A single evolutionarily divergent mutation determines the different FAD‐binding affinities of human and rat NQO1 due to site‐specific phosphorylation

Two new types of covalently linked, site-specific immunoprobes have been prepared using metal cluster labels, and used to stain components of cells. Combined fluorescein and 1.4 nm “Nanogold” labels were prepared by using the fluorescein-conjugated tris (aryl) phosphine ligand and the amino-substituted ligand in the synthesis of the Nanogold cluster. This cluster label was activated by reaction with a 60-fold excess of (sulfo-Succinimidyl-4-N-maleiniido-cyclohexane-l-carboxylate (sulfo-SMCC) at pH 7.5, separated from excess cross-linking reagent by gel filtration, and mixed in ten-fold excess with Goat Fab’ fragments against mouse IgG (obtained by reduction of F(ab’)2 fragments with 50 mM mercaptoethylamine hydrochloride). Labeled Fab’ fragments were isolated by gel filtration HPLC (Superose-12, Pharmacia). A combined Nanogold and Texas Red label was also prepared, using a Nanogold cluster derivatized with both and its protected analog: the cluster was reacted with an eight-fold excess of Texas Red sulfonyl chloride at pH 9.0, separated from excess Texas Red by gel filtration, then deprotected with HC1 in methanol to yield the amino-substituted label.

Download Full-text

The challenge of detecting modifications on proteins

Essays in Biochemistry ◽

10.1042/ebc20190055 ◽

2020 ◽

Vol 64 (1) ◽

pp. 135-153 ◽

Cited By ~ 1

Author(s):

Lauren Elizabeth Smith ◽

Adelina Rogowska-Wrzesinska

Keyword(s):

Mass Spectrometry ◽

Protein Function ◽

Chemical Properties ◽

Lysine Acetylation ◽

Mass Determination ◽

Post Translational Modifications ◽

Site Specific ◽

The Common

Abstract Post-translational modifications (PTMs) are integral to the regulation of protein function, characterising their role in this process is vital to understanding how cells work in both healthy and diseased states. Mass spectrometry (MS) facilitates the mass determination and sequencing of peptides, and thereby also the detection of site-specific PTMs. However, numerous challenges in this field continue to persist. The diverse chemical properties, low abundance, labile nature and instability of many PTMs, in combination with the more practical issues of compatibility with MS and bioinformatics challenges, contribute to the arduous nature of their analysis. In this review, we present an overview of the established MS-based approaches for analysing PTMs and the common complications associated with their investigation, including examples of specific challenges focusing on phosphorylation, lysine acetylation and redox modifications.

Download Full-text

Specificity determinants of phosphoprotein phosphatases controlling kinetochore functions

Essays in Biochemistry ◽

10.1042/ebc20190065 ◽

2020 ◽

Vol 64 (2) ◽

pp. 325-336 ◽

Cited By ~ 1

Author(s):

Dimitriya H. Garvanska ◽

Jakob Nilsson

Keyword(s):

Spindle Assembly Checkpoint ◽

Activity Level ◽

Phosphorylation Sites ◽

Binding Affinities ◽

Mitotic Kinases ◽

Anaphase Onset ◽

Phosphoprotein Phosphatases ◽

Linear Motifs ◽

Temporal And Spatial ◽

Kinetochore Function

Abstract Kinetochores are instrumental for accurate chromosome segregation by binding to microtubules in order to move chromosomes and by delaying anaphase onset through the spindle assembly checkpoint (SAC). Dynamic phosphorylation of kinetochore components is key to control these activities and is tightly regulated by temporal and spatial recruitment of kinases and phosphoprotein phosphatases (PPPs). Here we focus on PP1, PP2A-B56 and PP2A-B55, three PPPs that are important regulators of mitosis. Despite the fact that these PPPs share a very similar active site, they target unique ser/thr phosphorylation sites to control kinetochore function. Specificity is in part achieved by PPPs binding to short linear motifs (SLiMs) that guide their substrate specificity. SLiMs bind to conserved pockets on PPPs and are degenerate in nature, giving rise to a range of binding affinities. These SLiMs control the assembly of numerous substrate specifying complexes and their position and binding strength allow PPPs to target specific phosphorylation sites. In addition, the activity of PPPs is regulated by mitotic kinases and inhibitors, either directly at the activity level or through affecting PPP–SLiM interactions. Here, we discuss recent progress in understanding the regulation of PPP specificity and activity and how this controls kinetochore biology.

Download Full-text