Lifelong self-renewal of the adult intestinal epithelium requires the activity of stem cells located in mucosal crypts. Lgr5 and Bmi1 are two molecular markers of crypt-cell populations that replenish all lineages over time and hence function as stem cells. Intestinal stem cells require Wnt signaling, but the understanding of their cellular niche is incomplete. Lgr5-expressing crypt base columnar cells (CBCs) reside deep in the crypt, mingled among mature Paneth cells that are well positioned for short-range signaling. Partial lineage ablation previously had implied that Paneth cells are nonessential constituents of the stem-cell niche, but recently their absence was reported to interfere with Lgr5 + CBCs, resurrecting an appealing idea. However, previous mouse models failed to remove Paneth cells completely or permanently; defining the intestinal stem-cell niche requires clarity with respect to the Paneth cell role. We find that Lgr5 + cells with stem-cell activity cluster in future crypts early in life, before Paneth cells develop. We also crossed conditional Atoh1 −/− mice, which lack Paneth cells entirely, with Lgr5 GFP mice to visualize Lgr5 + CBCs and to track their stemcell function. In the sustained absence of Paneth cells, Lgr5 + CBCs occupied the full crypt base, proliferated briskly, and generated differentiated progeny over many months. Gene expression in fluorescence-sorted Lgr5 + CBCs reflected intact Wnt signaling despite the loss of Paneth cells. Thus, Paneth cells are dispensable for survival, proliferation, and stem-cell activity of CBCs, and direct contact with Lgr5-nonexpressing cells is not essential for CBC function. S tem cells in selected adult tissues, such as the bone marrow, skin, and digestive tract, play a vital role in replenishing multiple cell types throughout life, and their unique and potent capacity for self-renewal is replicated in cancer (1). These stem cells occupy specialized niches and respond to the local environment (2). The functions of such niches range from delivering trophic signals that control cell proliferation and prevent stemcell depletion to preventing unrestrained cell replication (3). Defining the cellular and molecular constituents of adult stem-cell niches therefore is an important challenge in biology and medicine.Intestinal stem cells reside in mucosal crypts and generate four distinct cell types. Enterocytes, goblet cells, and enteroendocrine cells line deep crypts in the colon and villi that project into the small bowel lumen; Paneth cells lie at the crypt base in the small intestine, increasing in number from duodenum to ileum, but are absent from the colon (4). Two small intestine crypt-cell populations are able to generate all four cell types over extended periods: Lgr5-expressing crypt base columnar cells (CBCs), which lie deep in the crypt, interspersed among Paneth cells (5), and Bmi1-expressing cells that occupy several crypt tiers, most notably the +4 position (6). Although recent evidence suggests that each of these cell populations can engende...

Activating mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) underlie the pathogenesis and chemoresistance of ∼30% of all human tumors, yet the development of high-affinity inhibitors that target the broad range of KRAS mutants remains a formidable challenge. Here, we report the development and validation of stabilized alpha helices of son of sevenless 1 (SAH-SOS1) as prototype therapeutics that directly inhibit wild-type and mutant forms of KRAS. SAH-SOS1 peptides bound in a sequence-specific manner to KRAS and its mutants, and dose-responsively blocked nucleotide association. Importantly, this functional binding activity correlated with SAH-SOS1 cytotoxicity in cancer cells expressing wild-type or mutant forms of KRAS. The mechanism of action of SAH-SOS1 peptides was demonstrated by sequencespecific down-regulation of the ERK-MAP kinase phosphosignaling cascade in KRAS-driven cancer cells and in a Drosophila melanogaster model of Ras85D V12 activation. These studies provide evidence for the potential utility of SAH-SOS1 peptides in neutralizing oncogenic KRAS in human cancer.R AS signaling is a critical control point for a host of cellular functions ranging from cellular survival and proliferation to cellular endocytosis and motility (1). The on or off state of RAS is dictated by nucleotide exchange. GTP-bound RAS is the activated form that engages its downstream effectors with high avidity. The endogenous GTPase activity of RAS hydrolyzes GTP to GDP and inactivates signaling. This biochemical process is further regulated by GTPase-activating proteins (GAPs) that impair RAS signaling through increasing endogenous GTPase activity and guanine-nucleotide exchange factors (GEFs) that enhance RAS signaling by facilitating GDP release and, thus, GTP association. Given the central roles of RAS in cellular growth and metabolism, it is not surprising that cancer cells usurp its prosurvival activities to achieve immortality.Activating mutations in KRAS represent the most frequent oncogenic driving force among the RAS homologs K-, N-, and H-RAS, and are associated with poor prognosis and chemoresistance (2). KRAS mutations are present in ∼30% of human tumors and at even higher frequencies in cancers of the pancreas, lung, thyroid gland, colon, and liver. For example, in pancreatic ductal adenocarcinomas (PDAC) that carry a 5-y survival rate of less than 5%, activating KRAS mutations are present in more than 90% of tumors (3). Thus, therapeutic inhibition of RAS is among the highest priority goals of the cancer field. Because oncogenic forms of KRAS typically harbor single-point mutants that stabilize its active GTP-bound form, a host of recent small molecule and peptide development efforts have been aimed at disarming this pathologic biochemical state. The extremely high affinity of KRAS for its GTP substrate has hampered the development of competitive GTP inhibitors. However, a GDP mimetic that covalently modifies the mutant cysteine of KRAS G12C represents a promising approach to plugging the nucleotid...

Understanding the molecular alterations that confer cancer cells with motile, metastatic properties is needed to improve patient survival. Here we report that p38γ MAPK regulates breast cancer cell motility and metastasis, in part by controlling expression of the metastasis-associated small GTPase RhoC. This p38γ-RhoC regulatory connection was mediated by a novel mechanism of modulating RhoC ubiquitination. This relationship persisted across multiple cell lines and in clinical breast cancer specimens. Using a computational mechanical model based on the finite element method, we demonstrated that p38γ-mediated cytoskeletal changes are sufficient to control cell motility. This model predicted novel dynamics of leading edge actin protrusions, which were experimentally verified and established to be closely related to cell shape and cytoskeletal morphology. Clinical relevance was supported by evidence that elevated expression of p38γ associated with lower overall survival of breast cancer patients. Taken together, our results offer a detailed characterization of how p38γ contributes to breast cancer progression, presents a new mechanics-based analysis of cell motility, and discovers a leading edge behavior in motile cells to accommodate modified cytoskeletal architecture. In summary, these findings not only identify a novel mechanism for regulating RhoC expression but also advance p38γ as a candidate therapeutic target.

MCL-1 is an anti-apoptotic BCL-2 family protein that has emerged as a major pathogenic factor in human cancer. Like BCL-2, MCL-1 bears a surface groove whose function is to sequester the BH3 killer domains of pro-apoptotic BCL-2 family members, a mechanism harnessed by cancer cells to establish formidable apoptotic blockades. Whereas drugging the BH3-binding groove has been achieved for BCL-2, translating this approach to MCL-1 has been challenging. Here, we report an alternative mechanism for MCL-1 inhibition by small molecule covalent modification of C286 at a novel interaction site distant from the BH3-binding groove. Our structure-function analyses revealed that the BH3-binding capacity of MCL-1 and its suppression of BAX are impaired by molecular engagement, a phenomenon recapitulated by C286W mutagenic mimicry in vitro and in cells. Thus, we characterize an allosteric mechanism for disrupting the anti-apoptotic, BH3-binding activity of MCL-1, informing a new strategy for disarming MCL-1 in cancer.

MCL-1 is a BCL-2 family protein implicated in the development and chemoresistance of human cancer. Unlike its anti-apoptotic homologs, Mcl-1 deletion has profound physiologic consequences, indicative of a broader role in homeostasis. We report that the BCL-2 homology 3 (BH3) α helix of MCL-1 can directly engage very long-chain acyl-CoA dehydrogenase (VLCAD), a key enzyme of the mitochondrial fatty acid β-oxidation (FAO) pathway. Proteomic analysis confirmed that the mitochondrial matrix isoform of MCL-1 (MCL-1) interacts with VLCAD. Mcl-1 deletion, or eliminating MCL-1 alone, selectively deregulated long-chain FAO, causing increased flux through the pathway in response to nutrient deprivation. Transient elevation in MCL-1 upon serum withdrawal, a striking increase in MCL-1 BH3/VLCAD interaction upon palmitic acid titration, and direct modulation of enzymatic activity by the MCL-1 BH3 α helix are consistent with dynamic regulation. Thus, the MCL-1 BH3 interaction with VLCAD revealed a separable, gain-of-function role for MCL-1 in the regulation of lipid metabolism.

Glucagon-like peptide 1 (GLP-1) is a natural peptide agonist of the GLP-1 receptor (GLP-1R) found on pancreatic β-cells. Engagement of the receptor stimulates insulin release in a glucosedependent fashion and increases β-cell mass, two ideal features for pharmacologic management of type 2 diabetes. Thus, intensive efforts have focused on developing GLP-1-based peptide agonists of GLP-1R for therapeutic application. A primary challenge has been the naturally short half-life of GLP-1 due to its rapid proteolytic degradation in vivo. Whereas mutagenesis and lipidation strategies have yielded clinical agents, we developed an alternative approach to preserving the structure and function of GLP-1 by all-hydrocarbon i, i + 7 stitching. This particular "stitch" is especially well-suited for reinforcing and protecting the structural fidelity of GLP-1. Lead constructs demonstrate striking proteolytic stability and potent biological activity in vivo. Thus, we report a facile approach to generating alternative GLP-1R agonists for glycemic control.

Very long-chain acyl-CoA dehydrogenase (VLCAD) is an inner mitochondrial membrane enzyme that catalyzes the first and rate-limiting step of long-chain fatty acid oxidation. Point mutations in human VLCAD can produce an inborn error of metabolism called VLCAD deficiency that can lead to severe pathophysiologic consequences, including cardiomyopathy, hypoglycemia, and rhabdomyolysis. Discrete mutations in a structurally-uncharacterized C-terminal domain region of VLCAD cause enzymatic deficiency by an incompletely defined mechanism. Here, we conducted a structure-function study, incorporating X-ray crystallography, hydrogen-deuterium exchange mass spectrometry, computational modeling, and biochemical analyses, to characterize a specific membrane interaction defect of full-length, human VLCAD bearing the clinically-observed mutations, A450P or L462P. By disrupting a predicted α-helical hairpin, these mutations either partially or completely impair direct interaction with the membrane itself. Thus, our data support a structural basis for VLCAD deficiency in patients with discrete mutations in an α-helical membrane-binding motif, resulting in pathologic enzyme mislocalization.

The search for efficient and environmentally friendly adsorbents has positioned lignocellulosic materials as attractive and low-cost alternatives instead of synthetic materials. Consequently, the present work investigates the efficacy of untreated lime peel (LM) and pineapple core (PP) as biosorbents for Cr(VI) removal. The maximum adsorption capacities (acquired at 24 h) of these sorbents were 9.20 and 4.99 mg/g, respectively. The use of these sorbents is expected to offer a rapid and efficient solution to treat effluents containing Cr(VI). Pineapple core showed the best biosorption properties and good distribution coefficients (distribution coefficient KD 8.35–99.20 mL/g) and the optimization of the adsorption was carried out by a response surface methodology using the Box–Behnken design. Thus, the effect of pH, biosorbent dosage, and temperature were assessed during the whole procedure. Three different responses were studied—Cr(VI) removal, Cr biosorption, and distribution coefficient—and the optimal conditions for maximizing the responses were identified by numerical optimization applying the desirability function. The resulting optimal conditions were: initial solution pH 2.01, biosorbent dosage 30 g/L, and temperature 30.05 °C. Finally, the process scale-up was evaluated by the simulation of the process working with a column of 100 L using the Fixed-bed Adsorption Simulation Tool (FASTv2.1). This research presents the obtained environmental benefits: i) reduction of pineapple waste, ii) Cr(VI) reduction and biosorption, iii) shortest sorption time for Cr, iv) properties that allow the biosorption process on the flow system, and v) low-cost process.