The carbohydrate-binding domain of overexpressed STBD1 is important for its stability and protein–protein interactions

STBD1 (starch-binding domain-containing protein 1) belongs to the CBM20 (family 20 carbohydrate binding module) group of proteins, and is implicated in glycogen metabolism and autophagy. However, very little is known about its regulation or interacting partners. Here, we show that the CBM20 of STBD1 is crucial for its stability and ability to interact with glycogen-associated proteins. Mutation of a conserved tryptophan residue (W293) in this domain abolished the ability of STBD1 to bind to the carbohydrate amylose. Compared with the WT (wild-type) protein, this mutant exhibited rapid degradation that was rescued upon inhibition of the proteasome. Furthermore, STBD1 undergoes ubiquitination when expressed in COS cells, and requires the N-terminus for this process. In contrast, inhibition of autophagy did not significantly affect protein stability. In overexpression experiments, we discovered that STBD1 interacts with several glycogen-associated proteins, such as GS (glycogen synthase), GDE (glycogen debranching enzyme) and Laforin. Importantly, the W293 mutant of STBD1 was unable to do so, suggesting an additional role for the CBM20 domain in protein–protein interactions. In HepG2 hepatoma cells, overexpressed STBD1 could associate with endogenous GS. This binding increased during glycogenolysis, suggesting that glycogen is not required to bridge this interaction. Taken together, our results have uncovered new insights into the regulation and binding partners of STBD1.     

Nonpeptide HIV protease inhibitors designed to replace a bound water

Cyclic nonpeptide molecules were designed and synthesized with the goal of displacing the conserved flap-associated water of HIV-1 protease. Several such molecules were competitive inhibitors with micromolar inhibition constants, and their structure-activity relationships were consistent with the design hypothesis.     

Fractionation of soluble proteins in Escherichia coli using DEAE-, SP-, and phenyl sepharose chromatographies.

In an effort to simplify a complex mixture of soluble proteins from Escherichia coli, methods to fractionate the samples prior to two-dimensional (2D) gel electrophoresis were developed. These methods involve the use of DEAE-Sepharose, SP-Sepharose, and phenyl Sepharose chromatographic columns and the fractionation of the protein mixtures based on differential anionic, cationic, and hydrophobic properties of the proteins, respectively. Fractionation of the soluble proteins from an E. coli extract with DEAE-Sepharose resulted in a threefold increase in the number of detectable 2D gel spots. These gel spots were amenable to protein identification by using in-gel trypsin digestions, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and peptide mass fingerprinting. Significantly, the DEAE-Sepharose column fractionation effectively partitioned the soluble proteins from the cell extracts. Similarly, an SP-Sepharose column was used to fractionate the soluble proteins from E. coli and resulted in over a twofold increase in the number of detectable gel spots. Lastly, fractionation of the cell extract with the phenyl Sepharose column resulted in a threefold increase in the number of detectable 2D gel spots. This work describes an easy, inexpensive way to fractionate the soluble proteins in E. coli and a way to better profile the E. coli proteome.     

Composition, Properties and Localisation of Pectins in Young and Mature Cells of the Mung Bean Hypocotyl

Two pectic fractions were extracted along the growth gradientof the mung bean hypocotyl. The first one (PF₁) contained pectinssoluble in boiling water and characterized by low uronic acids/cationsratio, high esterification degree and high neutral sugars/acidicsugars ratio. The second one (PF₂) contained pectins insolublein boiling water characterized by high uronic acids/cationsratio, low esterification degree, very low neutral sugar contentand a high selectivity for calcium. Water soluble pectins werepresent in all the wall area whereas the other ones were detectedmainly in the middle lamella. The PF₁/PF₂ ratio was high infast growing tissues but low in mature, slowly growing tissues.The development of the cell wall exchange properties along thegrowth gradient was related to the changes observed in the PF₁/PF₂balance. (Received July 31, 1985; Accepted January 20, 1986)     

In Situ and IAA-Induced Cell Elongation is Correlated to the Oleoyl Phosphatidylcholine Content along the Vigna radiata Hypocotyl

Successive segments of mung bean hypocotyls (Vigna radiata)show fatty acid changes according to the lengths of their cells.Only the accumulation of oleic acid was related to the growthprocess; its level was markedly increased all along the hypocotyl. When segments were treated with IAA in vitro, all that had incrementsin length also had increased oleic acid contents. These twofactors were highly correlated. Phosphatidylcholine, and toa less extent phosphatidylethanolamine, were the two phospholipidsinvolved in this phenomenon. Fusicoccin increased cell length but made little, if any, changein the fatty acid content. These results have been interpreted in terms of membrane synthesisand transfer of the membrane from the endoplasmic reticulumtoward the plasmalemma and tonoplast. (Received July 19, 1982; Accepted January 20, 1983)