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Biosynthesis is an environmentally benign and renewable approach that can be used to produce a broad range of natural and, in some cases, new-to-nature products. However, biology lacks many of the reactions that are available to synthetic chemists, resulting in a narrower scope of accessible products when using biosynthesis rather than synthetic chemistry. A prime example of such chemistry is carbene-transfer reactions1. Although it was recently shown that carbene-transfer reactions can be performed in a cell and used for biosynthesis2,3, carbene donors and unnatural cofactors needed to be added exogenously and transported into cells to effect the desired reactions, precluding cost-effective scale-up of the biosynthesis process with these reactions. Here we report the access to a diazo ester carbene precursor by cellular metabolism and a microbial platform for introducing unnatural carbene-transfer reactions into biosynthesis. The α-diazoester azaserine was produced by expressing a biosynthetic gene cluster in Streptomyces albus. The intracellularly produced azaserine was used as a carbene donor to cyclopropanate another intracellularly produced molecule—styrene. The reaction was catalysed by engineered P450 mutants containing a native cofactor with excellent diastereoselectivity and a moderate yield. Our study establishes a scalable, microbial platform for conducting intracellular abiological carbene-transfer reactions to functionalize a range of natural and new-to-nature products and expands the scope of organic products that can be produced by cellular metabolism.

Caveolin-1 (CAV1) enhanced migration, invasion, and metastasis of cancer cells is inhibited by co-expression of the glycoprotein E-cadherin. Although the two proteins form a multiprotein complex that includes β-catenin, it remained unclear how this would contribute to blocking the metastasis promoting function of CAV1. Here, we characterized by mass spectrometry the protein composition of CAV1 immunoprecipitates from B16F10 murine melanoma cells expressing or not E-cadherin. The novel protein tyrosine phosphatase PTPN14 was identified by mass spectrometry analysis exclusively in co-immunoprecipitates of CAV1 with E-cadherin. Interestingly, PTPN14 is implicated in controlling metastasis, but only few known PTPN14 substrates exist. We corroborated by western blotting experiments that PTPN14 and CAV1 co-inmunoprecipitated in the presence of E-cadherin in B16F10 melanoma and other cancer cells. Moreover, the CAV1(Y14F) mutant protein was shown to co-immunoprecipitate with PTPN14 even in the absence of E-cadherin, and overexpression of PTPN14 reduced CAV1 phosphorylation on tyrosine-14, as well as suppressed CAV1-enhanced cell migration, invasion and Rac-1 activation in B16F10, metastatic colon [HT29(US)] and breast cancer (MDA-MB-231) cell lines. Finally, PTPN14 overexpression in B16F10 cells reduced the ability of CAV1 to induce metastasis in vivo. In summary, we identify here CAV1 as a novel substrate for PTPN14 and show that overexpression of this phosphatase suffices to reduce CAV1-induced metastasis.

Close contacts between endoplasmic reticulum and mitochondria enable reciprocal Ca2+exchange, a key mechanism in the regulation of mitochondrial bioenergetics. During the early phase of endoplasmic reticulum stress, this inter-organellar communication increases as an adaptive mechanism to ensure cell survival. The signalling pathways governing this response, however, have not been characterized. Here we show that caveolin-1 localizes to the endoplasmic reticulum–mitochondria interface, where it impairs the remodelling of endoplasmic reticulum–mitochondria contacts, quenching Ca2+transfer and rendering mitochondrial bioenergetics unresponsive to endoplasmic reticulum stress. Protein kinase A, in contrast, promotes endoplasmic reticulum and mitochondria remodelling and communication during endoplasmic reticulum stress to promote organelle dynamics and Ca2+transfer as well as enhance mitochondrial bioenergetics during the adaptive response. Importantly, caveolin-1 expression reduces protein kinase A signalling, as evidenced by impaired phosphorylation and alterations in organelle distribution of the GTPase dynamin-related protein 1, thereby enhancing cell death in response to endoplasmic reticulum stress. In conclusion, caveolin-1 precludes stress-induced protein kinase A-dependent remodelling of endoplasmic reticulum–mitochondria communication.

Alkylidene cyclopropanes (ACPs) are valuable synthetic intermediates because of their constrained structure and opportunities for further diversification. Although routes to ACPs are known, preparations of ACPs with control of both the configuration of the cyclopropyl (Rvs S) group and the geometry of the alkene (Evs Z) are unknown. We describe enzymatic cyclopropanation of allenes with ethyl diazoacetate (EDA) catalyzed by an iridium-containing cytochrome (Ir(Me)-CYP119) that controls both stereochemical elements. Two mutants of Ir(Me)-CYP119 identified by 6-codon (6c, VILAFG) saturation mutagenesis catalyze the formation of (E)-ACPs with −93% to >99% ee and >99:1 E/Zratio with just three rounds of 96 mutants. By four additional rounds of mutagenesis, an enzyme variant was identified that forms (Z)-ACPs with up to 94% ee and a 28:72 E/Zratio. Computational studies show that the orientation of the carbene unit dictated by the mutated positions accounts for the stereoselectivity.

Single-molecule measurements combined with a novel mathematical strategy were applied to accurately characterize how bimolecular interactions respond to mechanical force, especially when protein purification is not possible. Specifically, we studied the effect of force on Thy-1/αvβ3 integrin interaction, a mediator of neuron-astrocyte communication.

Sarco/endoplasmic reticulum–mitochondria communication and mitochondrial dynamics are essential regulators of mitochondrial function, cellular metabolism, and calcium homeostasisSarco/endoplasmic reticulum–mitochondria contacts and mitochondrial dynamics modulate myocardial contractility and vascular smooth muscle cell differentiationAlterations in sarco/endoplasmic reticulum–mitochondria communication and in mitochondrial network morphology are implicated in several cardiovascular pathologies, including heart failure, coronary artery disease, and pulmonary hypertensionMore studies are required to define the role that alterations in sarco/endoplasmic–mitochondria contacts and mitochondrial dynamics have in the pathogenesis of cardiovascular diseases

Calcium (Ca2+) is a key player in the regulation of many cell functions. Just like Ca2+, mitochondria are ubiquitous, versatile, and dynamic players in determining both cell survival and death decisions. Given their ubiquitous nature, the regulation of both is deeply intertwined, whereby Ca2+regulates mitochondrial functions, while mitochondria shape Ca2+dynamics. Deregulation of either Ca2+or mitochondrial signaling leads to abnormal function, cell damage or even cell death, thereby contributing to muscle dysfunction or cardiac pathologies. Moreover, altered mitochondrial Ca2+homeostasis has been linked to metabolic diseases like cancer, obesity, and pulmonary hypertension. In this review article, we summarize the mechanisms that coordinate mitochondrial and Ca2+responses and how they affect human health. © 2017 American Physiological Society. Compr Physiol7:623-634, 2017.

We previously reported on enhanced susceptibility to death of lymphocytes from Alzheimer’s disease (AD) patients when exposed to hydrogen peroxide (H2O2)-induced oxidative stress and an increased resistance to death in those of patients with a history of skin cancer. This is consistent with our hypothesis proposing that the cellular machinery controlling cell death is deregulated in opposite directions in Alzheimer’s disease (AD) and cancer, to explain the inverse association observed in epidemiological studies. Here we investigated whether the observed increased susceptibility correlates with the degree of dementia severity. Peripheral lymphocytes from 23 AD patients, classified using the Clinical Dementia Rating (CDR) into severe dementia (CDR 3, n=10) and mild-to-moderate dementia (CDR 1- 2, n=13), and 15 healthy controls (HC) (CDR 0), were exposed to H2O2 for 20 hours. Lymphocyte death was determined by flow cytometry and propidium iodide staining. The greatest susceptibility to H2O2-induced death was observed for lymphocytes from severe dementia patients, whereas those with mild-to-moderate dementia exhibited intermediate values, compared to healthy controls. A significant increase in the apoptosis/necrosis ratio was found in AD patients. Poly (ADP-ribosyl) polymerase-1 (PARP-1) inhibition significantly protected from H2O2-induced death of lymphocytes, whereby a lower degree of protection was observed in severe AD patients. Moreover, inhibition of PARP-1 abolished the differences in apoptosis/necrosis ratios observed between the three groups of patients. These results support the notion that AD is a systemic disorder, whereby enhanced susceptibility to H2O2-induced death in peripheral lymphocytes correlates with dementia severity and enhanced death in AD patients is attributable to a PARP-dependent increase in the apoptosis/necrosis ratio.

Augmented expression of cyclooxygenase-2 (COX-2) and enhanced production of prostaglandin E2(PGE2) are associated with increased tumor cell survival and malignancy. Caveolin-1 is a scaffold protein that has been proposed to function as a tumor suppressor in human cancer cells, although mechanisms underlying this ability remain controversial. Intriguingly, the possibility that caveolin-1 regulates the expression of COX-2 has not been explored. Here we show that augmented caveolin-1 expression in cells with low basal levels of this protein, such as human colon cancer (HT29, DLD-1), breast cancer (ZR75), and embryonic kidney (HEK293T) cells reduced COX-2 mRNA and protein levels and β-catenin-Tcf/Lef and COX-2 gene reporter activity, as well as the production of PGE2and cell proliferation. Moreover, COX-2 overexpression or PGE2supplementation increased levels of the inhibitor of apoptosis protein survivin by a transcriptional mechanism, as determined by PCR analysis, survivin gene reporter assays and Western blotting. Furthermore, addition of PGE2to the medium prevented effects attributed to caveolin-1–mediated inhibition of β-catenin-Tcf/Lef–dependent transcription. Finally, PGE2reduced the coimmunoprecipitation of caveolin-1 with β-catenin and their colocalization at the plasma membrane. Thus, by reducing COX-2 expression, caveolin-1 interrupts a feedback amplification loop involving PGE2-induced signaling events linked to β-catenin/Tcf/Lef–dependent transcription of tumor survival genes including cox-2itself and survivin.

Augmented expression of cyclooxygenase-2 (COX-2) and enhanced production of prostaglandin E2(PGE2) are associated with increased tumor cell survival and malignancy. Caveolin-1 is a scaffold protein that has been proposed to function as a tumor suppressor in human cancer cells, although mechanisms underlying this ability remain controversial. Intriguingly, the possibility that caveolin-1 regulates the expression of COX-2 has not been explored. Here we show that augmented caveolin-1 expression in cells with low basal levels of this protein, such as human colon cancer (HT29, DLD-1), breast cancer (ZR75), and embryonic kidney (HEK293T) cells reduced COX-2 mRNA and protein levels and β-catenin-Tcf/Lef and COX-2 gene reporter activity, as well as the production of PGE2and cell proliferation. Moreover, COX-2 overexpression or PGE2supplementation increased levels of the inhibitor of apoptosis protein survivin by a transcriptional mechanism, as determined by PCR analysis, survivin gene reporter assays and Western blotting. Furthermore, addition of PGE2to the medium prevented effects attributed to caveolin-1–mediated inhibition of β-catenin-Tcf/Lef–dependent transcription. Finally, PGE2reduced the coimmunoprecipitation of caveolin-1 with β-catenin and their colocalization at the plasma membrane. Thus, by reducing COX-2 expression, caveolin-1 interrupts a feedback amplification loop involving PGE2-induced signaling events linked to β-catenin/Tcf/Lef–dependent transcription of tumor survival genes including cox-2 itself and survivin.

Apoptosis is a programmed form of cell death with well-defined morphological traits that are often associated with activation of caspases. More recently evidence has become available demonstrating that upon caspase inhibition alternative programs of cell death are executed, including ones with features characteristic of necrosis. These findings have changed our view of necrosis as a passive and essentially accidental form of cell death to that of an active, regulated and controllable process. Also necrosis has now been observed in parallel with, rather than as an alternative pathway to, apoptosis. Thus, cell death responses are extremely flexible despite being programmed. In this review, some of the hallmarks of different programmed cell death modes have been highlighted before focusing the discussion on necrosis. Obligatory events associated with this form of cell death include uncompensated cell swelling and related changes at the plasma membrane. In this context, representatives of the transient receptor channel family and their regulation are discussed. Also mechanisms that lead to execution of the necrotic cell death program are highlighted. Emphasis is laid on summarizing our understanding of events that permit switching between cell death modes and how they connect to necrosis. Finally, potential implications for the treatment of some disease states are mentioned.

Engagement of the Fas receptor promotes apoptosis by activation of caspases. In addition, alterations in plasma membrane lipid orientation and intracellular ceramide levels are often observed. In A20 B-lymphoma cells, FasL-induced cell death and phosphatidylserine (PS) externalization were completely prevented by the generic caspase inhibitor z-VAD-fmk. By contrast, the caspase-3 inhibitor Ac-DEVD-cho only partially restored cell viability and had no effect on surface exposure of PS. Flow cytometric analysis after FasL treatment identified two populations of dead cells. In one, death was dependent on caspase-3 and paralleled by DNA fragmentation and cell shrinkage. In the second, death occurred in the absence of caspase-3 activity and apoptotic features but was also blocked by zVAD-fmk. By morphological criteria these were identified as apoptotic and necrotic cells, respectively. Using fluorescent substrates, caspase-3 activity was detected only in the apoptotic cell population, whereas caspase-8 activity was detected in both. Both forms of caspase-8-dependent cell death were also detected downstream of Fas in Jurkat T-cells, where Fas-dependent PS externalization and delayed ceramide production, which is similar to results shown here in A20 cells, have been reported. However, for Raji B-cells, lacking lipid scrambling and ceramide production in response to Fas activation, only apoptosis was detected. Short-chain C2- or C6-ceramides, but not the respective inactive dihydro compounds or treatment with bacterial sphingomyelinase, induced predominantly necrotic rather than apoptotic cell death in A20 B-, Raji B- and Jurkat T-cells. Thus, delayed elevation of ceramide is proposed to promote necrosis in those Fas-stimulated cells where caspase-8 activation was insufficient to trigger caspase-3-dependent apoptosis.

Engagement of the Fas receptor promotes apoptosis by activation of caspases. In addition, alterations in plasma membrane lipid orientation and intracellular ceramide levels are often observed. In A20 B-lymphoma cells,FasL-induced cell death and phosphatidylserine (PS) externalization were completely prevented by the generic caspase inhibitor z-VAD-fmk. By contrast,the caspase-3 inhibitor Ac-DEVD-cho only partially restored cell viability and had no effect on surface exposure of PS. Flow cytometric analysis after FasL treatment identified two populations of dead cells. In one, death was dependent on caspase-3 and paralleled by DNA fragmentation and cell shrinkage. In the second, death occurred in the absence of caspase-3 activity and apoptotic features but was also blocked by zVAD-fmk. By morphological criteria these were identified as apoptotic and necrotic cells, respectively. Using fluorescent substrates, caspase-3 activity was detected only in the apoptotic cell population, whereas caspase-8 activity was detected in both. Both forms of caspase-8-dependent cell death were also detected downstream of Fas in Jurkat T-cells, where Fas-dependent PS externalization and delayed ceramide production, which is similar to results shown here in A20 cells, have been reported. However, for Raji B-cells, lacking lipid scrambling and ceramide production in response to Fas activation, only apoptosis was detected. Short-chain C2- or C6-ceramides, but not the respective inactive dihydro compounds or treatment with bacterial sphingomyelinase, induced predominantly necrotic rather than apoptotic cell death in A20 B-, Raji B- and Jurkat T-cells. Thus, delayed elevation of ceramide is proposed to promote necrosis in those Fas-stimulated cells where caspase-8 activation was insufficient to trigger caspase-3-dependent apoptosis.

In addition to the two monomer subunits of chicken brain-type creatine kinase (B-CK, EC, 2.7.3.2), termed Bb (basic) and Ba (acidic), another subspecies called Bb ∗was identified by chromatofocussing in the presence of 8 M urea (Quest et al., [20]). The latter low abundance protein species, isolated from tissue extracts, comigrated on 2D-gels with three minor species (Bbl-3), initially identified in immunoprecipitated, [ 35S]methionine labeled in vitro translation products of cDNA coding for the basic monomer Bb. During in vitro translation experiments in the presence of [ 32P]-γ -ATP, Bbl-3 were labeled while phosphatase treatment eliminated these minor species. It is concluded that Bb *is identical to Bbl-3 and represents phosphorylated derivatives of Bb. B-CK dimer populations from different tissues were separated by ion-exchange chromatography and the K mvalues of the resulting fractions were determined under phospho-creatine (CP)-limiting conditions. In fractions containing only Bb and Bb ∗two kinetically different enzyme species were detected ( K mvalues for CP = 1.6 mM and 0.8 mM), while fractions containing B-CK dimers composed of the major Ba and Bb monomers, but no Bb ∗, were homogeneous in this respect ( K mfor CP = 1.6 mM). Phosphorylation of Bb to yield Bb ∗is concluded to reduce the K mof B-CK dimers for CP by about 50%. This K mshift is within the range of CP concentrations found in tissues expressing the B-CK isoform and may therefore be of physiological relevance.

Regulatory domain elements of the non‐calcium‐dependent protein kinase Cδ (nPKCδ), including either or both of the cysteine‐rich regions Cys1(δ) and Cys2(δ), were expressed as fusion proteins with glutathione‐S‐transferase and characterized using liposomal or mixed micellar phorbol ester binding assays. Fusion proteins containing Cys2(δ) bound phorbol‐12,13‐dibutyrate (PDBu) efficiently in the assay employing phosphatidylserine (PS) vesicles, while no significant binding was seen for proteins containing only Cys1(δ). Likewise, in mixed micellar assays, fusion proteins with Cys2(δ) bound PDBu with high affinity (Kd: 14–37 nM) and to significant stoichiometric levels (0.23–0.66 mol/mol), but no binding could be detected for proteins with Cys1(δ) only. The PS dependence of PDBu binding to Cys2(δ) was highly cooperative with Hill numbers lying in the range of 2.5–5.2. These results demonstrate the presence of striking functional differences between the cysteine‐rich regions of nPKCδ and the calcium‐dependent isoform, cPKCγ, where both cysteine‐rich regions represent functional PDBu binding elements.

An inhibitory effect of several zinc chelators on endosome fusion reconstituted in anin vitrosystem has been recently reported (A. Aballayet al.,1995,Biochem. J.312, 919–923). The factor that requires zinc for its activity is still unknown. Since the regulatory domain of protein kinase C (PKC) contains cysteine-rich motifs which coordinate zinc, we suspected that PKC or a PKC-like protein might be that factor. To test this hypothesis, we studied the effect of calphostin C, a specific inhibitor of PKC that interacts with the cysteine-rich motif, and PMA (phorbol 12-myristate 13-acetate), an activator of several PKC isoforms that bind to the same region, on endosome fusion. Calphostin C inhibited endosome fusion in a zinc-regulated manner, whereas PMA enhanced endosome fusion. Moreover, fusion was strongly stimulated when both PMA and zinc were added together to zinc-depleted fusion reactions. Inhibitors of the catalytic domain of PKC had no effect on the assay suggesting that the kinase activity is not required. In contrast, a glutathioneS-transferase fusion protein containing a cysteine-rich region of the regulatory domain of PKCγ inhibited endosome fusion in a PMA-dependent manner. Western blot analysis demonstrated the presence of proteins containing PKC-like cysteine-rich regions that are released from endosomal fractions by zinc chelators. These results indicate that the previously proposed zinc-dependent factor required for endosome fusion could be either a PKC isoform or a protein containing the phorbol ester-binding domain of PKC.

Brain‐type creatine kinase B‐CK (EC 2.7.3.2) was purified from several chicken tissues, e.g. cardiac muscle, brain, gizzard and retina. Two major monomeric chicken B‐CK subunits, designated Bb (basic) and Ba (acidic), which differ in isoelectric point, were separated by chromatofocussing in the presence of 8 M urea on a MonoP column. The two subunits were shown by peptide mapping, amino acid analysis and partial sequencing, as well as by immunological criteria, to be distinct B‐CK polypeptides. The N‐tenninal sequence of 30 amino acid residues of Bb correspond entirely to data derived from a B‐CK c‐DNA clone termed H4 [(1986) Nucleic Acids Res. 14, 1449‐1463], whereas the N‐terminus of the acidic Ba species was blocked. Native dimeric B‐CK isoenzymes obtained from these tissues were separated by ion exchange chromatography on a MonoQ column yielding two B‐CK dimer populations, type‐I and type‐II B‐CK, varying in relative proportions. Quantitation of the CK activity peak ratios of these two populations revealed the existence of a tissue‐specific, post‐translational mechanism regulating B‐CK dimerization in neural tissues. Tissue‐specific dimerization of the two distinct B‐CK monomer species may represent a means of specifying the intracellular distribution of the dimeric B‐CK subspecies.

In addition to the two monomer subunits of chicken brain‐type creatine kinase (B‐CK, EC, 2.7.3.2), termed Bb (basic) and Ba (acidic), another subspecies called Bb∗was identified by chromatofocussing in the presence of 8 M urea (Quest et al., [20]). The latter low abundance protein species, isolated from tissue extracts, comigrated on 2D‐gels with three minor species (Bbl‐3), initially identified in immunoprecipitated, [35S]methionine labeled in vitro translation products of cDNA coding for the basic monomer Bb. During in vitro translation experiments in the presence of [32P]‐γ ‐ATP, Bbl‐3 were labeled while phosphatase treatment eliminated these minor species. It is concluded that Bb*is identical to Bbl‐3 and represents phosphorylated derivatives of Bb. B‐CK dimer populations from different tissues were separated by ion‐exchange chromatography and the Kmvalues of the resulting fractions were determined under phospho‐creatine (CP)‐limiting conditions. In fractions containing only Bb and Bb∗two kinetically different enzyme species were detected (Kmvalues for CP = 1.6 mM and 0.8 mM), while fractions containing B‐CK dimers composed of the major Ba and Bb monomers, but no Bb∗, were homogeneous in this respect (Kmfor CP = 1.6 mM). Phosphorylation of Bb to yield Bb∗is concluded to reduce the Kmof B‐CK dimers for CP by about 50%. This Kmshift is within the range of CP concentrations found in tissues expressing the B‐CK isoform and may therefore be of physiological relevance.

Brain-type creatine kinase B-CK (EC 2.7.3.2) was purified from several chicken tissues, e.g. cardiac muscle, brain, gizzard and retina. Two major monomeric chicken B-CK subunits, designated Bb (basic) and Ba (acidic), which differ in isoelectric point, were separated by chromatofocussing in the presence of 8 M urea on a MonoP column. The two subunits were shown by peptide mapping, amino acid analysis and partial sequencing, as well as by immunological criteria, to be distinct B-CK polypeptides. The N-tenninal sequence of 30 amino acid residues of Bb correspond entirely to data derived from a B-CK c-DNA clone termed H4 [(1986) Nucleic Acids Res. 14, 1449-1463], whereas the N-terminus of the acidic Ba species was blocked. Native dimeric B-CK isoenzymes obtained from these tissues were separated by ion exchange chromatography on a MonoQ column yielding two B-CK dimer populations, type-I and type-II B-CK, varying in relative proportions. Quantitation of the CK activity peak ratios of these two populations revealed the existence of a tissue-specific, post-translational mechanism regulating B-CK dimerization in neural tissues. Tissue-specific dimerization of the two distinct B-CK monomer species may represent a means of specifying the intracellular distribution of the dimeric B-CK subspecies.

Regulatory domain elements of the non-calcium-dependent protein kinase Cδ (nPKCδ), including either or both of the cysteine-rich regions Cys1(δ) and Cys2(δ), were expressed as fusion proteins with glutathione-S-transferase and characterized using liposomal or mixed micellar phorbol ester binding assays. Fusion proteins containing Cys2(δ) bound phorbol-12,13-dibutyrate (PDBu) efficiently in the assay employing phosphatidylserine (PS) vesicles, while no significant binding was seen for proteins containing only Cys1(δ). Likewise, in mixed micellar assays, fusion proteins with Cys2(δ) bound PDBu with high affinity ( Kd: 14–37 nM) and to significant stoichiometric levels (0.23–0.66 mol/mol), but no binding could be detected for proteins with Cys1(δ) only. The PS dependence of PDBu binding to Cys2(δ) was highly cooperative with Hill numbers lying in the range of 2.5–5.2. These results demonstrate the presence of striking functional differences between the cysteine-rich regions of nPKCδ and the calcium-dependent isoform, cPKCγ, where both cysteine-rich regions represent functional PDBu binding elements.

Following publication of the article, the authors wrote to point out the following mistake: