The Use of In Vitro Peptide-Library Screens in the Analysis of Phosphoserine/Threonine-Binding Domain Structure and Function

ABSTRACT Cep57 has been characterized as a component of a pericentriolar complex containing Cep63 and Cep152. Interestingly, Cep63 and Cep152 self-assemble into a pericentriolar cylindrical architecture, and this event is critical for the orderly recruitment of Plk4, a key regulator of centriole duplication. However, the way in which Cep57 interacts with the Cep63-Cep152 complex and contributes to the structure and function of Cep63-Cep152 self-assembly remains unknown. We demonstrate that Cep57 interacts with Cep63 through N-terminal motifs and associates with Cep152 via Cep63. Three-dimensional structured illumination microscopy (3D-SIM) analyses suggested that the Cep57-Cep63-Cep152 complex is concentrically arranged around a centriole in a Cep57-in and Cep152-out manner. Cep57 mutant cells defective in Cep63 binding exhibited improper Cep63 and Cep152 localization and impaired Sas6 recruitment for procentriole assembly, proving the significance of the Cep57-Cep63 interaction. Intriguingly, Cep63 fused to a microtubule (MT)-binding domain of Cep57 functioned in concert with Cep152 to assemble around stabilized MTs in vitro. Thus, Cep57 plays a key role in architecting the Cep63-Cep152 assembly around centriolar MTs and promoting centriole biogenesis. This study may offer a platform to investigate how the organization and function of the pericentriolar architecture are altered by disease-associated mutations found in the Cep57-Cep63-Cep152 complex.

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2072-P: Deletion of the Mitofusins 1 and 2 (Mfn1 and Mfn2) in the Pancreatic Beta Cell Disrupts Mitochondrial Structure and Function In Vitro and Strongly Impairs Glucose-Stimulated Insulin Secretion In Vivo

Diabetes ◽

10.2337/db20-2072-p ◽

2020 ◽

Vol 69 (Supplement 1) ◽

pp. 2072-P

Author(s):

ELENI GEORGIADOU ◽

PAULINE L. CHABOSSEAU ◽

ALEJANDRA TOMAS ◽

ISABELLE LECLERC ◽

GUY A. RUTTER

Keyword(s):

Insulin Secretion ◽

Beta Cell ◽

Pancreatic Beta Cell ◽

Structure And Function ◽

Insulin Secretion In Vivo ◽

Mitochondrial Structure ◽

Glucose Stimulated Insulin Secretion ◽

And Function

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Structure and Function of Myosin Light Chain Kinase of Smooth Muscle: N-Terminal Actin-Binding Domain.

The Kitakanto Medical Journal ◽

10.2974/kmj.47.57 ◽

1997 ◽

Vol 47 (2) ◽

pp. 57-65

Author(s):

Li-Hong Ye

Keyword(s):

Smooth Muscle ◽

Light Chain ◽

Myosin Light Chain Kinase ◽

Myosin Light Chain ◽

Structure And Function ◽

Binding Domain ◽

Actin Binding ◽

Actin Binding Domain ◽

And Function

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Interaction of chromium(III) or chromium(VI) with catalase and its effect on the structure and function of catalase: An in vitro study

Food Chemistry ◽

10.1016/j.foodchem.2017.10.062 ◽

2018 ◽

Vol 244 ◽

pp. 378-385 ◽

Cited By ~ 34

Author(s):

Linfeng Chen ◽

Jing Zhang ◽

Yaxian Zhu ◽

Yong Zhang

Keyword(s):

In Vitro Study ◽

Structure And Function ◽

Vitro Study ◽

Chromium Vi ◽

And Function

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Maintenance of Insect Ovarian Microtubular Structure and Function in vitro

Nature New Biology ◽

10.1038/newbio242253a0 ◽

1973 ◽

Vol 242 (121) ◽

pp. 253-254 ◽

Cited By ~ 1

Author(s):

H. STEBBINGS ◽

N. A. RATCLIFFE

Keyword(s):

Structure And Function ◽

And Function

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Differences in size, structure and function of free and membrane-bound polyribosomes of rat liver. Evidence for a single class of membrane-bound polyribosomes

Biochemical Journal ◽

10.1042/bj1680001 ◽

1977 ◽

Vol 168 (1) ◽

pp. 1-8 ◽

Cited By ~ 26

Author(s):

J C Ramsey ◽

W J Steele

Keyword(s):

Protein Synthesis ◽

Rat Liver ◽

Size Structure ◽

Large Fraction ◽

Liver Homogenate ◽

Structure And Function ◽

Structural And Functional Properties ◽

Membrane Bound ◽

And Function

Free loosely bound and tightly bound polyribosomes were separated from rat liver homogenate by salt extraction followed by differential centrifugation, and several of their structural and functional properties were compared to resolve the existence of loosely bound polyribosomes and verify the specificity of the separation. The free and loosely bound polyribosomes have similar sedimentation profiles and polyribosome contents, their subunit proteins have similar electrophoretic patterns and their products of protein synthesis in vitro show a close correspondence in size and amounts synthesized. In contrast, the tightly bound polyribosomes have different properties from those of the free and loosely bound polyribosomes; their average size is significantly smaller; their polyribosome content is higher; their 60 S-subunit proteins lack two components and contain four or more components not found elsewhere; their products of protein synthesis in vitro differ in size and amounts synthesized. These observations show that rat liver membranes entrap a large fraction of the free polyribosomes at low salt concentrations and that these polyribosomes are similar to those of the free-polyribosome fraction and are different from those of the tightly bound polyribosome fraction in size, structure and function.

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Multiplatform Physiologic and Metabolic Phenotyping Reveals Microbial Toxicity

mSystems ◽

10.1128/msystems.00123-18 ◽

2018 ◽

Vol 3 (6) ◽

Cited By ~ 2

Author(s):

Jingwei Cai ◽

Robert G. Nichols ◽

Imhoi Koo ◽

Zachary A. Kalikow ◽

Limin Zhang ◽

...

Keyword(s):

Mass Spectrometry ◽

Amino Acids ◽

Flow Cytometry ◽

Free Radical ◽

Gut Microbiota ◽

Structure And Function ◽

Metabolic Phenotyping ◽

And Function

ABSTRACTThe gut microbiota is susceptible to modulation by environmental stimuli and therefore can serve as a biological sensor. Recent evidence suggests that xenobiotics can disrupt the interaction between the microbiota and host. Here, we describe an approach that combinesin vitromicrobial incubation (isolated cecal contents from mice), flow cytometry, and mass spectrometry- and1H nuclear magnetic resonance (NMR)-based metabolomics to evaluate xenobiotic-induced microbial toxicity. Tempol, a stabilized free radical scavenger known to remodel the microbial community structure and functionin vivo, was studied to assess its direct effect on the gut microbiota. The microbiota was isolated from mouse cecum and was exposed to tempol for 4 h under strict anaerobic conditions. The flow cytometry data suggested that short-term tempol exposure to the microbiota is associated with disrupted membrane physiology as well as compromised metabolic activity. Mass spectrometry and NMR metabolomics revealed that tempol exposure significantly disrupted microbial metabolic activity, specifically indicated by changes in short-chain fatty acids, branched-chain amino acids, amino acids, nucleotides, glucose, and oligosaccharides. In addition, a mouse study with tempol (5 days gavage) showed similar microbial physiologic and metabolic changes, indicating that thein vitroapproach reflectedin vivoconditions. Our results, through evaluation of microbial viability, physiology, and metabolism and a comparison ofin vitroandin vivoexposures with tempol, suggest that physiologic and metabolic phenotyping can provide unique insight into gut microbiota toxicity.IMPORTANCEThe gut microbiota is modulated physiologically, compositionally, and metabolically by xenobiotics, potentially causing metabolic consequences to the host. We recently reported that tempol, a stabilized free radical nitroxide, can exert beneficial effects on the host through modulation of the microbiome community structure and function. Here, we investigated a multiplatform phenotyping approach that combines high-throughput global metabolomics with flow cytometry to evaluate the direct effect of tempol on the microbiota. This approach may be useful in deciphering how other xenobiotics directly influence the microbiota.

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Carbohydrate complexity structures stable diversity in gut-derived microbial consortia under high dilution pressure

10.1101/2020.01.31.929760 ◽

2020 ◽

Author(s):

Tianming Yao ◽

Ming-Hsu Chen ◽

Stephen R. Lindemann

Keyword(s):

Microbial Diversity ◽

Structural Complexity ◽

Structure And Function ◽

Microbial Consortia ◽

Carbohydrate Structure ◽

High Dilution ◽

Chemical Complexity ◽

Metabolic Interactions ◽

And Function

ABSTRACTDietary fibers are major substrates for the colonic microbiota, but the structural specificity of these fibers for the diversity, structure, and function of gut microbial communities are poorly understood. Here, we employed an in vitro sequential batch fecal culture approach to determine: 1) whether the chemical complexity of a carbohydrate structure influences its ability to maintain microbial diversity in the face of high dilution pressure and 2) whether substrate structuring or obligate microbe-microbe metabolic interactions (e.g. exchange of amino acids or vitamins) exert more influence on maintained diversity. Sorghum arabinoxylan (SAX, complex polysaccharide), inulin (low-complexity oligosaccharide) and their corresponding monosaccharide controls were selected as model carbohydrates. Our results demonstrate that complex carbohydrates stably sustain diverse microbial consortia. Further, very similar final consortia were enriched on SAX from the same individual’s fecal microbiota across a one-month interval, suggesting that polysaccharide structure is more influential than stochastic alterations in microbiome composition in governing the outcomes of sequential batch cultivation experiments. SAX-consuming consortia were anchored by Bacteroides ovatus and retained diverse consortia of >12 OTUs; whereas final inulin-consuming consortia were dominated either by Klebsiella pneumoniae or Bifidobacterium sp. and Escherichia coli. Furthermore, auxotrophic interactions were less influential in structuring microbial consortia consuming SAX than the less-complex inulin. These data suggest that carbohydrate structural complexity affords independent niches that structure fermenting microbial consortia, whereas other metabolic interactions govern the composition of communities fermenting simpler carbohydrates.IMPORTANCEThe mechanisms by which gut microorganisms compete for and cooperate on human-indigestible carbohydrates of varying structural complexity remain unclear. Gaps in this understanding make it challenging to predict the effect of a particular dietary fiber’s structure on the diversity, composition, or function of gut microbiomes, especially with inter-individual variability in diets and microbiomes. Here, we demonstrate that carbohydrate structure governs the diversity of gut microbiota under high dilution pressure, suggesting that such structures may support microbial diversity in vivo. Further, we also demonstrate that carbohydrate polymers are not equivalent in the strength by which they influence community structure and function, and that metabolic interactions among members arising due to auxotrophy exert significant influence on the outcomes of these competitions for simpler polymers. Collectively, these data suggest that large, complex dietary fiber polysaccharides structure the human gut ecosystem in ways that smaller and simpler ones may not.

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