Mariana Martín

Structural plasticity and catalysis regulation of a thermosensor histidine kinase

Temperature sensing is essential for the survival of living cells. A major challenge is to understand how a biological thermometer processes thermal information to optimize cellular functions. Using structural and biochemical approaches, we show that the thermosensitive histidine kinase, DesK, from Bacillus subtilis is cold-activated through specific interhelical rearrangements in its central four-helix bundle domain. As revealed by the crystal structures of DesK in different functional states, the plasticity of this helical domain influences the catalytic activities of the protein, either by modifying the mobility of the ATP-binding domains for autokinase activity or by modulating binding of the cognate response regulator to sustain the phosphotransferase and phosphatase activities. The structural and biochemical data suggest a model in which the transmembrane sensor domain of DesK promotes these structural changes through conformational signals transmitted by the membrane-connecting two-helical coiled-coil, ultimately controlling the alternation between output autokinase and phosphatase activities. The structural comparison of the different DesK variants indicates that incoming signals can take the form of helix rotations and asymmetric helical bends similar to those reported for other sensing systems, suggesting that a similar switching mechanism could be operational in a wide range of sensor histidine kinases.

Membrane Thickness Cue for Cold Sensing in a Bacterium

Thermosensors are ubiquitous integral membrane proteins found in all kinds of life. They are involved in many physiological roles, including membrane remodeling, chemotaxis, touch, and pain [1-3], but, the mechanism by which their transmembrane (TM) domains transmit temperature signals is largely unknown. The histidine kinase DesK from Bacillus subtilis is the paradigmatic example of a membrane-bound thermosensor suited to remodel membrane fluidity when the temperature drops below approximately 30°C [1, 4] providing, thus, a tractable system for investigating the mechanism of TM-mediated input-output control of thermal adaptation. Here we show that the multimembrane-spanning domain from DesK can be simplified into a chimerical single-membrane-spanning minimal sensor (MS) that fully retains, in vivo and in vitro, the sensing properties of the parental system. The MS N terminus contains three hydrophilic amino acids near the lipid-water interface creating an instability hot spot. Mutational analysis of this boundary-sensitive beacon revealed that membrane thickness controls the signaling state of the sensor by dictating the hydration level of the metastable hydrophilic spot. Guided by these results we biochemically demonstrated that the MS signal transmission activity is sensitive to bilayer thickness. Membrane thickness could be a general cue for sensing temperature in many organisms.

Functional in vitro assembly of the integral membrane bacterial thermosensor DesK

The Bacillus subtilis DesK histidine kinase (HK) is an integral membrane thermosensor that forms part of a regulatory circuit which controls the physical state of membrane lipids. In the pursuit of biochemical and structural approaches to study lipid fluidity-dependent DesK thermosensing, we found that standard expression methods failed to produce enough amounts of a fully functional protein. Here, we describe a high-yield purification method based in an Escherichia coliin vitro transcription-translation system. The enzymatic activities of the full-length protein, either solubilized with detergents or co-translationally inserted into liposomes, have been characterized and compared with those measured for the constitutively active cytoplasmic domain of DesK, lacking the transmembrane sensor domain. As expected, the autokinase activity of liposome-inserted DesK was greatly increased when the incubation temperature was decreased from 37 to 25 degrees C. This is the first report of the spontaneous in vitro membrane insertion of a fully functional bacterial HK thermosensor. Moreover, this single step procedure should greatly aid the isolation of a wide range of membrane-associated HKs for biochemical and biophysical studies.

Ectopic expression of mitochondrial gamma carbonic anhydrase 2 causes male sterility by anther indehiscence

Plant mitochondria include gamma-type carbonic anhydrases (γCAs) of unknown function. In Arabidopsis, the γCAs form a gene family of five members which all are attached to the NADH dehydrogenase complex (complex I) of the respiratory chain. Here we report a functional analysis of gamma carbonic anhydrase 2 (CA2). The gene encoding CA2 is constitutively expressed in all plant organs investigated but it is ten fold induced in flowers, particularly in tapetal tissue. Ectopic expression of CA2 in Arabidopsis causes male sterility in transgenic plants. In normal anther development, secondary thickenings of the endothecial cell wall cause anthers to open upon dehydration. Histological analyses revealed that abnormal secondary thickening prevents anther opening in 35S::CA2 transgenic plants. CA2 abundance in transgenic plants is increased 2–3 fold compared to wild-type plants as revealed by Western blotting analyses. Moreover, abundance of other members of the CA family, termed CA3 and CAL2, is increased in transgenic plants. Oxygen uptake measurements revealed that respiration in transgenic plants is mainly based on NADH reduction by the alternative NADH dehydrogenases present in plant mitochondria. Furthermore, the formation of reactive oxygen species (ROS) is very low in transgenic plants. We propose that reduction in ROS inhibits H2O2 dependent lignin polymerization in CA2 over-expressing plants, thereby causing male sterility.

A mitochondrial alkaline/neutral invertase isoform (A/N-InvC) functions in developmental energy-demanding processes in Arabidopsis

Recent findings demonstrate that alkaline/neutral invertases (A/N-Invs), enzymes that catalyze the breakdown of sucrose into glucose and fructose, are essential proteins in plant life. The fact that different isoforms are present in multiple locations makes them candidates for the coordination of metabolic processes. In the present study, we functionally characterized the encoding gene of a novel A/N-Inv (named A/N-InvC) from Arabidopsis, which localizes in mitochondria. A/N-InvC is expressed in roots, in aerial parts (shoots and leaves) and flowers. A detailed phenotypic analysis of knockout mutant plants (invc) reveals an impaired growth phenotype. Shoot growth was severely reduced, but root development was not affected as reported for A/N-InvA mutant (inva) plants. Remarkably, germination and flowering, two energy demanding processes, were the most affected stages. The effect of exogenous growth regulators led us to suggest that A/N-InvC may be modulating hormone balance in relation to the radicle emergence. We also show that oxygen consumption is reduced in inva and invc in comparison with wild-type plants, indicating that both organelle isoenzymes may play a fundamental role in mitochondrion functionality. Taken together, our results emphasize the involvement of mitochondrial A/N-Invs in developmental processes and uncover the possibility of playing different roles for the two isoforms located in the organelle.

Regulation of Bacillus subtilis DesK thermosensor by lipids

Temperature sensing is essential for the survival of living cells. The membrane-bound thermosensor DesK from Bacillus subtilis is a key representative of histidine kinases receptors able to remodel membrane lipid composition when the temperature drops below ~30°C. Although the receptor is well studied, a central issue remains: how does the compositional and functional diversity of the surrounding membrane modulate receptor function? Reconstituting full-length DesK into proteoliposomes of well-defined and controlled lipid composition represents a minimal synthetic approach to systematically address this question. Thus DesK has been reconstituted in a variety of phospholipid bilayers and its temperature-regulated autokinase activity determined as function of fatty acyl chain length, lipid head-group structure and phase preference. We show that the head group structure of lipids (both in vitro and in vivo) has little effect on DesK thermosensing, whereas properties determined by the acyl chain of lipids, such as membrane thickness and phase separation into coexisting lipid domains, exert a profound regulatory effect on kinase domain activation at low temperatures. These experiments suggest that the non-polar domain of glycerolipids is essential to regulate the allosteric structural transitions of DesK, by activating the autophosphorylation of the intracellular kinase domain in response to a decrease in temperature.

Polysaccharide-synthesizing glycosyltransferases and carbohydrate binding modules: the case of starch synthase III.

Glycosyltransferases (GTs) are a ubiquitous group of enzymes that catalyze the transfer of sugar moieties from activated donor molecules to specific acceptor molecules, forming glycosidic bonds. Nucleotide-sugars, lipid phosphate sugars and phosphate sugars can act as activated donor molecules while acceptor substrates involve carbohydrates, proteins, lipids, DNA and also, numerous small molecules (i. e. antibiotics, flavonols, steroids). GTs enzyme families are very ancient. They are founded in all the three domains of life and display three different folds (named GT-A, GTB and GT-C) which are a variant of a common α/β scaffold. In addition, several GTs contain an associated non-catalytic carbohydrate binding module (CBM) that could be critical for enzyme activity. This work reviews the current knowledge on the GTs structures and functions and highlights those enzymes that contain CBMs, particularly starch binding domains (SBDs). In addition, we also focus on A. thaliana starch synthase III enzyme, from the GT-5 family. This protein has a GT-B fold, and contains in its N-terminal region three in tandem SBDs, which are essential for the regulation of enzyme activity.

Improving the glycosyltransferase activity of Agrobacterium tumefaciens glycogen synthase by fusion of N-terminal starch binding domains (SBDs)

Glycogen and starch, the major storage carbohydrate in most living organisms, result mainly from the action of starch or glycogen synthases (SS or GS, respectively, EC 2.4.1.21). SSIII from Arabidopsis thaliana is an SS isoform with a particular modular organization: the C-terminal highly conserved glycosyltransferase domain is preceded by a unique specific region (SSIII-SD) which contains three in tandem starch binding domains (SBDs, named D1, D2 and D3) characteristic of polysaccharide degrading enzymes. N-terminal SBDs have a probed regulatory role in SSIII activity, showing starch binding ability and modulating the catalytic properties of the enzyme. On the other hand, GS from Agrobacterium tumefaciens has a simple primary structure organization, characterized only by the highly conserved glycosyltransferase domain and lacking SBDs. To further investigate the functional role of A. thaliana SSIII-SD, three chimeric proteins were constructed combining the SBDs from A. thaliana with the GS from A. tumefaciens. Recombinant proteins were expressed in and purified to homogeneity from Escherichia coli cells in order to be kinetically characterized. Furthermore, we tested the ability to restore in vivo glycogen biosynthesis in transformed E. coli glgA(-) cells, deficient in GS. Results show that the D3-GS chimeric enzyme showed increased capacity of glycogen synthesis in vivo with minor changes in its kinetics parameters compared to GS.

Abstract OT2-01-06: Phase III study of palbociclib (PD-0332991) in combination with endocrine therapy (exemestane or fulvestrant) versus chemotherapy (capecitabine) in hormonal receptor (HR) positive/HER2 negative metastatic breast cancer (MBC) patients with resistance to aromatase inhibitors. “The PEARL study” (GEICAM/2013-02)

Background: Endocrine therapy (ET) is the cornerstone treatment for HR–positive, HER2-negative breast cancer patients. However, endocrine resistance is a major clinical challenge.Treatment options at recurrence/progression to AIs include sequential endocrine-based therapiesin monotherapy or in combination with a targeted agent or chemotherapy. Preclinical data suggest that ER+/HER2- BC is dependent on cyclin-dependent kinases 4/6 (CDK4/6) function; the inhibition of this target may be effective in overcome endocrine resistance. Palbociclib (P) is an oral novel CDK4/6 inhibitor that is synergistic with ET in preclinical and clinical studies. Initially this study was designed to demonstrate the clinical benefit of P plus exemestane (E) vs capecitabine (C) (cohort1). Recent studies revealed that the acquisition of ESR1 mutations is a major mechanism of resistance to the treatment of AIs in the metastatic setting. Retrospective analyses show that patients with ESR1 mutation derived no benefit from sequential AI monotherapy. In contrast, preclinical and clinical data indicated that SERD, fulvestrant (F), is active in ESR1 mutant tumors. Hence the choice of endocrine partner with P is particularly important to create optimal synergy in endocrine resistance setting. The current design added cohort 2 of P plus F vs C. The overall study goal is to prospectively answer the question of optimal endocrine partner with P and its efficacy/safety over chemotherapy in endocrine resistant setting. Trial Design:This is an international (5 countries) randomized phase III study with 2 cohorts, patients will be randomized 1:1 to ET (cohort 1: E 25 mg daily, cohort 2: F 500mg days 1 and 15 cycle 1 and then day 1 every 4 weeks) plus P (125 mg daily x3 weeks every 4 weeks) vs. C (1,250 mg/m2 twice daily x2 weeks every 3 weeks). Postmenopausal patients with HR+/HER2- MBC are eligible if resistant to previous AI (letrozole or anastrozole in cohort 1 or any AI in cohort 2) defined as:recurrence while on or within 12 months after the end of adjuvant treatment orprogression while on or within 1 month after the end of treatment for MBC. Previous chemotherapy is permitted either in the (neo)adjuvant setting and/or as first line for MBC. Patients must have measurable disease according to RECIST 1.1 or bone lesions, lytic or mixed, in the absence of measurable disease. The primary objectives are to demonstrate that P plus F is superior to C and that P plus ET is superior to C inwomen whose tumor had estrogen receptor ESR1 wild type in terms of Progression-Free Survival (PFS); secondary objectives are PFS regardless the ESR1 mutational status, overall survival, response rate, clinical benefit rate, response duration, safety, quality of life and biomarkers. The study will recruit approximately 302 patients in cohort 1 and 300 patients in cohort 2. The study started recruitment in March 2014; 290 patients have been included so far (289 cohort1 + 1 cohort 2). Analysis of primary endpoint is planned in Sep2019 (ClinTrials.gov reference NCT02028507. Citation Format: Martin M, Kahan Z, Carrasco E, Bartlett CH, Casas M, Gil-Gil M, Munoz M, Ciruelos EM, Ruiz-Borrego M, Margeli M, Anton A, Bermejo B, Morales S, Gal-Yam E, Koehler M, Garcia-Saenz JA, de la Haba J, Chacon I, Zielinski C. Phase III study of palbociclib (PD-0332991) in combination with endocrine therapy (exemestane or fulvestrant) versus chemotherapy (capecitabine) in hormonal receptor (HR) positive/HER2 negative metastatic breast cancer (MBC) patients with resistance to aromatase inhibitors. “The PEARL study” (GEICAM/2013-02) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr OT2-01-06.

Development of fast and simple chromogenic methods for glucan phosphatases in-gel activity assays

Glucan phosphatases are essential for normal starch degradation in plants and glycogen metabolism in mammals. Here we develop two chromogenic methods for the detection of glucan phosphatase activity in situ after non denaturing poliacrylamide gel electrophoresis; one method uses pNPP and the second one applies BCIP/NBT. The assays are sensitive, fast, simple, reliable and cost-effective preventing the use of radioactive or fluorogenic compounds. Taking advantage of an efficient separation method combined with the reported assays it is possible to obtain information about oligomeric state of the active enzymes as well as to simultaneously detect glucan substrate binding and phosphatase activity.