Biology subjects

Schmidt, W. K.

Publications and source records attributed to Schmidt, W. K..

5 recordsLinked to original sources

bioRxiv2024

Comprehensive analysis of CXXX sequence space reveals that S. cerevisiae GGTase-I mainly relies on a2X substrate determinants

Many proteins undergo a post-translational lipid attachment, which increases their hydrophobicity, thus strengthening their membrane association properties or aiding in protein interactions. Geranylgeranyltransferase-I (GGTase-I) is an enzyme involved in a three-step post-translational modification (PTM) pathway that attaches a 20-carbon lipid group called geranylgeranyl at the carboxy-terminal cysteine of proteins ending in a canonical CaaL motif (C - cysteine, a - aliphatic, L - often leucine, but can be phenylalanine, isoleucine, methionine, or valine). Genetic approaches involving two distinct reporters were employed in this study to assess S. cerevisiae GGTase-I specificity, for which limited data exists, towards all 8000 CXXX combinations. Orthogonal biochemical analyses and structure-based alignments were also performed to better understand the features required for optimal target interaction. These approaches indicate that yeast GGTase-I best modifies the Cxa[L/F/I/M/V] sequence that resembles but is not an exact match for the canonical CaaL motif. We also observed that minor modification of non-canonical sequences is possible. A consistent feature associated with well-modified sequences was the presence of a non-polar a2 residue and a hydrophobic terminal residue, which are features recognized by mammalian GGTase-I. These results thus support that mammalian and yeast GGTase-I exhibit considerable shared specificity. Article SummaryThis work investigates yeast GGTase-I specificity through genetics, high throughput sequencing, and two distinct reporter systems. This approach allows for comprehensive evaluation of all CXXX sequence space, which has not been possible with earlier approaches. We identified CXXX sequences supporting geranylgeranylation that differ from the historically defined CaaL sequence often cited in the literature as the GGTase-I target motif, and our results indicate that the last two amino acids of the target motif largely dictate GGTase-I specificity.

genetics↗

bioRxiv2023

A Humanized Yeast System for Evaluating the Protein Prenylation of a Wide Range of Human and Viral CaaX Sequences

The C-terminal CaaX sequence (cysteine-aliphatic-aliphatic-any of several amino acids) is subject to isoprenylation on the conserved cysteine and is estimated to occur in 1-2% of proteins within yeast and human proteomes. Recently, non-canonical CaaX sequences in addition to shorter and longer length CaX and CaaaX sequences have been identified that can be prenylated. Much of the characterization of prenyltransferases has relied on the yeast system because of its genetic tractability and availability of reporter proteins, such as the a-factor mating pheromone, Ras GTPase, and Ydj1 Hsp40 chaperone. To compare the properties of yeast and human prenyltransferases, including the recently expanded target specificity of yeast farnesyltransferase, we have developed yeast strains that express human farnesyltransferase or geranylgeranyltransferase-I in lieu of their yeast counterparts. The humanized yeast strains display robust prenyltransferase activity that functionally replaces yeast prenyltransferase activity in a wide array of tests, including the prenylation of a wide variety of canonical and non-canonical human CaaX sequences, virus encoded CaaX sequences, non-canonical length sequences, and heterologously expressed human proteins HRas and DNAJA2. These results reveal highly overlapping substrate specificity for yeast and human farnesyltransferase, and mostly overlapping substrate specificity for GGTase-I. This yeast system is a valuable tool for further defining the prenylome of humans and other organisms, identifying proteins for which prenylation status has not yet been determined. Summary StatementWe report yeast engineered to express human prenylation enzymes with which prenylation can be investigated for established and novel CaaX sequences associated with proteins involved in human disease.

biochemistry↗

bioRxiv2023

Yeast Farnesyltransferase has broad reactivity with CXXX sequences

The current understanding of farnesyltransferase (FTase) specificity was pioneered through investigations of reporters like Ras and Ras-related proteins that possess a C-terminal CaaX motif that consists of 4 amino acid residues: Cysteine - aliphatic1 - aliphatic2 - variable (X). These studies led to the finding that proteins with the CaaX motif are subject to a 3-step post-translational modification pathway involving farnesylation, proteolysis, and carboxylmethylation. Emerging evidence indicates, however, that FTase can farnesylate sequences outside the CaaX motif and that these sequences do not undergo the canonical 3-step pathway. In this work, we report a comprehensive evaluation of all possible CXXX sequences as FTase targets using the reporter Ydj1, an Hsp40 chaperone that only requires farnesylation for its activity. Our genetic and high throughput sequencing approach reveals an unprecedented profile of sequences that yeast FTase can recognize in vivo, which effectively expands the potential target space of FTase within the yeast proteome. We also document that yeast FTase specificity is majorly influenced by restrictive amino acids at a2 and X positions as opposed to the resemblance of CaaX motif as previously regarded. This first complete evaluation of CXXX space expands the complexity of protein isoprenylation and marks a key step forward in understanding the potential scope of targets for this isoprenylation pathway.

genetics↗

bioRxiv2021

Functional classification and validation of yeast prenylation motifs using machine learning and genetic reporters

Protein prenylation by farnesyltransferase (FTase) is often described as the targeting of a cysteine-containing motif (CaaX) that is enriched for aliphatic amino acids at the a1 and a2 positions, while quite flexible at the X position. Prenylation prediction methods often rely on these features despite emerging evidence that FTase has broader target specificity than previously considered. Using a machine learning approach and training sets based on canonical (prenylated, proteolyzed, and carboxymethylated) and recently identified shunted motifs (prenylation only), this study aims to improve prenylation predictions with the goal of determining the full scope of prenylation potential among the 8000 possible Cxxx sequence combinations. Further, this study aims to subdivide the prenylated sequences as either shunted (i.e., uncleaved) or cleaved (i.e., canonical). Predictions were determined for Saccharomyces cerevisiae FTase and compared to results derived using currently available prenylation prediction methods. In silico predictions were further evaluated using in vivo methods coupled to two yeast reporters, the yeast mating pheromone a-factor and Hsp40 Ydj1p, that represent proteins with canonical and shunted CaaX motifs, respectively. Our machine learning based approach expands the repertoire of predicted FTase targets and provides a framework for functional classification.

biochemistry↗

bioRxiv2018

NRSF-mediated repression of neuronal genes in developing brain persists in the absence of NRSF-Sin3 interaction

Repression of target genes by the transcriptional repressor neuronal restrictive silencing factor (NRSF)/repressor element 1 silencing transcription factor (REST) contributes to enduring plasticity in the developing brain. However, the cofactor(s) interacting with NRSF to enable target gene repressor are not well understood, and may vary among neuronal populations and brain regions as well as with different contexts. Here we employed the novel designer drug mS-11 to block the interactions of the cofactor Sin3 with NRSF. We tested if NRSF-Sin3 interaction is required for repression of NRSF target genes in developing hypothalamus after activity-dependent modulation of NRSF function. In the hypothalamus in vitro, blocking glutamatergic neurotransmission robustly increased NRSF binding to the target gene Crh, resulting in its repression. Blocking the binding of NRSF to the chromatin with decoy NRSE-oligodeoxynucleotides abrogated this repression. In contrast, mS-11 at several concentrations did not impede Crh repression. NRSF-mediated repression may underlie disease processes such as the onset of epilepsy. Therefore, identifying small-molecule antagonists of NRSF is crucial for the development of disease-preventing or modifying interventions.

neuroscience↗