To metastasize, a tumor cell must acquire abilities such as the capacity to colonize new tissue and evade immune surveillance. Recent evidence suggests that microRNAs can promote the evolution of ...malignant behaviors by regulating multiple targets. We performed a microRNA analysis of human melanoma, a highly invasive cancer, and found that miR-30b/30d upregulation correlates with stage, metastatic potential, shorter time to recurrence, and reduced overall survival. Ectopic expression of miR-30b/30d promoted the metastatic behavior of melanoma cells by directly targeting the GalNAc transferase GALNT7, resulted in increased synthesis of the immunosuppressive cytokine IL-10, and reduced immune cell activation and recruitment. These data support a key role of miR-30b/30d and GalNAc transferases in metastasis, by simultaneously promoting cellular invasion and immunosuppression.
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► miR-30b/d levels associate with advanced stage and poor outcome in melanoma patients ► miR-30d promotes metastasis and immunosuppressive features in vivo ► miR-30d enhances invasion and IL-10 secretion by targeting GalNAc transferases ► miR-30d upregulation leads to GALNT7-dependent alterations in cellular glycosylation
Course-based undergraduate research experiences (CUREs) are laboratory courses that integrate broadly relevant problems, discovery, use of the scientific process, collaboration, and iteration to ...provide more students with research experiences than is possible in individually mentored faculty laboratories. Members of the national Malate dehydrogenase CUREs Community (MCC) investigated the differences in student impacts between traditional laboratory courses (control), a short module CURE within traditional laboratory courses (mCURE), and CUREs lasting the entire course (cCURE). The sample included approximately 1,500 students taught by 22 faculty at 19 institutions. We investigated course structures for elements of a CURE and student outcomes including student knowledge, student learning, student attitudes, interest in future research, overall experience, future GPA, and retention in STEM. We also disaggregated the data to investigate whether underrepresented minority (URM) outcomes were different from White and Asian students. We found that the less time students spent in the CURE the less the course was reported to contain experiences indicative of a CURE. The cCURE imparted the largest impacts for experimental design, career interests, and plans to conduct future research, while the remaining outcomes were similar between the three conditions. The mCURE student outcomes were similar to control courses for most outcomes measured in this study. However, for experimental design, the mCURE was not significantly different than either the control or cCURE. Comparing URM and White/Asian student outcomes indicated no difference for condition, except for interest in future research. Notably, the URM students in the mCURE condition had significantly higher interest in conducting research in the future than White/Asian students.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Carbohydrates are the most difficult class of biological molecules to study by high-throughput methods owing to the chemical similarities between the constituent monosaccharide building blocks, ...template-less biosynthesis, and the lack of clearly identifiable consensus sequences for the glycan modification of cohorts of glycoproteins. These molecules are crucial for a wide variety of cellular processes ranging from cell-cell communication to immunity, and they are altered in disease states such as cancer and inflammation. Thus, there has been a dedicated effort to develop glycan analysis into a high-throughput analytical field termed glycomics. Herein we highlight major advances in applying separation, mass spectrometry, and microarray methods to the fields of glycomics and glycoproteomics. These new analytical techniques are rapidly advancing our understanding of the importance of glycosylation in biology and disease.
Cell surface glycans form a critical interface with the biological milieu, informing diverse processes from the inflammatory cascade to cellular migration. Assembly of discrete carbohydrate ...structures requires the coordinated activity of a repertoire of proteins, including glycosyltransferases and glycosidases. Little is known about the regulatory networks controlling this complex biosynthetic process. Recent work points to a role for microRNA (miRNA) in the regulation of specific glycan biosynthetic enzymes. Herein we take a unique systems-based approach to identify connections between miRNA and the glycome. By using our glycomic analysis platform, lectin microarrays, we identify glycosylation signatures in the NCI-60 cell panel that point to the glycome as a direct output of genomic information flow. Integrating our glycomic dataset with miRNA data, we map miRNA regulators onto genes in glycan biosynthetic pathways (glycogenes) that generate the observed glycan structures. We validate three of these predicted miRNA/glycogene regulatory networks: high mannose, fucose, and terminal β-GalNAc, identifying miRNA regulation that would not have been observed by traditional bioinformatic methods. Overall, our work reveals critical nodes in the global glycosylation network accessible to miRNA regulation, providing a bridge between miRNA-mediated control of cell phenotype and the glycome.
Many members of the mechanistically diverse enolase superfamily have unknown functions. In this report we use both genome (operon) context and screening of a library of acid sugars to assign the ...l-fuconate dehydratase (FucD) function to a member of the mandelate racemase (MR) subgroup of the superfamily encoded by the Xanthomonas campestris pv. campestris str. ATCC 33913 genome (GI:21233491). Orthologues of FucD are found in both bacteria and eukaryotes, the latter including the rTS beta protein in Homo sapiens that has been implicated in regulating thymidylate synthase activity. As suggested by sequence alignments and confirmed by high-resolution structures in the presence of active site ligands, FucD and MR share the same active site motif of functional groups: three carboxylate ligands for the essential Mg2+ located at the ends of the third, fourth, and fifth β-strands in the (β/α)7β-barrel domain (Asp 248, Glu 274, and Glu 301, respectively), a Lys-x-Lys motif at the end of the second β-strand (Lys 218 and Lys 220), a His-Asp dyad at the end of the seventh and sixth β-strands (His 351 and Asp 324, respectively), and a Glu at the end of the eighth β-strand (Glu 382). The mechanism of the FucD reaction involves initial abstraction of the 2-proton by Lys 220, acid catalysis of the vinylogous β-elimination of the 3-OH group by His 351, and stereospecific ketonization of the resulting enol, likely by the conjugate acid of Lys 220, to yield the 2-keto-3-deoxy-l-fuconate product. Screening of the library of acid sugars revealed substrate and functional promiscuity: In addition to l-fuconate, FucD also catalyzes the dehydration of l-galactonate, d-arabinonate, d-altronate, l-talonate, and d-ribonate. The dehydrations of l-fuconate, l-galactonate, and d-arabinonate are initiated by abstraction of the 2-protons by Lys 220. The dehydrations of l-talonate and d-ribonate are initiated by abstraction of the 2-protons by His 351; however, protonation of the enediolate intermediates by the conjugate acid of Lys 220 yields l-galactonate and d-arabinonate in competition with dehydration. The functional promiscuity discovered for FucD highlights possible structural mechanisms for evolution of function in the enolase superfamily.
The l-rhamnonate dehydratase (RhamD) function was assigned to a previously uncharacterized family in the mechanistically diverse enolase superfamily that is encoded by the genome of Escherichia coli ...K-12. We screened a library of acid sugars to discover that the enzyme displays a promiscuous substrate specificity: l-rhamnonate (6-deoxy-l-mannonate) has the “best” kinetic constants, with l-mannonate, l-lyxonate, and d-gulonate dehydrated less efficiently. Crystal structures of the RhamDs from both E. coli K-12 and Salmonella typhimurium LT2 (95% sequence identity) were obtained in the presence of Mg2+; the structure of the RhamD from S. typhimurium was also obtained in the presence of 3-deoxy-l-rhamnonate (obtained by reduction of the product with NaBH4). Like other members of the enolase superfamily, RhamD contains an N-terminal α + β capping domain and a C-terminal (β/α)7β-barrel (modified TIM-barrel) catalytic domain with the active site located at the interface between the two domains. In contrast to other members, the specificity-determining “20s loop” in the capping domain is extended in length and the “50s loop” is truncated. The ligands for the Mg2+ are Asp 226, Glu 252 and Glu 280 located at the ends of the third, fourth and fifth β-strands, respectively. The active site of RhamD contains a His 329-Asp 302 dyad at the ends of the seventh and sixth β-strands, respectively, with His 329 positioned to function as the general base responsible for abstraction of the C2 proton of l-rhamnonate to form a Mg2+-stabilized enediolate intermediate. However, the active site does not contain other acid/base catalysts that have been implicated in the reactions catalyzed by other members of the MR subgroup of the enolase superfamily. Based on the structure of the liganded complex, His 329 also is expected to function as the general acid that both facilitates departure of the 3-OH group in a syn-dehydration reaction and delivers a proton to carbon-3 to replace the 3-OH group with retention of configuration.
The d-mannonate dehydratase (ManD) function was assigned to a group of orthologous proteins in the mechanistically diverse enolase superfamily by screening a library of acid sugars. Structures of the ...wild type ManD from Novosphingobium aromaticivorans were determined at pH 7.5 in the presence of Mg2+ and also in the presence of Mg2+ and the 2-keto-3-keto-d-gluconate dehydration product; the structure of the catalytically active K271E mutant was determined at pH 5.5 in the presence of the d-mannonate substrate. As previously observed in the structures of other members of the enolase superfamily, ManD contains two domains, an N-terminal α+β capping domain and a (β/α)7β-barrel domain. The barrel domain contains the ligands for the essential Mg2+, Asp 210, Glu 236, and Glu 262, at the ends of the third, fourth, and fifth β-strands of the barrel domain, respectively. However, the barrel domain lacks both the Lys acid/base catalyst at the end of the second β-strand and the His-Asp dyad acid/base catalyst at the ends of the seventh and sixth β-strands, respectively, that are found in many members of the superfamily. Instead, a hydrogen-bonded dyad of Tyr 159 in a loop following the second β-strand and Arg 147 at the end of the second β-strand are positioned to initiate the reaction by abstraction of the 2-proton. Both Tyr 159 and His 212, at the end of the third β-strand, are positioned to facilitate both syn-dehydration and ketonization of the resulting enol intermediate to yield the 2-keto-3-keto-d-gluconate product with the observed retention of configuration. The identities and locations of these acid/base catalysts as well as of cationic amino acid residues that stabilize the enolate anion intermediate define a new structural strategy for catalysis (subgroup) in the mechanistically diverse enolase superfamily. With these differences, we provide additional evidence that the ligands for the essential Mg2+ are the only conserved residues in the enolase superfamily, establishing the primary functional importance of the Mg2+-assisted strategy for stabilizing the enolate anion intermediate.
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