugs to the availability of nucleotide salvage pathway substrates and verified others for compounds that seemingly elicit off-target anticancer effects. Our findings establish generalizable strategies for exploiting conditional lethality in HPLM to reveal therapeutic candidates and mechanisms of action.

Cancer cells adapt their metabolic processes to support rapid proliferation, but less is known about how cancer cells alter metabolism to promote cell survival in a poorly vascularized tumor microenvironment1–3. Here, we identify a key role for serine and glycine metabolism in the survival of brain cancer cells within the ischemic zones of gliomas. In human glioblastoma multiforme (GBM), mitochondrial serine hydroxymethyltransferase (SHMT2) and glycine decarboxylase (GLDC) are highly expressed in the pseudopalisading cells that surround necrotic foci. We find that SHMT2 activity limits that of pyruvate kinase (PKM2) and reduces oxygen consumption, eliciting a metabolic state that confers a profound survival advantage to cells in poorly vascularized tumor regions. GLDC inhibition impairs cells with high SHMT2 levels as the excess glycine not metabolized by GLDC can be converted to the toxic molecules aminoacetone and methylglyoxal. Thus, SHMT2 is required for cancer cells to adapt to the tumor environment, but also renders these cells sensitive to glycine cleavage system inhibition.