Nonspecific phospholipase C4 hydrolyzes phosphosphingolipids and sustains plant root growth during phosphate deficiency

Phosphate is a vital macronutrient for plant growth, and its availability in soil is critical for agricultural sustainability and productivity. A substantial amount of cellular phosphate is used to synthesize phospholipids for cell membranes. Here, we identify a key enzyme, nonspecific phospholipase C4 (NPC4) that is involved in phosphosphingolipid hydrolysis and remodeling in Arabidopsis during phosphate starvation. The level of glycosylinositolphosphorylceramide (GIPC), the most abundant sphingolipid in Arabidopsis thaliana, decreased upon phosphate starvation. NPC4 was highly induced by phosphate deficiency, and NPC4 knockouts in Arabidopsis decreased the loss of GIPC and impeded root growth during phosphate starvation. Enzymatic analysis showed that NPC4 hydrolyzed GIPC and displayed a higher activity toward GIPC as a substrate than toward the common glycerophospholipid phosphatidylcholine. NPC4 was associated with the plasma membrane lipid rafts in which GIPC is highly enriched. These results indicate that NPC4 uses GIPC as a substrate in planta and the NPC4-mediated sphingolipid remodeling plays a positive role in root growth in Arabidopsis response to phosphate deficiency.

Nonspecific phospholipase C4 (NPC4), which is induced by phosphate deficiency, hydrolyzes the common phosphosphingolipid glycosylinositolphosphorylceramide and mediates sphingolipid remodeling that supports root growth in Arabidopsis response to phosphate deficiency.     

DHHC2 is a protein S-acyltransferase for Lck

Abstract

Lck is a non-receptor tyrosine kinase of the Src family that is essential for T cell activation. Dual N-terminal acylation of Lck with myristate (N-acylation) and palmitate (S-acylation) is essential for its membrane association and function. Reversible S-acylation of Lck is observed in vivo and may function as a control mechanism. Here we identify the DHHC family protein S-acyltransferase DHHC2 as an enzyme capable of palmitoylating of Lck in T cells. Reducing the DHHC2 level in Jurkat T cells using siRNA causes decreased Lck S-acylation and partial dislocation from membranes, and conversely overexpression of DHHC2 increases S-acylation of an Lck surrogate, LckN10-GFP. DHHC2 localizes primarily to the endoplasmic reticulum and Golgi apparatus suggesting that it is involved in S-acylation of newly-synthesized or recycling Lck involved in T cell signalling.     

Identification of acylation products in SHAPE chemistry

SHAPE chemistry (selective 2′-hydroxyl acylation analyzed by primer extension) has been developed to specifically target flexible nucleotides (often unpaired nucleotides) independently to their purine or pyrimidine nature for RNA secondary structure determination. However, to the best of our knowledge, the structure of 2′-O-acylation products has never been confirmed by NMR or X-ray data. We have realized the acylation reactions between cNMP and NMIA under SHAPE chemistry conditions and identified the acylation products using standard NMR spectroscopy and LC–MS/MS experiments. For cAMP and cGMP, the major acylation product is the 2′-O-acylated compound (>99%). A trace amount of N-acylated cAMP has also been identified by LC–UV–MS². While for cCMP, the isolated acylation products are composed of 96% of 2′-O-acylated, 4% of N,O-diacylated, and trace amount of N-acylated compounds. In addition, the characterization of the major 2′-O-acylated compound by NMR showed slight differences in the conformation of the acylated sugar between the three cyclic nucleotides. This interesting result should be useful to explain some unexpected reactivity of the SHAPE chemistry.     

Non‐specific phospholipase C5 and diacylglycerol promote lateral root development under mild salt stress in Arabidopsis

Developing a robust root system is crucial to plant survival and competition for soil resources. Here we report that the non‐specific phospholipase C5 (NPC5) and its derived lipid mediator diacylglycerol (DAG) mediate lateral root (LR) development during salt stress in Arabidopsis thaliana. T‐DNA knockout mutant npc5‐1 produced few to no LR under mild NaCl stress, whereas overexpression of NPC5 increased LR number. Roots of npc5‐1 contained a lower level of DAG than wild type, whereas NPC5 overexpressor exhibited an increase in DAG level. Application of DAG, but not phosphatidic acid, fully restored LR growth of npc5‐1 to that of wild type under NaCl stress. NPC5 expression was significantly induced in Arabidopsis seedlings treated with NaCl. Npc5‐1 was less responsive to auxin‐mediated root growth than the wild type. These results indicate that NPC5 mediates LR development in response to salt stress and suggest that DAG functions as a lipid mediator in the stress signalling.     

The Golgi S-acylation machinery comprises zDHHC enzymes with major differences in substrate affinity and S-acylation activity

S-acylation, the attachment of fatty acids onto cysteine residues, regulates protein trafficking and function and is mediated by a family of zDHHC enzymes. The S-acylation of peripheral membrane proteins has been proposed to occur at the Golgi, catalyzed by an S-acylation machinery that displays little substrate specificity. To advance understanding of how S-acylation of peripheral membrane proteins is handled by Golgi zDHHC enzymes, we investigated interactions between a subset of four Golgi zDHHC enzymes and two S-acylated proteins—synaptosomal-associated protein 25 (SNAP25) and cysteine-string protein (CSP). Our results uncover major differences in substrate recognition and S-acylation by these zDHHC enzymes. The ankyrin-repeat domains of zDHHC17 and zDHHC13 mediated strong and selective interactions with SNAP25/CSP, whereas binding of zDHHC3 and zDHHC7 to these proteins was barely detectable. Despite this, zDHHC3/zDHHC7 could S-acylate SNAP25/CSP more efficiently than zDHHC17, whereas zDHHC13 lacked S-acylation activity toward these proteins. Overall the results of this study support a model in which dynamic intracellular localization of peripheral membrane proteins is achieved by highly selective recruitment by a subset of zDHHC enzymes at the Golgi, combined with highly efficient S-acylation by other Golgi zDHHC enzymes.     

Different effects of phospholipase Dζ2 and non‐specific phospholipase C4 on lipid remodeling and root hair growth in Arabidopsis response to phosphate deficiency

Phosphate (Pi) deficiency in soils is a major limiting factor for plant growth. In response to Pi deprivation, one prominent metabolic adaptation in plants is the decrease in membrane phospholipids that consume approximately one‐third cellular Pi. The level of two phospholipid‐hydrolyzing enzymes, phospholipase Dζ2 (PLDζ2) and non‐specific phospholipase C4 (NPC4), is highly induced in Pi‐deprived Arabidopsis. To determine the role of PLDζ2 and NPC4 in plant growth under Pi limitation, Arabidopsis plants deficient in both PLDζ2 and NPC4 (npc4pldζ2) were generated and characterized. Lipid remodeling in leaves and roots was analyzed at three different durations of Pi deficiency. NPC4 affected lipid changes mainly in roots at an early stage of Pi deprivation, whereas PLDζ2 exhibited a more overt effect on lipid remodeling in leaves at a later stage of Pi deprivation. Pi deficiency‐induced galactolipid increase and phospholipid decrease were impeded in pldζ2 and npc4pldζ2 plants. In addition, seedlings of npc4pldζ2 had the same root hair density as pldζ2 but shorter root hair length than pldζ2 in response to Pi deficiency. The loss of NPC4 decreased root hair length but had no effect on root hair density. These data suggest that PLDζ2 and NPC4 mediate the Pi deprivation‐induced lipid remodeling in a tissue‐ and time‐specific manner. PLDζ2 and NPC4 have distinctively different roles in root hair growth and development in response to Pi deprivation; PLDζ2 negatively modulates root hair density and length, whereas NPC4 promotes root hair elongation.     

The cholesterol trafficking protein NPC1 is required for Drosophila spermatogenesis

Niemann–Pick C (NPC) disease is a lethal neurodegenerative disorder affecting cellular sterol trafficking. Besides neurodegeneration, NPC patients also exhibit other pleiotropic conditions, indicating that NPC protein is required for other physiological processes. Previous studies indicated that a sterol shortage that in turn leads to a shortage of steroid hormones (for example, ecdysone in Drosophila) is likely to be the cause of NPC disease pathology. We have shown that mutations in Drosophila npc1, one of the two NPC disease-related genes, leads to larval lethal and male infertility. Here, we reported that npc1 mutants are defective in spermatogenesis and in particular in the membrane-remodeling individualization process. Interestingly, we found that ecdysone, the steroid hormone responsible for the larval lethal phenotype in npc1 mutants, is not required for individualization. However, supplying 7-dehydrocholesterol can partially rescue the male infertility of npc1 mutants, suggesting that a sterol shortage is responsible for the spermatogenesis defects. In addition, the individualization defects of npc1 mutants were enhanced at high temperature, suggesting that the sterol shortage may lead to temperature-sensitive defects in the membrane-remodeling process. Together, our study reveals a sterol-dependent, ecdysone-independent mechanism of NPC1 function in Drosophila spermatogenesis.     

S-Nitrosylation Inhibits Pannexin 1 Channel Function

S-Nitrosylation is a post-translational modification on cysteine(s) that can regulate protein function, and pannexin 1 (Panx1) channels are present in the vasculature, a tissue rich in nitric oxide (NO) species. Therefore, we investigated whether Panx1 can be S-nitrosylated and whether this modification can affect channel activity. Using the biotin switch assay, we found that application of the NO donor S-nitrosoglutathione (GSNO) or diethylammonium (Z)-1–1(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA NONOate) to human embryonic kidney (HEK) 293T cells expressing wild type (WT) Panx1 and mouse aortic endothelial cells induced Panx1 S-nitrosylation. Functionally, GSNO and DEA NONOate attenuated Panx1 currents; consistent with a role for S-nitrosylation, current inhibition was reversed by the reducing agent dithiothreitol and unaffected by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a blocker of guanylate cyclase activity. In addition, ATP release was significantly inhibited by treatment with both NO donors. To identify which cysteine residue(s) was S-nitrosylated, we made single cysteine-to-alanine substitutions in Panx1 (Panx1^C40A, Panx1^C346A, and Panx1^C426A). Mutation of these single cysteines did not prevent Panx1 S-nitrosylation; however, mutation of either Cys-40 or Cys-346 prevented Panx1 current inhibition and ATP release by GSNO. This observation suggested that multiple cysteines may be S-nitrosylated to regulate Panx1 channel function. Indeed, we found that mutation of both Cys-40 and Cys-346 (Panx1^C40A/C346A) prevented Panx1 S-nitrosylation by GSNO as well as the GSNO-mediated inhibition of Panx1 current and ATP release. Taken together, these results indicate that S-nitrosylation of Panx1 at Cys-40 and Cys-346 inhibits Panx1 channel currents and ATP release.     

Quantitative role of LAL, NPC2, and NPC1 in lysosomal cholesterol processing defined by genetic and pharmacological manipulations

Lipoprotein cholesterol taken up by cells is processed in the endosomal/lysosomal (E/L) compartment by the sequential action of lysosomal acid lipase (LAL), Niemann-Pick C2 (NPC2), and Niemann-Pick C1 (NPC1). Inactivation of NPC2 in mouse caused sequestration of unesterified cholesterol (UC) and expanded the whole animal sterol pool from 2,305 to 4,337 mg/kg. However, this pool increased to 5,408 and 9,480 mg/kg, respectively, when NPC1 or LAL function was absent. The transport defect in mutants lacking NPC2 or NPC1, but not in those lacking LAL, was reversed by cyclodextrin (CD), and the ED₅₀ values for this reversal varied from ∼40 mg/kg in kidney to >20,000 mg/kg in brain in both groups. This reversal occurred only with a CD that could interact with UC. Further, a CD that could interact with, but not solubilize, UC still overcame the transport defect. These studies showed that processing and export of sterol from the late E/L compartment was quantitatively different in mice lacking LAL, NPC2, or NPC1 function. In both npc2^−/− and npc1^−/− mice, the transport defect was reversed by a CD that interacted with UC, likely at the membrane/bulk-water interface, allowing sterol to move rapidly to the export site of the E/L compartment.     

Arabidopsis PROTEIN S‐ACYL TRANSFERASE4 mediates root hair growth

Polar growth of root hairs is critical for plant survival and requires fine‐tuned Rho of plants (ROP) signaling. Multiple ROP regulators participate in root hair growth. However, protein S‐acyl transferases (PATs), mediating the S‐acylation and membrane partitioning of ROPs, are yet to be found. Using a reverse genetic approach, combining fluorescence probes, pharmacological drugs, site‐directed mutagenesis and genetic analysis with related root‐hair mutants, we have identified and characterized an Arabidopsis PAT, which may be responsible for ROP2 S‐acylation in root hairs. Specifically, functional loss of PAT4 resulted in reduced root hair elongation, which was rescued by a wild‐type but not an enzyme‐inactive PAT4. Membrane‐associated ROP2 was significantly reduced in pat4, similar to S‐acylation‐deficient ROP2 in the wild type. We further showed that PAT4 and SCN1, a ROP regulator, additively mediate the stability and targeting of ROP2. The results presented here indicate that PAT4‐mediated S‐acylation mediates the membrane association of ROP2 at the root hair apex and provide novel insights into dynamic ROP signaling during plant tip growth.     

cDNA cloning and characterization of human and mouse Ca-independent phosphatidylethanolamine N-acyltransferases

The formation of N-acylphosphatidylethanolamine by N-acylation of phosphatidylethanolamine (PE) is the initial step in the biosynthetic pathway of bioactive N-acylethanolamines, including the endocannabinoid anandamide and the anti-inflammatory substance N-palmitoylethanolamine. We recently cloned a rat enzyme capable of catalyzing this reaction, and referred to the enzyme as Ca²⁺-independent N-acyltransferase (iNAT). Here we report cDNA cloning and characterization of human and mouse iNATs. We cloned iNAT-homologous cDNAs from human and mouse testes, and overexpressed them in COS-7 cells. The purified recombinant proteins abstracted an acyl group from both sn-1 and sn-2 positions of phosphatidylcholine, and catalyzed N-acylation of PE as well as phospholipase A₁/A₂-like hydrolysis. The iNAT activity was mainly detected in soluble rather than particulate fractions, and was only slightly increased by Ca²⁺. These results demonstrated that the human and mouse homologues function as iNAT. As for the organ distribution of iNAT, human testis and pancreas and mouse testis exhibited by far the highest expression level, suggesting its physiological importance in the specific organs. Moreover, mutagenesis studies showed crucial roles of His-154 and Cys-241 of rat iNAT in the catalysis and a possible role of the N-terminal domain in membrane association or protein–protein interaction.     

Molecular Basis of Regulating High Voltage-Activated Calcium Channels by S-Nitrosylation

Nitric oxide (NO) is involved in a variety of physiological processes, such as vasoregulation and neurotransmission, and has a complex role in the regulation of pain transduction and synaptic transmission. We have shown previously that NO inhibits high voltage-activated Ca²⁺ channels in primary sensory neurons and excitatory synaptic transmission in the spinal dorsal horn. However, the molecular mechanism involved in this inhibitory action remains unclear. In this study, we investigated the role of S-nitrosylation in the NO regulation of high voltage-activated Ca²⁺ channels. The NO donor S-nitroso-N-acetyl-dl-penicillamine (SNAP) rapidly reduced N-type currents when Cav2.2 was coexpressed with the Cavβ1 or Cavβ3 subunits in HEK293 cells. In contrast, SNAP only slightly inhibited P/Q-type and L-type currents reconstituted with various Cavβ subunits. SNAP caused a depolarizing shift in voltage-dependent N-type channel activation, but it had no effect on Cav2.2 protein levels on the membrane surface. The inhibitory effect of SNAP on N-type currents was blocked by the sulfhydryl-specific modifying reagent methanethiosulfonate ethylammonium. Furthermore, the consensus motifs of S-nitrosylation were much more abundant in Cav2.2 than in Cav1.2 and Cav2.1. Site-directed mutagenesis studies showed that Cys-805, Cys-930, and Cys-1045 in the II-III intracellular loop, Cys-1835 and Cys-2145 in the C terminus of Cav2.2, and Cys-346 in the Cavβ3 subunit were nitrosylation sites mediating NO sensitivity of N-type channels. Our findings demonstrate that the consensus motifs of S-nitrosylation in cytoplasmically accessible sites are critically involved in post-translational regulation of N-type Ca²⁺ channels by NO. S-Nitrosylation mediates the feedback regulation of N-type channels by NO.     

GLUT1CBP(TIP2/GIPC1) interactions with GLUT1 and myosin VI: evidence supporting an adapter function for GLUT1CBP

We identified a novel interaction between myosin VI and the GLUT1 transporter binding protein GLUT1CBP(GIPC1) and first proposed that as an adapter molecule it might function to couple vesicle-bound proteins to myosin VI movement. This study refines the model by identifying two myosin VI binding domains in the GIPC1 C terminus, assigning respective oligomerization and myosin VI binding functions to separate N- and C-terminal domains, and defining a central region in the myosin VI tail that binds GIPC1. Data further supporting the model demonstrate that 1) myosin VI and GIPC1 interactions do not require a mediating protein; 2) the myosin VI binding domain in GIPC1 is necessary for intracellular interactions of GIPC1 with myosin VI and recruitment of overexpressed myosin VI to membrane structures, but not for the association of GIPC1 with such structures; 3) GIPC1/myosin VI complexes coordinately move within cellular extensions of the cell in an actin-dependent and microtubule-independent manner; and 4) blocking either GIPC1 interactions with myosin VI or GLUT1 interactions with GIPC1 disrupts normal GLUT1 trafficking in polarized epithelial cells, leading to a reduction in the level of GLUT1 in the plasma membrane and concomitant accumulation in internal membrane structures.     

Selective Acylation of Plasma Membrane Proteins of Mycoplasma agalactiae: The Causal Agent of Agalactia

Revealed by in vivo labeling with ¹⁴C-palmitic acid, about 15 acylated proteins were identified in the plasma membrane of Mycoplasma agalactiae (type strain PG2), including the major component p40. Triton X-114 phase partitioning and Western blotting demonstrated the amphiphilic properties of the acyl proteins and showed that they were also antigenic components. Chemical analyses of fatty acids bound to proteins revealed the following selectivity order within acylation: stearic acid (18:0) > linoleic acid (18:2c) ≈ palmitic acid (16:0) > oleic acid (18:1c) > myristic acid (14:0), with 16:0 and 18:1c preferred for the O-acylation and 18:0 for the N-acylation. The ratio [O-ester- + amide-bound acyl chains]/O-ester-linked chains being close to 1.4 as well as the presence of S-glycerylcysteine suggest that acyl proteins in M. agalactiae are true lipoproteins containing N-acyl diacyl glycerylcysteine, probably processed by a mechanism analogous to that described for Gram-negative eubacteria. Received: 22 June 1999 / Accepted: 2 August 1999     

The apple palmitoyltransferase MdPAT16 influences sugar content and salt tolerance via an MdCBL1–MdCIPK13–MdSUT2.2 pathway

Protein S-acyltransferases (PATs) are a category of eukaryotic transmembrane proteins that mediate the S-acylation of their target proteins. S-acylation, commonly known as palmitoylation, is a reversible protein modification that regulates the membrane association and function of target proteins. However, the functions and mechanisms of PATs in apple (Malus domestica) remain poorly understood. In this study, an MdPAT family member, MdPAT16, was identified and shown to have palmitoyltransferase activity. We demonstrated that this gene responds to salt stress and that its expression improves plant salt stress resistance. In addition, its overexpression significantly promotes the accumulation of soluble sugars. The same phenotypes were observed in transgenic tissue culture seedlings, transgenic roots, and Arabidopsis thaliana that ectopically expressed MdPAT16. MdPAT16 was shown to interact with MdCBL1 and stabilize MdCBL1 protein levels through palmitoylation. The N-terminal sequence of MdCBL1 contains a palmitoylation site, and its N-terminal deletion led to changes in MdCBL1 protein stability and subcellular localization. The phenotypes of MdCBL1 transgenic roots and transiently injected apple fruits were fully consistent with the sugar accumulation phenotype of MdPAT16. Mutation of the palmitoylation site interfered with this phenotype. These findings suggest that MdPAT16 palmitoylates its downstream target proteins, improving their stability. This may be a missing link in the plant salt stress response pathway and have an important impact on fruit quality.     

Multiple Surface Regions on the Niemann-Pick C2 Protein Facilitate Intracellular Cholesterol Transport

The cholesterol storage disorder Niemann-Pick type C (NPC) disease is caused by defects in either of two late endosomal/lysosomal proteins, NPC1 and NPC2. NPC2 is a 16-kDa soluble protein that binds cholesterol in a 1:1 stoichiometry and can transfer cholesterol between membranes by a mechanism that involves protein-membrane interactions. To examine the structural basis of NPC2 function in cholesterol trafficking, a series of point mutations were generated across the surface of the protein. Several NPC2 mutants exhibited deficient sterol transport properties in a set of fluorescence-based assays. Notably, these mutants were also unable to promote egress of accumulated intracellular cholesterol from npc2^−/− fibroblasts. The mutations mapped to several regions on the protein surface, suggesting that NPC2 can bind to more than one membrane simultaneously. Indeed, we have previously demonstrated that WT NPC2 promotes vesicle-vesicle interactions. These interactions were abrogated, however, by mutations causing defective sterol transfer properties. Molecular modeling shows that NPC2 is highly plastic, with several intense positively charged regions across the surface that could interact favorably with negatively charged membrane phospholipids. The point mutations generated in this study caused changes in NPC2 surface charge distribution with minimal conformational changes. The plasticity, coupled with membrane flexibility, probably allows for multiple cholesterol transfer routes. Thus, we hypothesize that, in part, NPC2 rapidly traffics cholesterol between closely appositioned membranes within the multilamellar interior of late endosomal/lysosomal proteins, ultimately effecting cholesterol egress from this compartment.     

The phosphatidylcholine-hydrolysing phospholipase C NPC4 plays a role in response of Arabidopsis roots to salt stress

Phosphatidylcholine-hydrolysing phospholipase C, also known as non-specific phospholipase C (NPC), is a new member of the plant phospholipase family that reacts to environmental stresses such as phosphate deficiency and aluminium toxicity, and has a role in root development and brassinolide signalling. Expression of NPC4, one of the six NPC genes in Arabidopsis, was highly induced by NaCl. Maximum expression was observed from 3?h to 6?h after the salt treatment and was dependent on salt concentration. Results of histochemical analysis of P(NPC4):GUS plants showed the localization of salt-induced expression in root tips. On the biochemical level, increased NPC enzyme activity, indicated by accumulation of diacylglycerol, was observed as early as after 30?min of salt treatment of Arabidopsis seedlings. Phenotype analysis of NPC4 knockout plants showed increased sensitivity to salinity as compared with wild-type plants. Under salt stress npc4 plants had shorter roots, lower fresh weight, and reduced seed germination. Expression levels of abscisic acid-related genes ABI1, ABI2, RAB18, PP2CA, and SOT12 were substantially reduced in salt-treated npc4 plants. These observations demonstrate a role for NPC4 in the response of Arabidopsis to salt stress.     

S-acylation regulates Kv1.5 channel surface expression

The number of ion channels expressed on the cell surface shapes the complex electrical response of excitable cells. An imbalance in the ratio of inward and outward conducting channels is unfavorable and often detrimental. For example, over- or underexpression of voltage-gated K⁺ (Kv) channels can be cytotoxic and in some cases lead to disease. In this study, we demonstrated a novel role for S-acylation in Kv1.5 cell surface expression. In transfected fibroblasts, biochemical evidence showed that Kv1.5 is posttranslationally modified on both the NH₂ and COOH termini via hydroxylamine-sensitive thioester bonds. Pharmacological inhibition of S-acylation, but not myristoylation, significantly decreased Kv1.5 expression and resulted in accumulation of channel protein in intracellular compartments and targeting for degradation. Channel protein degradation was rescued by treatment with proteasome inhibitors. Time course experiments revealed that S-acylation occurred in the biosynthetic pathway of nascent channel protein and showed that newly synthesized Kv1.5 protein, but not protein expressed on the cell surface, is sensitive to inhibitors of thioacylation. Sensitivity to inhibitors of S-acylation was governed by COOH-terminal, but not NH₂-terminal, cysteines. Surprisingly, although intracellular cysteines were required for S-acylation, mutation of these residues resulted in an increase in Kv1.5 cell surface channel expression, suggesting that screening of free cysteines by fatty acylation is an important regulatory step in the quality control pathway. Together, these results show that S-acylation can regulate steady-state expression of Kv1.5. quality control; potassium; channels; palmitoylation; posttranslational     

Enzymatic acylation of polar dipeptides: Influence of reaction media and molecular environment of functional groups

The enzymatic acylation of polar dipeptides was investigated. First, the Novozym435^®-catalyzed acylation of Lys-Ser, HCl exhibiting three potential acylable sites was carried out in organic media (2-methyl-2-butanol, oleic acid) and in an ionic liquid ([Bmim]⁺[PF₆]^?). In these reactions, the chemo-selectivity of the acylation was exclusively in favour of the N?-lysine acylation and the efficiency (substrate conversion) was demonstrated to be under control of the peptide solubility. The use of [Bmim]⁺[PF₆]^? permitted to significantly improve the dipeptide solubility, and to enhance both substrates conversion and initial rates of acylation reaction. In the three reaction media used, the O-acylated derivative of the dipeptide was never detected suggesting a weak reactivity of the serine hydroxyl group due to its molecular environment and particularly to the presence of a free carboxylic group known for its electroattractor property.Last, the acylation of a natural dipeptide (carnosine), exhibiting a very low solubility in organic solvents, was also performed. Carnosine was successfully N-acylated in 2-methyl-2-butanol, and a yield of 39% was obtained when improving the substrate solubility: a better dispersibility was obtained by application of a high pressure on the reaction medium just before starting the reaction.