All about that fat: Lipid modification of proteins in Cryptococcus neoformans

A new generation of artificial proteins, derived from alpha-helicoidal HEAT-like repeat protein scaffolds (αRep), was previously characterized as an effective source of intracellular interfering proteins. In this work, a phage-displayed library of αRep was screened on a region of HIV-1 Gag polyprotein encompassing the C-terminal domain of the capsid, the SP1 linker and the nucleocapsid. This region is known to be essential for the late steps of HIV-1 life cycle, Gag oligomerization, viral genome packaging and the last cleavage step of Gag, leading to mature, infectious virions. Two strong αRep binders were isolated from the screen, αRep4E3 (32 kDa; 7 internal repeats) and αRep9A8 (28 kDa; 6 internal repeats). Their antiviral activity against HIV-1 was evaluated in VLP-producer cells and in human SupT1 cells challenged with HIV-1. Both αRep4E3 and αRep9A8 showed a modest but significant antiviral effects in all bioassays and cell systems tested. They did not prevent the proviral integration reaction, but negatively interfered with late steps of the HIV-1 life cycle: αRep4E3 blocked the viral genome packaging, whereas αRep9A8 altered both virus maturation and genome packaging. Interestingly, SupT1 cells stably expressing αRep9A8 acquired long-term resistance to HIV-1, implying that αRep proteins can act as antiviral restriction-like factors.

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“…1c ). The coding sequence of an N-myristoylation signal 22 , 52 was inserted to the 5′-end of the αRep-GFP genes (Fig. 1c ).…”

Section: Resultsmentioning

confidence: 99%

Alpha-helicoidal HEAT-like Repeat Proteins (αRep) Selected as Interactors of HIV-1 Nucleocapsid Negatively Interfere with Viral Genome Packaging and Virus Maturation

Hadpech

Nangola

Chupradit

et al. 2017

Sci Rep

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“…The procedures for using the chemical reporter system were similar to those used to identify palmitoylated proteins in various organisms, including the fungus Cryptococcus neoformans (52, 53). Briefly, palmitoylated proteins were purified from conidia through acyl-biotin exchange following the same procedure as previously described (54, 55).…”

Section: Methodsmentioning

confidence: 99%

Interactions between Melanin Enzymes and Their Atypical Recruitment to the Secretory Pathway by Palmitoylation

Upadhyay

Lin

2016

mBio

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Melanins are biopolymers that confer coloration and protection to the host organism against biotic or abiotic insults. The level of protection offered by melanin depends on its biosynthesis and its subcellular localization. Previously, we discovered that Aspergillus fumigatus compartmentalizes melanization in endosomes by recruiting all melanin enzymes to the secretory pathway. Surprisingly, although two laccases involved in the late steps of melanization are conventional secretory proteins, the four enzymes involved in the early steps of melanization lack a signal peptide or a transmembrane domain and are thus considered “atypical” secretory proteins. In this work, we found interactions among melanin enzymes and all melanin enzymes formed protein complexes. Surprisingly, the formation of protein complexes by melanin enzymes was not critical for their trafficking to the endosomal system. By palmitoylation profiling and biochemical analyses, we discovered that all four early melanin enzymes were strongly palmitoylated during conidiation. However, only the polyketide synthase (PKS) Alb1 was strongly palmitoylated during both vegetative hyphal growth and conidiation when constitutively expressed alone. This posttranslational lipid modification correlates the endosomal localization of all early melanin enzymes. Intriguingly, bioinformatic analyses predict that palmitoylation is a common mechanism for potential membrane association of polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) in A. fumigatus. Our findings indicate that protein-protein interactions facilitate melanization by metabolic channeling, while posttranslational lipid modifications help recruit the atypical enzymes to the secretory pathway, which is critical for compartmentalization of secondary metabolism.

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“…The S ‐acylation of proteins involves the reversible addition of an acyl group to a cysteine residue, and the covalent attachment of fatty acids to a protein can increase the hydrophobicity and interaction of the protein with membranes (Santiago‐Tirado and Doering, 2016; Zheng et al, 2019). Our results indicate that the S ‐acylation and the resulting membrane association are required for NPC4 to function in plant stress responses.…”

Section: Discussionmentioning

confidence: 99%

Acylation of non‐specific phospholipase C4 determines its function in plant response to phosphate deficiency

et al. 2021

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Non-specific phospholipase C (NPC) is involved in plant growth, development and stress responses. To elucidate the mechanism by which NPCs mediate cellular functions, here we show that NPC4 is S-acylated at the C terminus and that acylation determines its plasma membrane (PM) association and function. The acylation of NPC4 was detected using NPC4 isolated from Arabidopsis and reconstituted in vitro. The C-terminal Cys-533 was identified as the S-acylation residue, and the mutation of Cys-533 to Ala-533 in NPC4 (NPC4 C533A ) led to the loss of S-acylation and membrane association of NPC4. The knockout of NPC4 impeded the phosphate deficiency-induced decrease of the phosphosphingolipid glycosyl inositol phosphoryl ceramide (GIPC), but introducing NPC4 C533A to npc4-1 failed to complement this defect, thereby supporting the hypothesis that the non-acylated NPC4 C533A fails to hydrolyze GIPC during phosphate deprivation. Moreover, NPC4 C533A failed to complement the primary root growth in npc4-1 under stress. In addition, NPC4 in Brassica napus was S-acylated and mutation of the S-acylating cysteine residue of BnaC01.NPC4 led to the loss of S-acylation and its membrane association. Together, our results reveal that S-acylation of NPC4 in the C terminus is conserved and required for its membrane association, phosphosphingolipid hydrolysis and function in plant stress responses.

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All about that fat: Lipid modification of proteins in Cryptococcus neoformans

Cited by 10 publications

References 82 publications

Alpha-helicoidal HEAT-like Repeat Proteins (αRep) Selected as Interactors of HIV-1 Nucleocapsid Negatively Interfere with Viral Genome Packaging and Virus Maturation

Alpha-helicoidal HEAT-like Repeat Proteins (αRep) Selected as Interactors of HIV-1 Nucleocapsid Negatively Interfere with Viral Genome Packaging and Virus Maturation

Interactions between Melanin Enzymes and Their Atypical Recruitment to the Secretory Pathway by Palmitoylation

Acylation of non‐specific phospholipase C4 determines its function in plant response to phosphate deficiency

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