Identification and purification of a soluble region of BubR1: a critical component of the mitotic checkpoint complex

The mitotic checkpoint complex (MCC) ensures the fidelity of chromosomal segregation, by delaying the onset of anaphase until all sister chromatids have been properly attached to the mitotic spindle. In essence, this MCC-induced delay is achieved via the inhibition of the anaphase-promoting complex (APC). Among the components of the MCC, BubR1 plays two major roles in the functions of the mitotic checkpoint. First, BubR1 is able to inhibit APC activity, either by itself or as a component of the MCC, by sequestering a APC coactivator, known as Cdc20. Second, BubR1 activates mitotic checkpoint signaling cascades by binding to the centromere-associated protein E, a microtubule motor protein. Obtaining highly soluble BubR1 is a prerequisite for the study of its structure. BubR1 is a multi-domain protein, which includes a KEN box motif, a mad3-like region, a Bub3 binding domain, and a kinase domain. We obtained a soluble BubR1 construct using a three-step expression strategy. First, we obtained two constructs from BLAST sequence homology searches, both of which were expressed abundantly in the inclusion bodies. We then adjusted the lengths of the two constructs by secondary structure prediction, thereby generating partially soluble constructs. Third, we optimized the solubility of the two constructs by either chopping or adding a few residues at the C-terminus. Finally, we obtained a highly soluble BubR1 construct via the Escherichia coli expression system, which allowed for a yield of 10.8 mg/L culture. This report may provide insight into the design of highly soluble constructs of insoluble multi-domain proteins

Differential Proteome Profiling Using iTRAQ in Microalbuminuric and Normoalbuminuric Type 2 Diabetic Patients

Diabetic nephropathy (DN) is a long-term complication of diabetes mellitus that leads to end-stage renal disease. Microalbuminuria is used for the early detection of diabetic renal damage, but such levels do not reflect the state of incipient DN precisely in type 2 diabetic patients because microalbuminuria develops in other diseases, necessitating more accurate biomarkers that detect incipient DN. Isobaric tags for relative and absolute quantification (iTRAQ) were used to identify urinary proteins that were differentially excreted in normoalbuminuric and microalbuminuric patients with type 2 diabetes where 710 and 196 proteins were identified and quantified, respectively. Some candidates were confirmed by 2-DE analysis, or validated by Western blot and multiple reaction monitoring (MRM). Specifically, some differentially expressed proteins were verified by MRM in urine from normoalbuminuric and microalbuminuric patients with type 2 diabetes, wherein alpha-1-antitrypsin, alpha-1-acid glycoprotein 1, and prostate stem cell antigen had excellent AUC values (0.849, 0.873, and 0.825, resp.). Moreover, we performed a multiplex assay using these biomarker candidates, resulting in a merged AUC value of 0.921. Although the differentially expressed proteins in this iTRAQ study require further validation in larger and categorized sample groups, they constitute baseline data on preliminary biomarker candidates that can be used to discover novel biomarkers for incipient DN

An Exploratory Pilot Study with Plasma Protein Signatures Associated with Response of Patients with Depression to Antidepressant Treatment for 10 Weeks.

Major depressive disorder (MDD) is a leading cause of global disability with a chronic and recurrent course. Recognition of biological markers that could predict and monitor response to drug treatment could personalize clinical decision-making, minimize unnecessary drug exposure, and achieve better outcomes. Four longitudinal plasma samples were collected from each of ten patients with MDD treated with antidepressants for 10 weeks. Plasma proteins were analyzed qualitatively and quantitatively with a nanoflow LC-MS/MS technique. Of 1153 proteins identified in the 40 longitudinal plasma samples, 37 proteins were significantly associated with response/time and clustered into six according to time and response by the linear mixed model. Among them, three early-drug response markers (PHOX2B, SH3BGRL3, and YWHAE) detectable within one week were verified by liquid chromatography-multiple reaction monitoring/mass spectrometry (LC-MRM/MS) in the well-controlled 24 patients. In addition, 11 proteins correlated significantly with two or more psychiatric measurement indices. This pilot study might be useful in finding protein marker candidates that can monitor response to antidepressant treatment during follow-up visits within 10 weeks after the baseline visit

Higher Plasma Stromal Cell-Derived Factor 1 Is Associated with Lower Risk for Sarcopenia in Older Asian Adults

Background Despite the protective effects of stromal cell-derived factor 1 (SDF-1) in stimulating muscle regeneration shown in experimental research, there is a lack of clinical studies linking circulating SDF-1 concentrations with muscle phenotypes. In order to elucidate the role of SDF-1 as a potential biomarker reflecting human muscle health, we investigated the association of plasma SDF-1 levels with sarcopenia in older adults. Methods This cross-sectional study included 97 community-dwelling participants who underwent a comprehensive geriatric assessment at a tertiary hospital in South Korea. Sarcopenia was defined by specific cutoff values applicable to the Asian population, whereas plasma SDF-1 levels were determined using an enzyme immunoassay. Results After accounting for sex, age, and body mass index, participants with sarcopenia and low muscle mass exhibited plasma SDF-1 levels that were 21.8% and 18.3% lower than those without these conditions, respectively (P=0.008 and P=0.009, respectively). Consistently, higher plasma SDF-1 levels exhibited a significant correlation with higher skeletal muscle mass index (SMI) and gait speed (both P=0.043), and the risk of sarcopenia and low muscle mass decreased by 58% and 55% per standard deviation increase in plasma SDF-1 levels, respectively (P=0.045 and P=0.030, respectively). Furthermore, participants in the highest SDF-1 tertile exhibited significantly higher SMI compared to those in the lowest tertile (P=0.012). Conclusion These findings clinically corroborate earlier experimental discoveries highlighting the muscle anabolic effects of SDF-1 and support the potential role of circulating SDF-1 as a biomarker reflecting human muscle health in older adults

Crystal structure of YrrB: a TPR protein with an unusual peptide-binding site

YrrB is a hypothetical protein containing a tetratricopeptide repeat (TPR) domain from a Gram-positive bacterium, Bacillus subtilis. We determined YrrB structure in the C2 space group to 2.5A resolution, which is the first TPR structure of the Gram-positive bacterium B. subtilis. In contrast to other known TPR structures, the concave surface of the YrrB TPR domain is composed of the putative peptide-binding pocket lined with positively-charged residues. This unique charge distribution reveals that YrrB can interact with partner proteins via an unusual TPR-mediated interaction mode, compared to that of other TPR-containing structures. Functional annotation using genomics analysis suggested that YrrB may be an interacting mediator in the complex formation among RNA sulfuration components. No proteins containing a TPR domain have been identified in the biosynthesis of sulfur-containing biomolecules. Thus, YrrB could play a new role as a connecting module among those proteins in the conserved gene cluster for RNA sulfuration

Crystal structure of PilF: functional implication in the type 4 pilus biogenesis in Pseudomonas aeruginosa

PilF is a requisite protein involved in the type 4 pilus biogenesis system from the Gram-negative human pathogenic bacteria, Pseudomonas aeruginosa. We determined the PilF structure at a 2.2A resolution; this includes six tandem tetratrico peptide repeat (TPR) units forming right-handed superhelix. PilF structure was similar to the heat shock protein organizing protein, which interacts with the C-terminal peptide of Hsp90 and Hsp70 via a concave Asn ladder in the inner groove of TPR superhelix. After simulated screening, the C-terminal pentapeptides of PilG, PilU, PilY, and PilZ proved to be a likely candidate binding to PilF, which are ones of 25 necessary components involved in the type 4 pilus biogenesis system. We proposed that PilF would be critical as a bridgehead in protein-protein interaction and thereby, PilF may bind a necessary molecule in type 4 pilus biogenesis system such as PilG, PilU, PilY, and PilZ

Crystal Structure of the N-terminal Domain of Anaphase-promoting Complex Subunit 7

Anaphase-promoting complex or cyclosome (APC/C) is an unusual E3 ubiquitin ligase and an essential protein that controls mitotic progression. APC/C includes at least 13 subunits, but no structure has been determined for any tetratricopeptide repeat (TPR)-containing subunit (Apc3 and -6-8) in the TPR subcomplex of APC/C. Apc7 is a TPR-containing subunit that exists only in vertebrate APC/C. Here we report the crystal structure of quad mutant of nApc7 (N-terminal fragment, residues 1-147) of human Apc7 at a resolution of 2.5 angstrom. The structure of nApc7 adopts a TPR-like motif and has a unique dimerization interface, although the protein does not contain the conserved TPR sequence. Based on the structure of nApc7, in addition to previous experimental findings, we proposed a putative homodimeric structure for full-length Apc7. This model suggests that TPR-containing subunits self-associate and bind to adaptors and substrates via an IR peptide in TPR-containing subunits of APC/C.This work was supported by the 21C Frontier Functional Proteomics Project of the Korean Ministry of Science and Technology Grant FPR 08-A2-110 and by the Innovative Research Institute for Cell Therapy, Republic of Korea, Grant A062260.Holm L, 2008, BIOINFORMATICS, V24, P2780, DOI 10.1093/bioinformatics/btn507Han D, 2008, PROTEINS, V70, P900, DOI 10.1002/prot.21597Ohi MD, 2007, MOL CELL, V28, P871, DOI 10.1016/j.molcel.2007.10.003Nyarko A, 2007, BIOCHEMISTRY-US, V46, P11331, DOI 10.1021/bi700735tKrissinel E, 2007, J MOL BIOL, V372, P774, DOI 10.1016/j.jmb.2007.05.022Pal M, 2007, J CELL SCI, V120, P3238, DOI 10.1242/jcs.004762Thornton BR, 2006, GENE DEV, V20, P3069, DOI 10.1101/gad.1478306Peters JM, 2006, NAT REV MOL CELL BIO, V7, P644, DOI 10.1038/nrm1988Wu YK, 2006, NAT STRUCT MOL BIOL, V13, P589, DOI 10.1038/nsmb1106Hayes MJ, 2006, NAT CELL BIOL, V8, P607, DOI 10.1038/ncb1410Painter J, 2006, ACTA CRYSTALLOGR D, V62, P439, DOI 10.1107/S0907444906005270Thornton BR, 2006, GENE DEV, V20, P449, DOI 10.1101/gad.1396906Eytan E, 2006, P NATL ACAD SCI USA, V103, P2081, DOI 10.1073/pnas.0510695103Tanaka T, 2006, BIOPOLYMERS, V84, P161, DOI 10.1002/hip.20361Passmore LA, 2005, MOL CELL, V20, P855, DOI 10.1016/j.molcel.2005.11.003Dube P, 2005, MOL CELL, V20, P867, DOI 10.1016/j.molcel.2005.11.008Turnell AS, 2005, NATURE, V438, P690, DOI 10.1038/nature04151Kraft C, 2005, MOL CELL, V18, P543, DOI 10.1016/j.molcel.2005.04.023Wilson CGM, 2005, FEBS J, V272, P166, DOI 10.1111/j.1432-1033.2004.04397.xPark KH, 2005, BREAST CANCER RES, V7, pR238, DOI 10.1186/bcr978Emsley P, 2004, ACTA CRYSTALLOGR D, V60, P2126, DOI 10.1107/S0907444904019158Krissinel E, 2004, ACTA CRYSTALLOGR D, V60, P2256, DOI 10.1107/S0907444904026460van Roessel P, 2004, CELL, V119, P707, DOI 10.1016/j.cell.2004.11.028Jinek M, 2004, NAT STRUCT MOL BIOL, V11, P1001, DOI 10.1038/nsmb833Passmore LA, 2004, BIOCHEM J, V379, P513, DOI 10.1042/BJ20040198Konishi Y, 2004, SCIENCE, V303, P1026, DOI 10.1126/science.1093712Terwilliger T, 2004, J SYNCHROTRON RADIAT, V11, P49, DOI 10.1107/S0909049503023938Kraft C, 2003, EMBO J, V22, P6598, DOI 10.1093/emboj/cdg627Gonzalez A, 2003, ACTA CRYSTALLOGR D, V59, P1935, DOI 10.1107/S0907444903017700Vodermaier HC, 2003, CURR BIOL, V13, P1459, DOI 10.1016/S0960-9822(03)00581-5Main ERG, 2003, STRUCTURE, V11, P497, DOI 10.1016/S0969-2126(03)00076-5Terwilliger TC, 2003, METHOD ENZYMOL, V374, P22, DOI 10.1016/S0076-6879(03)74002-6Yoon HJ, 2002, CURR BIOL, V12, P2048, DOI 10.1016/S0960-9822(02)01331-3Winn MD, 2002, ACTA CRYSTALLOGR D, V58, P1929, DOI 10.1107/S0907444902016116Harper JW, 2002, GENE DEV, V16, P2179, DOI 10.1101/gad.1013102Peters JM, 2002, MOL CELL, V9, P931, DOI 10.1016/S1097-2765(02)00540-3Gieffers C, 2001, MOL CELL, V7, P907, DOI 10.1016/S1097-2765(01)00234-9Winn MD, 2001, ACTA CRYSTALLOGR D, V57, P122, DOI 10.1107/S0907444900014736Pfleger CM, 2000, GENE DEV, V14, P655Visintin R, 1997, SCIENCE, V278, P460, DOI 10.1126/science.278.5337.460Garnier J, 1996, METHOD ENZYMOL, V266, P540THOMPSON JD, 1994, NUCLEIC ACIDS RES, V22, P4673, DOI 10.1093/nar/22.22.4673LAMB JR, 1994, EMBO J, V13, P4321JONES TA, 1991, ACTA CRYSTALLOGR A, V47, P110, DOI 10.1107/S0108767390010224GLOTZER M, 1991, NATURE, V349, P132, DOI 10.1038/349132a0MATTHEWS BW, 1968, J MOL BIOL, V33, P491, DOI 10.1016/0022-2836(68)90205-2

Online monitoring of immunoaffinity-based depletion of high-abundance blood proteins by UV spectrophotometry using enhanced green fluorescence protein and FITC-labeled human serum albumin

<p>Abstract</p> <p>Background</p> <p>The removal of high-abundance proteins from plasma is an efficient approach to investigating flow-through proteins for biomarker discovery studies. Most depletion methods are based on multiple immunoaffinity methods available commercially including LC columns and spin columns. Despite its usefulness, high-abundance depletion has an intrinsic problem, the sponge effect, which should be assessed during depletion experiments. Concurrently, the yield of depletion of high-abundance proteins must be monitored during the use of the depletion column. To date, there is no reasonable technique for measuring the recovery of flow-through proteins after depletion and assessing the capacity for capture of high-abundance proteins.</p> <p>Results</p> <p>In this study, we developed a method of measuring recovery yields of a multiple affinity removal system column easily and rapidly using enhanced green fluorescence protein as an indicator of flow-through proteins. Also, we monitored the capture efficiency through depletion of a high-abundance protein, albumin labeled with fluorescein isothiocyanate.</p> <p>Conclusion</p> <p>This simple method can be applied easily to common high-abundance protein depletion methods, effectively reducing experimental variations in biomarker discovery studies.</p