Biochemical functional predictions for protein structures of unknown or uncertain function

Mills, Caitlyn L.; Beuning, Penny J.; Ondrechen, Mary Jo

doi:10.1016/j.csbj.2015.02.003

Cited by 91 publications

(72 citation statements)

References 162 publications

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“…Determining the cellular, molecular and biological characteristics and functions of proteins of unknown function represents a challenge that can be addressed using computational and biochemical methods [21]. In the current study we have employed such complementary approaches to show that CPX-1 is a secreted collagen-binding glycoprotein.…”

Section: Discussionmentioning

confidence: 99%

Carboxypeptidase X-1 (CPX-1) is a secreted collagen-binding glycoprotein

Kim

O’Neill

Whitehead

2015

Biochemical and Biophysical Research Communications

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Carboxypeptidase X-1 (CPX-1) is an atypical member of the carboxypeptidase (CP) family of proteins involved in a variety of physiological and pathological processes. However, unlike most other family members CPX-1 lacks catalytic activity making its biological function unclear. CPX-1 contains a 160 amino acid discoidin domain (DSD) that serves as a binding domain in other proteins prompting us to investigate a putative functional role for this domain in CPX-1.Sequence alignment confirmed the overarching homology between the DSD of CPX-1 and other DSDs whilst more detailed analysis revealed conservation of the residues known to form the collagen-binding trench within the DSD of the discoidin domain receptors (DDRs) 1 and 2.Biochemical characterisation of transiently expressed human CPX-1 revealed that CPX-1 was secreted in an N-glycosylation-dependent manner as treatment with the N-glycosylation inhibitor tunicamycin inhibited secretion concomitant with a reduction in CPX-1 mobility on Western blot. Using a collagen pull-down assay we found that secreted CPX-1 bound collagen and this appeared independent of N-glycosylation as treatment with PNGaseF did not affect binding. Further analysis under non-reducing and reducing (+DTT) conditions revealed that CPX-1 was secreted in both monomeric and dimeric forms and only the former bound collagen.Finally, mutation of a key residue situated within the putative collagen-binding trench within the DSD of CPX-1 (R192A) significantly reduced secretion and collagen-binding by 40% and 60%, respectively. Collectively these results demonstrate that CPX-1 is a secreted collagen-binding glycoprotein and provide a foundation for future studies investigating the function of CPX-1.

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Section: Discussionmentioning

confidence: 99%

Carboxypeptidase X-1 (CPX-1) is a secreted collagen-binding glycoprotein

Kim

O’Neill

Whitehead

2015

Biochemical and Biophysical Research Communications

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“…Bioinformatic tools have been developed to predict the important moieties for catalysis, as well as their local spatial arrangements that can be used to identify protein function. In addition, the combination of different types of methods can help obtain more data and better predictions of the possible roles of proteins of unknown function (Mills, Beuning, & Ondrechen, ).…”

Section: Introductionmentioning

confidence: 99%

Structural modeling and mRNA expression of epididymal β‐defensins in GnRH immunized boars: A model for secondary hypogonadism in man

Weber

Alves

Abujamra

et al. 2018

Molecular Reproduction Devel

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Human secondary hypogonadism is associated with impaired testicular function, however, little is known about its impact on sperm epididymal maturation. Endocrine disruption in the epididymis could impair the secretion of key proteins, such as β‐defensins, responsible for spermatozoa maturation during epididymal transit. This study evaluated the sequence and structural similarities between porcine epididymal β‐defensins porcine β‐defensins (pBD3), pBD4, pBD125, and pEP2C and their human homologs using bioinformatics integrated with information derived from protein databanks. We then verified whether the expression of pBD3, pBD4, pBD125, and pEP2C genes in the testis and epididymis are influenced by disruption of the hypothalamic‐pituitary‐testicular (HPT) axis in a pig model for male human secondary hypogonadism. Upon modeling porcine β‐defensins, structural and functional analysis confirmed the presence of motifs associated with β‐defensin function, validating the models generated in silico. pBD3 and pBD4 showed acceptable structural alignments with human β‐defensins BDEF103 and BDEF110, respectively. In addition, evaluation of hormonal regulation of β‐defensins was assessed by analyzing the expression of these four β‐defensins in adult boars immunized against gonadotropin‐releasing hormone (GnRH). Our results indicate that HPT axis disruption modifies the expression of pBD3, pBD4, pBD125, and pEP2C in boar testis and epididymis, suggesting an endocrine‐dependent regulation of β‐defensins in swine epididymis. In conclusion, sequence and structural homology between pBD3 and pBD4 and their human homologs provide a basis for using the pig as a model for the study of human secondary hypogonadism. Further investigation of the human homologs in hypogonadal men could elucidate the connections between fertility and epididymal expression of β‐defensins.

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“…Studies with native enzymes originating from specialized or pure cultures help to reliably assign function to unknown proteins, thereby improving database annotations (Mills et al., 2015) and mechanistic understanding of degradation processes (Prior et al., 2009, Xu et al., 2015). However, pure or enriched laboratory cultures cannot provide reliable information about the diversity and activity of the enzymes actually involved in micropollutant degradation under environmental conditions.…”

Section: Introductionmentioning

confidence: 99%

Micropollutant degradation via extracted native enzymes from activated sludge

et al. 2016

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A procedure was developed to assess the biodegradation of micropollutants in cell-free lysates produced from activated sludge of a municipal wastewater treatment plant (WWTP). This proof-of-principle provides the basis for further investigations of micropollutant biodegradation via native enzymes in a solution of reduced complexity, facilitating downstream protein analysis. Differently produced lysates, containing a variety of native enzymes, showed significant enzymatic activities of acid phosphatase, β-galactosidase and β-glucuronidase in conventional colorimetric enzyme assays, whereas heat-deactivated controls did not. To determine the enzymatic activity towards micropollutants, 20 compounds were spiked to the cell-free lysates under aerobic conditions and were monitored via LC-ESI-MS/MS. The micropollutants were selected to span a wide range of different biodegradabilities in conventional activated sludge treatment via distinct primary degradation reactions. Of the 20 spiked micropollutants, 18 could be degraded by intact sludge under assay conditions, while six showed reproducible degradation in the lysates compared to the heat-deactivated negative controls: acetaminophen, N-acetyl-sulfamethoxazole (acetyl-SMX), atenolol, bezafibrate, erythromycin and 10,11-dihydro-10-hydroxycarbamazepine (10-OH-CBZ). The primary biotransformation of the first four compounds can be attributed to amide hydrolysis. However, the observed biotransformations in the lysates were differently influenced by experimental parameters such as sludge pre-treatment and the addition of ammonium sulfate or peptidase inhibitors, suggesting that different hydrolase enzymes were involved in the primary degradation, among them possibly peptidases. Furthermore, the transformation of 10-OH-CBZ to 9-CA-ADIN was caused by a biologically-mediated oxidation, which indicates that in addition to hydrolases further enzyme classes (probably oxidoreductases) are present in the native lysates. Although the full variety of indigenous enzymatic activity of the activated sludge source material could not be restored, experimental modifications, e.g. different lysate filtration, significantly enhanced specific enzyme activities (e.g. >96% removal of the antibiotic erythromycin). Therefore, the approach presented in this study provides the experimental basis for a further elucidation of the enzymatic processes underlying wastewater treatment on the level of native proteins.

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Biochemical functional predictions for protein structures of unknown or uncertain function

Cited by 91 publications

References 162 publications

Carboxypeptidase X-1 (CPX-1) is a secreted collagen-binding glycoprotein

Carboxypeptidase X-1 (CPX-1) is a secreted collagen-binding glycoprotein

Structural modeling and mRNA expression of epididymal β‐defensins in GnRH immunized boars: A model for secondary hypogonadism in man

Micropollutant degradation via extracted native enzymes from activated sludge

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