Biology subjects

Kornitzer, D.

Publications and source records attributed to Kornitzer, D..

5 recordsLinked to original sources

bioRxiv2026

A fungal hemophore relay mediates heme transfer via transient protein interactions

The fungal pathobiont Candida albicans acquires heme from host proteins via a set of soluble and anchored extracellular CFEM-type hemophores that can capture the bound heme and exchange it, eventually delivering it to the cell membrane for endocytosis into the yeast cell. Yet the molecular mechanism by which the heme is transferred through this protein cascade across the cell envelope remains unclear. To address this mechanism, we developed a set a fusions of three C. albicans hemophores with fluorescent proteins. Fluorescence of these fusion proteins is strongly quenched when heme is bound to the hemophore moiety, enabling to measure heme transfer instantaneously. Kinetic analysis of the different transfer reactions reveals that heme transfer from the host protein to the CFEM hemophores and between the CFEM hemophores are governed by different regimes. Kinetics of heme transfer from hemoglobin or serum albumin to the CFEM hemophores is mainly first-order, suggesting that heme stochastically released from host proteins is captured by the hemophores. In contrast, transfer of heme between the hemophores is near second-order, consistent with a mechanism requiring protein-protein interactions. To confirm this, we show that CFEM hemophores can interact in homodimeric and heterodimeric complexes. Furthermore, while dimerization-defective mutants of the soluble hemophore Csa2 are proficient in heme binding and extraction, they are defective in heme transfer. This supports a model of heme transfer by direct interaction between the members of the fungal hemophore cascade. SIGNIFICANCEAcquisition of extracellular heme as iron or heme sources is common in microorganisms, and particularly prevalent among pathogenic organisms that must contend with an iron-poor host environment. To extract heme from host proteins, microorganisms deploy various systems that include extracellular soluble and cell-anchored hemophores. Here we describe a new approach for monitoring heme binding and transfer in real time, based on the development of fluorescent derivatives of fungal hemophores. These novel reagents open a new window on the study of a common virulence factor of microbial pathogens.

microbiology↗

bioRxiv2022

Regulation of heme utilization and homeostasis in Candida albicans

Heme (iron-protoporphyrin IX) is an essential but potentially toxic cellular cofactor. While most organisms are heme prototrophs, many microorganisms can utilize environmental heme as iron source. The pathogenic yeast Candida albicans can utilize host heme in the iron-poor host environment, using an extracellular cascade of soluble anchored hemophores, and plasma membrane ferric reductase-like proteins. To gain additional insight into the C. albicans heme uptake pathway, we performed an unbiased genetic selection for mutants resistant to the toxic heme analog Ga3+-protoporphyrin IX at neutral pH, and a secondary screen for inability to utilize heme as iron source. Among the mutants isolated were the genes of the pH-responsive RIM pathway, and a zinc finger transcription factor, HUF1. In the presence of hemin in the medium, HUF1 is induced, the Huf1 protein is stabilized and Huf1-GFP localizes to the nucleus. In the huf1 mutant, cytoplasmic heme levels are elevated, while influx of extracellular heme is lower. Gene expression analysis indicated that in the presence of extracellular hemin, Huf1 activates the heme oxygenase HMX1, which breaks down excess cytoplasmic heme, while at the same time it also activates all the known heme uptake genes. These results indicate that Huf1 is a heme-responsive transcription factor that plays a role both in cytoplasmic heme homeostasis and in utilization of extracellular heme. The induction of heme uptake genes by Huf1 under iron satiety indicates that preferential utilization of host heme can be a dietary strategy in a heme prototroph.

microbiology↗

bioRxiv2022

Ferric reductase-related proteins mediate fungal heme acquisition

Heme can serve as iron source in many environments, including the iron-poor animal host environment. The fungal pathobiont Candida albicans expresses a family of extracellular CFEM hemophores that capture heme from host proteins and transfer it across the cell wall to the cell membrane, to be endocytosed and utilized as heme or iron source. Here we identified Frp1 and Frp2, two ferric reductase (FRE)-related proteins that lack an extracellular N-terminal substrate-binding domain, as being required for hemoglobin heme utilization and for sensitivity to toxic heme analogs. Frp1/2 redistribute from the ER to the plasma membrane in the presence of hemoglobin, consistent with a direct role in heme trafficking. Expression of Frp1 with the CFEM hemophore Pga7 can promote heme utilization in Saccharomyces cerevisiae as well, confirming the functional interaction between these proteins. Sequence and structure comparison reveals that the CFEM hemophores are related to the FRE substrate-binding domain that is missing in Frp1/2. We conclude that Frp1/2 and the CFEM hemophores form a functional complex that evolved from ferric reductases to enable extracellular heme uptake.

microbiology↗

bioRxiv2020

Using genetically encoded heme sensors to probe the mechanisms of heme uptake and homeostasis in Candida albicans

Candida albicans is a major fungal pathogen that can utilize hemin and hemoglobin as iron sources in the iron-scarce host environment. While C. albicans is a heme prototroph, we show here that it can also efficiently utilize external heme as a cellular heme source. Using genetically encoded ratiometric fluorescent heme sensors, we show that heme extracted from hemoglobin and free hemin enter the cells with different kinetics. Heme supplied as hemoglobin is taken up via the CFEM (Common in Fungal Extracellular Membrane) hemophore cascade, and reaches the cytoplasm over several hours, whereas entry of free hemin via CFEM-dependent and independent pathways is much faster, less than an hour. To prevent an influx of extracellular heme from reaching toxic levels in the cytoplasm, the cells deploy Hmx1, a heme oxygenase. Hmx1 was previously suggested to be involved in utilization of hemoglobin and hemin as iron sources, but we find that it is primarily required to prevent heme toxicity. Taken together, the combination of novel heme sensors with genetic analysis revealed new details of the fungal mechanisms of heme import and homeostasis, necessary to balance the uses of heme as essential cofactor and potential iron source against its toxicity.

microbiology↗

bioRxiv2020

Rub1/NEDD8, a ubiquitin-like modifier, is also a ubiquitin modifier

Of all ubiquitin-like small protein modifiers, Rub1/NEDD8 is the closest kin of ubiquitin in sequence and in structure. Despite their profound similarities, prevalence of ubiquitin and of Rub1 is starkly different: targets of ubiquitin modification reach into the thousands, whereas unique targets of Rub1/NEDD8 appear limited to one family of proteins, Cullins. This distinction is likely due to dedicated E1 activating enzymes that select either one or the other and relay the modifier until it is covalently attached to a target. To convert typical neddylation targets for modification by ubiquitin, and vice versa, we designed reciprocal substitutions at position 72 of Rub1 and of ubiquitin to render them substrates for activation by their non-cognate E1 activating enzymes. We found that this single amino acid is sufficient to distinguish between Ub and Rub1 in living cells, and determine their targets. Thus, modification of Cullins by UbR72T could compensate for loss of Rub1, even as it maintained its ability to polymerize and direct conjugates for degradation. Conversely, Rub1T72R activated by ubiquitin-activating enzyme entered into the ubiquitination cascade, however was not efficiently polymerized, essentially capping polyubiquitin chains. Upon shortage of free ubiquitin under stress, even native Rub1 spilled-over into the ubiquitinome suppressing polyubiquitination. By contrast, the need to maintain monomeric modifications on unique targets is a likely explanation for why the Rub1-activating enzyme strictly discriminates against ubiquitin. Swapping Rub1 and ubiquitin signals uncovered a reason for maintaining two separate pathways across eukaryotic kingdom.

biochemistry↗