Experimental evolution of TetX2: Correlating changes in fitness to their structural and functional origins

The study of protein evolution and adaptation resides at the junction between the disciplines of biological chemistry and evolutionary biology. We chose the B. thetaiotaomicron tetracycline resistant enzyme TetX2, as our model system to study the biophysical basis for adaptation to antibiotics; a phenomenon that continuously poses global health challenges. In the work presented here, experimental evolution and biophysical characterization were used to identify and link the physicochemical properties of TetX2 and its adaptive mutants to increased resistance to minocycline. Bacteroides thetaiotaomicron TetX2 was previously identified as a novel oxidoreductase with broad activity against tetracyclines. Experimental evolution of E. coli expressing a chromosomal copy of tet(X2) was used to identify an adaptive mutation (TetX2 T280A ) that confers higher resistance to minocycline and tigecycline. In addition to TetX2 T280A , a family of variants of TetX2 with single amino acid changes in TetX2 sequence that conferred equal or higher resistance towards MCN was identified by error-prone mutagenesis. Changes in fitness of E. coli carrying a single chromosomal copy of either wild-type or one of the mutant alleles were assessed by growth rate assays over a range of minocycline concentrations. Despite similar in vivo performances of TetX2 T280A and two other variants (TetX2 N371I and TetX2 N371T ), TetX2 T280A was the only successful mutant in the adaption experiment suggesting that mutational supply may play an important role in evolutionary dynamics of populations undergoing adaptation. The most surprising result is that the differences in growth rates among TetX2 variants arise from small changes in in vitro catalytic activity and in vivo protein expression. The steady-state kinetic studies with minocycline and NADPH suggest a binary mechanism for antibiotic inactivation by TetX2 which is supported by the structural characteristics of the enzyme. The atomic structures of the best adaptive mutant TetX2 T280A in complex with minocycline and tigecycline reveal the details of substrate recognition and show that the site of the mutation is ∼18 Å away from the active site suggesting an indirect mechanism for improved catalysis. Taken together, our data show that very small changes in the in vitro biochemical properties and expression levels can have surprisingly large fitness effects and are important during adaption. In addition, a promising preliminary mathematical model suggests that based on kinetic activity and in vivo expression levels the success of bacteria undergoing adaptation to antibiotics can be predicted

Small changes in enzyme function can lead to surprisingly large fitness effects during adaptive evolution of antibiotic resistance

In principle, evolutionary outcomes could be largely predicted if all of the relevant physicochemical variants of a particular protein function under selection were known and integrated into an appropriate physiological model. We have tested this principle by generating a family of variants of the tetracycline resistance protein TetX2 and identified the physicochemical properties most correlated with organismal fitness. Surprisingly, small changes in the Km(MCN), less than twofold, were sufficient to produce highly successful adaptive mutants over clinically relevant drug concentrations. We then built a quantitative model directly relating the in vitro physicochemical properties of the mutant enzymes to the growth rates of bacteria carrying a single chromosomal copy of the tet(X2) variants over a wide range of minocycline (MCN) concentrations. Importantly, this model allows the prediction of enzymatic properties directly from cellular growth rates as well as the physicochemical-fitness landscape of TetX2. Using experimental evolution and deep sequencing to monitor the allelic frequencies of the seven most biochemically efficient TetX2 mutants in 10 independently evolving populations, we showed that the model correctly predicted the success of the two most beneficial variants tet(X2)T280A and tet(X2)N371I. The structure of the most efficient variant, TetX2T280A, in complex with MCN at 2.7 Å resolution suggests an indirect effect on enzyme kinetics. Taken together, these findings support an important role for readily accessible small steps in protein evolution that can, in turn, greatly increase the fitness of an organism during natural selection

Statins and colorectal cancer

Statins are naturally occurring compounds that inhibit the enzyme 3-hydroxy-3-methyl-glutaryl-CoA reductase. Through their beneficial management of the body’s lipid metabolism, they are widely used medicinal drugs employed extensively in the primary and secondary prevention of cardiovascular disease. In addition, many studies to date have shown the therapeutic advantages derived from using statins in conditions such as endometriosis, osteoporosis, polycystic ovary syndrome and rheumatic disease. Due to the steady increase of cancer morbidity rates, as demonstrated by epidemiological data, the putative role of statins in treating or preventing cancer has been ever more frequently investigated; including for colorectal cancer. This paper attempts to bring together current knowledge/ evidence on statin therapy targeted at the development, disease course and treatment of colorectal cancer, both in terms of epidemiological findings and clinical observations. Because of the reported link between metabolic disorders and the development of colorectal cancer, particular focus is given to the effects of statins on signalling pathways involving insulin-like growth factors (IGFs)

Statyny w raku jelita grubego

Statyny należą do związkow chemicznych pochodzenia naturalnego, wykazujących aktywność inhibicyjną wobec enzymu redukatazy 3-hydroksy-3-metylo-glutarylo-CoA. Tym samym, poprzez wywieranie korzystnego efektu na gospodarkę lipidową organizmu, należą do powszechnie stosowanych lekow w prewencji pierwotnej i wtornej chorob układu sercowo-naczyniowego. Ponadto w wielu dotychczasowych badaniach podejmowano temat korzyści terapii statynami w takich schorzeniach jak: endometrioza, osteoporoza, zespoł policystycznych jajnikow i choroby reumatyczne. Z uwagi na niekorzystne dane epidemiologiczne, wskazujące na stały wzrost zachorowalności na nowotwory złośliwe, coraz częściej podejmowane są badania na temat ewentualnej pozytywnej roli statyn w profilaktyce i leczeniu chorob onkologicznych, w tym raka jelita grubego. W prezentowanej pracy autorzy podjęli probę syntezy dotychczasowej wiedzy na temat znaczenia terapii statynami w rozwoju, przebiegu i leczeniu raka jelita grubego, z uwzględnieniem danych epidemiologicznych oraz obserwacji klinicznych. Z uwagi na udokumentowany związek występowania zaburzeń metabolicznych z rozwojem raka jelita grubego szczegolny nacisk położono na analizę udziału statyn w szlakach sygnałowych związanych z insulinopodobnymi czynnikami wzrostu

Aktywność rybocyklibu i hormonoterapii u chorych na zaawansowanego, luminalnego HER2-ujemnego raka piersi w praktyce klinicznej — opisy przypadków

Selektywne inhibitory kinaz zależnych od cyklin CDK4 i CDK 6 (iCDK4/6, cyclin-dependent kinase inhibitors) — palbocyklib, rybocyklib i abemacyklib — stanowią nową grupę leków zmniejszających proliferację przez zahamowanie przejścia komórek w cyklu komórkowym z fazy G1 do S. Leki te są obecnie standardem postępowania u chorych na miejscowo zaawansowanego lub uogólnionego raka piersi z ekspresją receptorów estrogenowych (ER, estrogen receptor) i progesteronowych (PR, progesterone receptor) bez nadekspresji receptora naskórkowego czynnika wzrostu typu 2 (HER2, human epidermal growth factor receptor type 2) w skojarzeniu z jednym z inhibitorów aromatazy lub fulwestrantem w pierwszej linii leczenia lub po niepowodzeniu wcześniejszej hormonoterapii. W niniejszym opracowaniu przedstawiono opisy przypadków chorych na raka piersi leczonych rybocyklibem w skojarzeniu z hormonoterapią w ramach programu rozszerzonego dostępu (MAP, managed access program)

Regulation of the PI3K pathway through a p85α monomer–homodimer equilibrium

© Cheung et al. The canonical action of the p85α regulatory subunit of phosphatidylinositol 3-kinase (PI3K) is to associate with the p110α catalytic subunit to allow stimuli-dependent activation of the PI3K pathway. We elucidate a p110α-independent role of homodimerized p85α in the positive regulation of PTEN stability and activity. p110α-free p85α homodimerizes via two intermolecular interactions (SH3:proline-rich region and BH:BH) to selectively bind unphosphorylated activated PTEN. As a consequence, homodimeric but not monomeric p85α suppresses the PI3K pathway by protecting PTEN from E3 ligase WWP2-mediated proteasomal degradation. Further, the p85α homodimer enhances the lipid phosphatase activity and membrane association of PTEN. Strikingly, we identified cancer patient-derived oncogenic p85α mutations that target the homodimerization or PTEN interaction surface. Collectively, our data suggest the equilibrium of p85α monomerdimers regulates the PI3K pathway and disrupting this equilibrium could lead to disease development.Link_to_subscribed_fulltex

Clinical Characteristics of Colorectal Cancer Patients in terms of Selected Platelet Indices

Mounting evidence suggests that inflammation, immune response, and coagulation status determine many processes during the carcinogenesis pathway in colorectal cancer (CRC). Inflammation strongly promotes tumor formation, progression, and metastasis. The systemic inflammatory response (SIR) may be reflected by simple indicators evaluated on the basis of peripheral blood morphology parameters. The indices are easily obtained by the peripheral blood test and could be promising biomarkers for CRC. We present the results of the retrospective study evaluating the potential relation between the platelet indices (platelet count (PC), platelet-to-lymphocyte ratio (PLR), neutrophil platelet score (NPS), mean platelet volume (MPV), and MPV/PC ratio) and the clinicopathological features of CRC patients. The study included 247 patients (104 males and 143 females) aged 39-87 years with CRC stages II-IV. The complete blood counts with the automated differential counts were performed prior to the qualification to systemic treatment. High PC, high PLR, and NPS 0 were associated with older age and higher BMI of the patients. No link between the analyzed platelet indices and histological grade of the tumor, primary tumor location, and gender was noted. The patients aged ≥65 years were characterized by the higher MPV/PC ratio than the younger population. We observed a trend to the higher MPV/PC ratio among the patients with excessive body weight defined by BMI compared to BMI within normal limits. A higher frequency of PC>400, NPS 1 and 2, and a trend to more frequent PLR≥150 were observed in the subgroup with metastatic disease compared to individuals with CRC stages II and III. The presented results expand the knowledge on potential association between SIR parameters and other clinicopathological factors that should be considered during interpreting the prognostic and predictive value of the inflammation parameters