Synthesis and screening of a CaaL peptide library versus FTase reveals a surprising number of substrates

Krzysiak, Amanda J.; Aditya, Animesh; Hougland, James L.; Fierke, Carol A.; Gibbs, Richard A.

doi:10.1016/j.bmcl.2009.11.011

Cited by 29 publications

(29 citation statements)

References 23 publications

(36 reference statements)

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“…In this study, sequences for which product conversion was detected by HPLC were labeled as ‘true’ substrates, while sequences for which no conversion was detected were labeled as ‘false’ substrates [23]. Using the threshold of -0.4 results in predictions with 87.5% TPR and 17.5% FPR, consistent with the performance on other peptide libraries (Figure 2C).…”

Section: Resultssupporting

confidence: 68%

Identification of a Novel Class of Farnesylation Targets by Structure-Based Modeling of Binding Specificity

London¹,

Lamphear

Hougland

et al. 2011

PLoS Comput Biol

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Farnesylation is an important post-translational modification catalyzed by farnesyltransferase (FTase). Until recently it was believed that a C-terminal CaaX motif is required for farnesylation, but recent experiments have revealed larger substrate diversity. In this study, we propose a general structural modeling scheme to account for peptide binding specificity and recapitulate the experimentally derived selectivity profile of FTase in vitro. In addition to highly accurate recovery of known FTase targets, we also identify a range of novel potential targets in the human genome, including a new substrate class with an acidic C-terminal residue (CxxD/E). In vitro experiments verified farnesylation of 26/29 tested peptides, including both novel human targets, as well as peptides predicted to tightly bind FTase. This study extends the putative range of biological farnesylation substrates. Moreover, it suggests that the ability of a peptide to bind FTase is a main determinant for the farnesylation reaction. Finally, simple adaptation of our approach can contribute to more accurate and complete elucidation of peptide-mediated interactions and modifications in the cell.

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Section: Resultssupporting

confidence: 68%

Identification of a Novel Class of Farnesylation Targets by Structure-Based Modeling of Binding Specificity

London¹,

Lamphear

Hougland

et al. 2011

PLoS Comput Biol

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“…Of course, several other proteins can become isoprenylated, for example, Rheb proteins, nuclear lamins, Rac and Cdc42. 12,13 Finally, we investigated whether the observed findings also hold true for primary human AML cells. Heterogeneity in the cytotoxic effects of simvastatin, as well as differences in the degree of rescue by mevalonate, GGPP and FPP, were found in primary AML cells (Supplementary Table S1).…”

Section: Letters To the Editormentioning

confidence: 99%

Heterogeneity in simvastatin-induced cytotoxicity in AML is caused by differences in Ras-isoprenylation

et al. 2011

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polymerase in the process of PCR amplification, releasing the reporter dyes. Following PCR, plates were read using the 7900HT Fast Real-Time PCR system, and the data were analyzed using Allele Discrimination software (Applied Biosystems).The median age at multiple myeloma diagnosis was 66 years and the median year of diagnosis was 1992 (Table 1). For multiple myeloma patients who subsequently developed MDS, in accord with prior studies, 9 the median time between the two diagnoses was 3.9 years (range: 1.8-14.3). When we assessed the results from the genotyping, we found 4 out of 15 (27%) of the multiple myeloma patients who developed MDS had the G/G genotype, whereas only 2 out of 17 (12%) of the patients who did not develop MDS showed the G/G genotype. The G/T genotype was found in 47% of both groups; multiple myeloma patients who did not develop MDS had a higher fraction of T/T genotype (41% vs 27%).Our novel findings support a role for susceptibility genes in the development of second malignancies following the diagnosis of multiple myeloma, likely in combination with multiple myeloma therapies. Future studies are needed to replicate and expand on our observations, with the overall goal of characterizing the role for treatment-and nontreatmentrelated factors underlying the development of secondary malignancies subsequent to multiple myeloma. Conflict of interestThe authors declare no conflict of interest. AcknowledgementsThe study was approved by the Mayo Clinic Institutional Review Board; informed consent was obtained from all participants in this study. An exemption from institutional review board review was obtained from the National Institutes of Health Office of Human Subjects Research because we used anonymous data/samples. Letters to the Editor 845Leukemia susceptible to the cytotoxic actions of these drugs.1 Therefore, interfering with the cholesterol metabolism by using statins to suppress cholesterol synthesis has been proposed as a method to improve antileukemic treatment strategies.3 Statins (for example, simvastatin) are widely used plasma cholesterol-lowering drugs that inhibit cholesterol synthesis at the level of HMG-CoA reductase (HMG-CoAR) by blocking the conversion of HMG-CoA to mevalonate. Mevalonate is also the precursor of isoprenoids such as farnesyldiphosphate (FPP) and geranylgeranyldiphosphate (GGPP). Isoprenylation, catalyzed by farnesyltransferase or geranylgeranyltransferase, is required for the attachment of small GTPases (for example, Rho, Ras) to the plasma membrane and their subsequent participation in signal transduction pathways that regulate growth and survival. Statins induce cell death in various human AML cell lines, as well as in primary human AML cells in vitro.1,4 Different mechanisms of action have been proposed for simvastatinmediated cell death, including the direct consequences of suppressed cholesterol synthesis and inhibition of isoprenylation and thereby the actions of signaling molecules such as Ras. 1,5 Ras is frequently activated in AML cells by mutatio...

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“…Using this approach, Krzysiak and colleagues demonstrated that FTase readily catalyzes farnesylation of a large number of peptides terminating in Leu (-CaaL), contrary to the CaaX paradigm which describes these as canonical GGTase-I substrate sequences [4]. This result expands the pool of FTase substrates and demonstrates additional cross-reactivity with GGTase-I.…”

Section: Substrate Identificationmentioning

confidence: 99%

Recent advances in protein prenyltransferases: substrate identification, regulation, and disease interventions

Zverina

Lamphear

Wright

et al. 2012

Current Opinion in Chemical Biology

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Protein post-translational modifications increase the functional diversity of the proteome by covalently adding chemical moieties onto proteins thereby changing their activation state, cellular localization, interacting partners, and life cycle. Lipidation is one such modification that enables membrane association of naturally cytosolic proteins. Protein prenyltransferases irreversibly install isoprenoid units of varying length via a thioether linkage onto proteins that exert their cellular activity at membranes. Substrates of prenyltransferases are involved in countless signaling pathways and processes within the cell. Identification of new prenylation substrates, prenylation pathway regulators, and dynamic trafficking of prenylated proteins are all avenues of intense, ongoing research that are challenging, exciting, and have the potential to significantly advance the field in the near future.

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Synthesis and screening of a CaaL peptide library versus FTase reveals a surprising number of substrates

Cited by 29 publications

References 23 publications

Identification of a Novel Class of Farnesylation Targets by Structure-Based Modeling of Binding Specificity

Identification of a Novel Class of Farnesylation Targets by Structure-Based Modeling of Binding Specificity

Heterogeneity in simvastatin-induced cytotoxicity in AML is caused by differences in Ras-isoprenylation

Recent advances in protein prenyltransferases: substrate identification, regulation, and disease interventions

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